In recent decades, with a growing world population, aquaculture has increased its activity to levels where environmental sustainability is compromised. In response to this problem, Biofloc technology has emerged. This technology consists of the use of microbial aggregates with the capacity to maintain water quality and provide food for cultured aquatic organisms. In this way, the toxic forms of nitrogen are reused while acting as a probiotic, providing a partial replacement of commercial food. The availability of stable mature Bioflocs for the reactivation of production tanks represents a clear competitive advantage. For this, it is essential to know the most appropriate cryopreservation technique for these microbial inoculums. Under this perspective, the present work has focused on the study of the microbiota of mature Biofloc subjected to 5 different cryopreservation methods.
Material and Methods
The effectiveness of refrigeration, freezing, freezing with addition of glycerol (10%), freezing with addition of glucose (10%) and lyophilization of the samples in skimmed milk was evaluated. First, samples of mature and concentrated Biofloc were generated. Subsequently, they were subjected to the different cryopreservation scenarios and analyzed by cultivar microbiological techniques. Total, heterotrophic and nitrifying bacterial populations were evaluated in this study. Total bacteria were measured with the Guava Muse® Cell Analyzer cytometer (Luminex Corporation, USA); Heterotrophic bacteria were cultured at 30 °C for 48 h in Marine Broth medium (Panreac AppliChem); Nitrifying bacteria, were cultured at room temperature for 14 days in a medium with specific components with a minimum mineral composition described by Rodriguez et al., 2017. Finally, we performed a metataxonomic analysis, in order to know the biodiversity and metataxome present in the Biofloc of each cryopreservation treatment. To know the uncultured microbiota of the samples, sequencing of the variable regions V3-V4 16S rRNA gene was carried out. DNA extraction was performed with the Power Soil Kit, Quiagen (Qiagen N.V., Hilden, Germany). After amplification of the genetic material by PCR, sequencing of the samples was performed with a MiSeq PE300 kit (Illumina Inc., San Diego, CA, United States). The quality of the demultiplexed FASTQ files was checked through FastQC software; and pairwise assembly of R1 and R2 reads was done with FLASH. Sequences were analyzed using QIIME2 software version 2021-04. Regarding biodiversity indices, two alpha biodiversity indices were calculated: the Chao1 index (richness of samples) and the Shannon index (diversity of samples).
Results and discusión
When analyzing the culture, it was observed that both freeze-drying and refrigeration resulted in a reduction of the microbial load, and, therefore, it was not an effective method when trying to preserve the viability of the microbiome (Figure 1). Freezing at -80 °C, both with the addition of 10% glucose and 10% glycerol, ensured the maintenance of a high microbial diversity, with a predominance of marine heterotrophic bacteria over nitrifying bacteria.
On the other hand, meta-taxonomic analysis revealed that the preservation methods that provided the highest richness to the biofloc were freeze-drying (Figure 2), followed by treatment with 10% glycerol. The result of the meta-taxonomic analysis coincided with the fact that refrigeration was not an optimal method for preserving the total microbiota, but also indicated that the use of 10% glucose generated a lower richness in the microbial community. The biodiversity present in the mature Biofloc was high, being constituted by a high number of diverse species. The Flavobacteriaceae family was the most representative microbial group, presenting a higher relative abundance in half of the preservation methods analyzed.
Rodríguez, A., Mau, S., Piedra, L., Jiménez, R., Herrera, J. P. (2017). Isolation of ammonium- and nitriteoxidizing bacterial strains from soil, and their potential use in the reduction of nitrogen in household waste water. International Journal of Tropical Biology, 64, 1527-1539.
These results are part of the I+D+i Research Project: “Optimizing shrimp feeding and nutrition in biofloc system (BioFlango)” (PID2020-114574RB-C21), and the research contract of J. Gómez-Aguilera was supported by European Union Next Generation-Plan of Recuperación-Ministerio de Ciencia e Innovación-Gobierno de España, Conselleria d’innovació, Universitats, Ciència i Societat Digital of Generalitat Valenciana (INVEST/2022/434).