Scaling down – Evaluation of start-up and microbial stabilisation dynamics in five parallel laboratory-scale CSTR biogas system

Laboratory-scale continuous stirred tank reactors (CSTRs) are commonly used to study and optimise anaerobic digestion (AD), yet the time required for such systems to become representative of full-scale processes remains unclear. This study investigated start-up and stabilisation dynamics in five parallel 10-L CSTRs operated as direct scale-downs of a full-scale agricultural biogas plant, using identical inoculum, substrate composition, organic loading rate (2.57 g VS L −1 day −1 ), hydraulic retention time (55 days), and temperature (37 °C). The reactors were monitored for 213 days (3.9 HRTs), with one reactor operated for an additional 330 days. All reactors showed highly reproducible behaviour but experienced a transient disturbance, characterised by volatile fatty acid (VFA) accumulation and fluctuating methane production after approximately one HRT. This disturbance was likely caused by a higher effective daily organic loading rate compared to full scale, resulting from once-daily feeding excluding weekends. Increasing the feeding frequency led to VFA degradation and recovery, and stable process performance was achieved after approximately three HRTs, with methane yields of 285 ± 21 NL CH 4 kg −1 VS. The microbial community showed pronounced dynamics during start-up, including major genus-level shifts within Cloacimonadia and a transition in aceticlastic methanogens from Methanothrix to Methanosarcina compared to the full-scale system. Although microbial composition stabilised after about three HRTs, it remained dynamic during prolonged operation and diverged from the original inoculum. These results demonstrate that even direct scale-down reactors require extended stabilisation and may develop microbial communities that differ from the full-scale process, raising questions about their representativeness.