Cultivation via classical agar plate (CAP) approaches are widely used to study microbiomes yet are time-consuming. An alternative approach is the application of single-cell dispensing (SCD), which allows high-throughput, label-free sorting of microscopic particles. We aimed to benchmark a new anaerobic SCD workflow to determine its efficiency in cultivating bacteria from the human gut. Speed, efficacy, cultured fractions, and retrieved bacterial diversity were compared to a CAP workflow using faecal communities on three rich culture media. The SCD approach decreased the experimental time to obtain pure cultures from 17 +/- 4 to 5 +/- 0 days. Growth output for the SCD approach ranged from 19% to 51% of 800 sorted cells depending on the sample and the culture medium. The diversity of isolates obtained by SCD was comparable to the CAP method (21 +/- 3 species out of 45 colonies analysed per sample). The fraction of molecular species (16S rRNA gene amplicons) covered by the isolates in each sample was comparable for both methods (29.3 +/- 5.8% and 29.8 +/- 3.1% for SCD and CAP, respectively, representing a cumulative relative abundance of 48.1 +/- 12.0% and 51.5 +/- 17.8%). This cultured fraction increased to 43.5 +/- 15.4% (73.5 +/- 13.8% rel. abundance) for the SCD approach when sequencing the sorted bacteria directly after growth as bulk biomass from 2,400 dispensed single cells without downstream identification of single strains. SCD-based cultivation also captured species not detected by sequencing (16 +/- 5 per sample, including seven novel taxa). From this work, 82 human gut bacterial species across five phyla (Actinobacteriota, Bacteroidota, Desulfobacterota, Firmicutes, Proteobacteria) and 24 families were obtained, including the first cultured member of 11 novel genera, and 11 novel species that were fully characterized taxonomically. In this work we show that SCD accelerates the recovery of pure cultures of anaerobic microbes. With the increasing awareness of the significance of inter-individual microbiota diversity, this SCD approach will facilitate the establishment of personalised culture collections in future.
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