The change (on day 40 of the operating period) from gentle continuous mixing by biogas recirculation to vigorous continuous mixing by liquid recirculation with a peristaltic pump showed a very different outcome. The vigorously mixed bioreactor showed an immediate and steady decline in the VMPRs (Figure 4.5A). Feeding had to be terminated on day 53 to prevent complete digester failure because the VFA levels in the effluent had increased from an average of 256.3 mg acetate/L on day 40 to 2846 mg acetate/L on day 52. Biomass washout was partly to blame, because biomass levels in the effluent had increased from 10 gVS/L on day 40 to 12gVS/L on day 41, resulting in a steady loss in VS biomass in the vigorously mixed bioreactor (the data for the control bioreactor remained constant; Figure 4.5B). In addition, slot-blot hybridization techniques showed that a severe loss in the concentration of the predominant methanogens was the result of the change in mixing intensity. That is, the relative 16S rRNA levels for M. concilii and for the order Methanomicrobiales decreased to levels lower than 1% on day 52 of the operating period (Figure 4.5C, D). This result supports the hypothesis postulated by Stroot et al. (2001) and McMahon et al. (2001) that vigorous, continuous mixing inhibited relationships between syntrophs and their methanogenic partners by destroying their juxtaposed position. In addition, it is also in agreement with Hoffmann et al. (2008) , who found that the filamentous concentrations of M. concilii was negatively affected by increased mixing intensities. Thus, vigorous mixing decreased the concentration of methanogens, which had been slowly built up during the long start-up periods (described in the ASBRs section). With the loss of methanogenic activity, the high-rate system became severely overloaded, and combined with a declining biomass concentration, unstable condi­tions emerged within weeks.