Two start-ups with a nitrifying trickling filter carrying different substratum densities (157 and 226 m2 m−3) have been made during winter time. The biofilm development time in both cases was approximately 100 days (nitrification rates between 0.6–1.6 gN m−2 d−1 when fully developed) and the pattern of the increase of the nitrification rate was similar during both the biofilm development periods. The substratum density did not affect the nitrification rate, and thus the total amount of ammonium nitrified in the denser material was higher. Microbial parameters were measured during biofilm development in the denser material. The bacteria were more loosely attached to the surface at the beginning of the biofilm development (50% firmly attached), while 80-100% were firmly attached after 20 days. Irreversible attachment was achieved after 20 days. The bacterial size decreased during the initial phase. Microscopic examination showed that the film consisted of both densely populated and empty areas during the biofilm development period, which indicates that the area was not fully used by the microorganisms. The maximum nitrification rate was reached at around 10 gram biofilm dry weight per square metre.

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