Fulvic acid: a novel biofilm denitrification enhancer

Abstract

ABSTRACT To address the challenges of prolonged start-up cycles and poor operational stability in biofilm reactors for treating high ammonia nitrogen pollution in aquaculture effluent, this study adopted fulvic acid (FA) as a novel biofilm enhancer to systematically analyze its regulatory mechanisms on denitrification performance and biofilm formation in sequencing batch biofilm reactors (SBBR). Through microplate biofilm formation experiments and SBBR process validation, researchers found that the biofilm formation index reached 1.4 at an FA concentration of 1200 mg/L. FA improved biofilm structural stability by promoting extracellular polymer secretion, yielding a 22-fold increase in biofilm thickness compared with the control group (the biofilm thickness peaked at 2208.3 μm). Additionally, high FA concentrations (>500 mg/L) strengthened denitrification processes by facilitating carbon source supply and electron transfer, enriched denitrifying functional bacteria such as Comamonadaceae, and inhibited Gram-positive bacteria (e.g., Actinobacteriota). The removal efficiencies of NH4+-N, NO₃−-N, and total nitrogen reached 79.67%, 80% and 62.4%, respectively. This study reveals that FA enhances SBBR denitrification through dual mechanisms of carbon source supplementation and physicochemical property regulation, providing an efficient and cost-effective regulatory strategy for aquaculture effluent treatment. GRAPHICAL ABSTRACTEight visuals including charts, diagrams, and a chemical structure summarize fulvic acid effects on biofilm growth and nitrogen removal.The figure shows a collage of eight visuals summarizing an experiment on fulvic acid and nitrogen removal. In the upper left, a tray of ninety six wells is labeled 96 well plates. To its right, a line chart labeled Effects of fulvic acid concentrations on biofilm formation plots fulvic acid concentration in milligrams per liter on the horizontal axis from 0 to 6000 with irregular tick intervals and biofilm formation index on the vertical axis from 0.0 to about 1.6; the curve rises sharply at low concentrations, then fluctuates with several peaks and valleys. Near the center right, two schematic illustrations compare biofilm on a flat surface: the left sketch shows sparse colonies labeled R1 control group, and the right sketch shows a tall block of dense thick biofilm labeled thickness 2208.3 micrometers. At the lower left, a ball and stick chemical structure is labeled fulvic acid. Along the bottom, two circular labels marked ammonium nitrogen and nitrate nitrogen flank arrows pointing toward a circle labeled nitrogen, representing nitrification followed by denitrification. In the lower center, a pie chart labeled phylum level community pie chart shows multiple colored slices with one large sector labeled Proteobacteria 42.14 percent and another labeled Bacteroidota 17.17 percent; smaller slices represent many other taxa. Next to it, a heat map grid labeled intergroup analysis of key nitrogen metabolism lists various nitrogen transformation pathways down the side with squares shaded from low to high intensity. In the lower right, a line graph labeled total nitrogen removal rate plots time in days on the horizontal axis from 0 to 42 and total nitrogen removal rate in percent on the vertical axis from 0 to 100 with tick marks every 10 percent. Multiple colored lines labeled R1 through R10 oscillate over time; some lines remain low while others increase to moderate or higher removal before declining. Text at the bottom states total nitrogen removal rate reached 62.4 percent. All data are approximate.