Effects of modifications to retain protozoa in continuous-culture fermenters on ruminal fermentation, microbial populations, and microbial biomass assessed by two different methods

An important limitation of continuous-culture fermenters is their inability of maintaining microbial populations similar to those observed in the rumen, especially protozoa numbers, which usually decrease markedly or even disappear. Two approaches (a polyurethane-sponge (SP) and a filter system (FIL) for additionally retaining protozoa were tested in continuous culture system already designed to retain protozoa (Muetzel et al., 2009), and their effects on microbial populations, fermentation parameters and microbial biomass were assessed. Two 14-day incubation runs were carried out with 6 fermenters, and in each run two fermenters were randomly assigned to each of the experimental treatments (control, SP and FIL). Total protozoa numbers assessed by microscopic counting were 1.7 and 2.1 times greater in SP and FIL fermenters than in control ones on day 14, although differences did not reach the significance level (P = 0.855). Protozoal DNA concentration on day 14 were 1.6 and 1.4 times greater in SP and FIL fermenters, respectively, than in control ones, but differences were not significant (P = 0.524). Results from protozoal DNA concentrations determined in each fermenter on the different sampling days (n = 48) were positively correlated (P < 0.001) with the numbers of total (r = 0.826), entodiniomorphid (r = 0.824) and holotrich (r = 0.675) protozoa determined by microscopic counting, indicating that both methods are valid to assess protozoa populations. The proportion of holotrich in FIL-fermenters was relatively constant over the incubation period (ranging from 10.5 to 13.3% of total protozoa), but decreased with time in control (from 10.9% at day 2 to 6.7% at day 14) and SP (from 10.9 to 6.9%) fermenters. Neither the bacterial DNA concentration nor the relative abundance of fungal and archaeal DNA were influenced by any of the modifications tested, but values changed over the sampling period (days 10-14). Bacterial DNA concentration increased (P < 0.001) from day 10 to 14 in all fermenters, whereas the relative abundance of fungal and archaeal DNA decreased (P < 0.001). The tested modifications did not affect (P > 0.05) fermentation parameters, which reached a steady-state after 6 days of incubation. Values of microbial biomass determined using purine bases as a microbial marker were significantly correlated with the amount of bacterial plus protozoal DNA (r = 0.794; P = 0.002; n = 12) in each fermenter. In conclusion, the two tested modifications increased the protozoa numbers in continuous-culture fermenters, and the FIL allowed maintaining a proportion of holotrich protozoa similar to that in the ruminal fluid used as inoculum.