Chicken-manure digestate contains phytohormones with potential biostimulant activity in lettuce

Liquid digestate from anaerobic digestion is a potential liquid fertilizer, but its high water content complicates storage, transport, and application. Beyond basic nutrients, its profile of biostimulants, particularly phytohormones, remains insufficiently characterized. This study used widely targeted metabolomics and targeted UPLC-MS/MS to compare chicken-manure digestate collected before membrane pretreatment (BK) with the final concentrate produced by an industrial U-NRO system (MC). BK and MC represented one composite industrial sample pair, with three independently prepared analytical replicates per matrix rather than independent process replicates. In total, 1465 metabolites were annotated and 49 phytohormone-related analytes were quantified. MC contained higher measured concentrations of several auxin-, salicylic acid-, jasmonic acid-, gibberellin-, cytokinin-, and melatonin-related compounds, whereas the summed abscisic acid-related pool decreased markedly. Pathway annotation based on the detected metabolites suggested several possible metabolic routes associated with the occurrence of phytohormone-related compounds in the digestate. The co-detection of tryptophan and several indole-related metabolites was consistent with possible tryptophan-associated transformations, while phenylalanine- and salicylic-acid-related metabolites suggested a possible association with phenylalanine- and phenylpropanoid-related pathways. In germination assays, 0.33% MC increased seedling length without reducing germination speed, whereas 3.3% MC was phytotoxic. In the pot experiment, no harvest-stage phenotypic trait differed significantly between the full-fertilizer control and the selected 70% fertilizer plus MC. Overall, the study establishes a molecular inventory of an industrial digestate concentrate and defines a preliminary concentration-dependent plant-response range.