## 3 Results and Discussion
### Biofilm Formation on the Perlite Carrier  
Actinobacteria were visually inspected to ensure they formed biofilms after incubation on perlite. When moving the tube containing perlite beads, it was observed that they adhered to each other through the biofilm and were no longer loose in the liquid medium. These characteristics provide the first evidence of biofilm formation on carriers. Next, random beads were removed from each tube to visualize them under a bright field microscope using a Gram stain, looking for the pellets and hyphae on the perlite crystals. Finally, high-vacuum SEM was used to visualize the biofilm inoculant samples at 18.0 kV (Figure 1) and to corroborate the biofilm formation by actinobacteria before the introduction of the bioincolulant in the pots. Biofilm formation was successful in all the treatments (Figure 1), verified by the viable count. Figure 1A-C shows how the perlite grain is entirely covered by the biofilm (indicated by number 1 in such micrographs), indicating that the biofilm induction process is successful under these experimental conditions. Furthermore, it was also possible to corroborate that the actinobacteria hyphae penetrated the carrier internally and externally in all bioincolulant samples, as exemplified by the strain ARI_A20-5 (Figure 1D). Only small areas on the outer surface of the entire perlite without biofilm coating were observed (indicated on the micrographs of Figure 1 with number 2). Only nine actinobacteria genera exhibit the ability to produce biofilms, and even though actinobacteria are widely distributed, there are few studies about biofilm development in the phylum [25]. [PERSON] et al. performed the same CV methodology used in this study [12], testing the biofilm formation with a thermophilic actinobacteria _Thermobifida fusca_. In their research, the formation of biofilms with this bacterium was examined on microscopic glass slides, plastic coverslips, metal wires, Teflon seal tape, and cellulose surfaces. Cell growth was macroscopically visible as mucoid-attached agglomerates, unlike the planktonic mycelial pellets, which did not bind surfaces. They concluded that _T. fusca_ could \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Strain** & **BLAST Analysisa** & **\% Similarity** & **Accession Number** \\ \hline ARI\_A20-3 & _Streptomyces griseorubens_ & 98.16 & KP718552.1 \\ ARI\_A20-5 & _Streptomyces flaveolus_ & 99.7 & KU647253.1 \\ ARI\_A30-32 & _Streptomyces aureus_ & 99.56 & EU841615.1 \\ \hline \hline \end{tabular} \({}^{\text{a}}\)Nearest match.  
\end{table} 
Table 2: Identification of the actinobacterial isolates based on partial sequencing of the 1500-pb region of the 168 rDNA gene. produce biofilm on nutritive surfaces such as chitin and cellulose, but on non-nutritive surfaces; it appeared to be molded by filamentous cells in an unconventional \"spaghetti-like\" form. [12]. Studying biofilm formation in actinobacteria is necessary, especially for the _Streptomyces_ genus. The contributions of the components to matrix integrity are poorly understood at the molecular level, and extract and molecular interactions of extracellular matrix in the actinobacterial biofilm have not been thoroughly described [23; 25; 69]. However, it has been reported that the _Streptomyces_ biofilm producers in a single species (mono-biofilm) preferred rough surfaces as a substrate for adhesion and were more suitable for forming biofilm than in a smooth surface [25], which is the case of the use of perlite beads in this study. Perlite contributes to the stability of the biofilm. It has been documented that some _Streptomyces_ consortia biofilizers occur when fungi serve as the biotic surface to which the bacteria attach [21]. In this work, the potential use of a novel biofilmed solid inoculant on perlite as a carrier using _Streptomyces_ as mono-biofilm or as consortia species that were different from those reported previously [67; 70], has been exposed as an improved biofilmed biofertilizer for uses in agriculture.