# Investigating the Effects of Fish Effluents as Organic Fertilisers on Basil (_Ocimum basilicum_)
## 2 Materials and Methods
### Measurements and Analyses  
Plant yields were measured, and soil analyses were conducted to reveal the composition of the substrate before and after fertilisation with fish effluents. The effects of fish effluent fertilisation on plant growth, health, and quality were assessed by means of vegetable tissue analysis, growth rate calculations, and the overall health and appearance of the crop. The plant yield measurements were undertaken on all plants, and the weight measurements were performed in batches per replicate. The concentration of nitrogen in leaf samples was measured using a CHN analyser (model: Thermo Scientific FlashEA 1112, Waltham, MA, USA) at the University of Greenwich, Medway Campus. The whole leaf mass from each replicate was harvested, freeze-dried, and powdered using a mortar; three samples of the powdered product from each replicate were then analysed.  
The analyses of the macro- and micronutrients in the fish effluent and substrate samples were performed by NRM Labs, the UK's largest independent provider of agronomic and environmental waste analysis for land-based industries, based in Bracknell, UK. The methods are described in Appendix A.  
For soil microbial analysis, three initial substrate samples were taken from each substrate type prior to the beginning of the experiment, and four samples per treatment were taken after three weeks, halfway through the experiment, and on the day the experiment ended. The vials were then frozen at \(-21\)\({}^{\circ}\)C until shipped for analysis by Biome Makers (Sacramento, California). DNA extraction was performed with the DNEasy PowerLyzer PowerSoil kit from Qiagen, Venlo, the Netherlands. To characterise the bacterial, archaeal, and fungal microbial communities associated with bulk soils and rhizosphere samples, the 16S rRNA and internal transcribed spacer (ITS) marker regions were selected. The 16S primer was used for sequencing bacteria and archaea, and the ITS primer was used for sequencing fungi. Primers were removed from paired-end reads using Cutadapt 3.5, and the trimmed reads were merged with a minimum overlapping of 100 nucleotides. Next, the sequences were quality-filtered by Expected Error with a maximum value of 1.0. Because of their highly conserved length, 16S reads were subjected to an additional filtering step in order to remove extreme sequence lengths. After quality pre-processing, reads with single nucleotide differences were iteratively clustered together to form ASVs (Amplicon Sequencing Variants) using Swarm v3. De novo chimaeras and remaining singletons were subsequently removed. Finally, the ASVs were compared against Biome Makers' proprietary internal reference database of amplicons using a global alignment, with 97% identity to select the best hit; in cases of multiple best hits with identical qualities, the pipeline automatically adjusted the ASV result resolution to the nearest common ancestor of the hits' taxonomies, which can decrease it to genus or family level. The reference database of amplicons was built using internal manually curated taxonomies from the latest version available of SILVA 138.1 for 16S sequences.