A novel method to estimate biofilm activity based on enzymatic oxygen release from hydrogen peroxide decomposition

Biofilm is central for biological water treatment processes in recirculating aquaculture systems (RAS). A lack of suitable methods for quantifying biofilm activity, however, makes it difficult to assess and compare the microbial status of biofilm. This type of information of the biofilm will be useful to assess the colonization status of nitrifying biocarriers or to evaluate the effect of disinfectants on the biofilm activity. Here we introduce a novel assay for rapid assessment of microbial activities in the biofilm attached on bioelements from a RAS biofilter. The assay consisted of an intermittent respirometer platform where biofilter elements were exposed to 10 mg/L hydrogen peroxide (H₂O₂) for 1 h, following concurrent measurements of oxygen release from the decomposition of H₂O₂ caused by biofilm-associated enzymes. A different number of colonized, mature bioelements from a moving bed biofilter in a freshwater RAS were tested with repeated H₂O₂ exposure, and compared against their autoclaved forms. A substantial increase in dissolved oxygen (DO) concentration (0.92-2.31 mg O₂/L) occurred with mature bioelements during 1 h of H₂O₂ exposure, compared to small amounts of DO release (≤0.27 mg O₂/L) with autoclaved bioelements. This substantiates that H₂O₂ decomposition by biofilm is mainly governed by microbial enzymatic activities. A monomolecular model fitted well with the observed oxygen release profiles of tested mature bioelements after H₂O₂ exposure (R² > 0.98). The kinetic rate constant of net oxygen release (k , h^-1) was proportional (R² for linear fit = 0.99) to the number of mature bioelements tested. Repeated exposure of H₂O₂ to the same bioelements did not change k , which indicates that 10 mg/L H₂O₂ with an exposure time of 1 h does not suppress enzymatic activity in biofilm. Our study provides a new rapid method that allows simple quantification of microbial activity in biofilm samples from aquaculture systems, which could potentially be also applied to study biofilm from wastewater treatment plants and other industries.