Thickness determines microbial community structure and function in nitrifying biofilms via deterministic assembly

Abstract

Microbial biofilms are ubiquitous in aquatic environments where they provide important ecosystem functions. A key property believed to influence the community structure and function of biofilms is thickness. However, since biofilm thickness is inextricably linked to external factors such as water flow, temperature, development age and nutrient conditions, its importance is difficult to quantify. Here, we designed an experimental system in a wastewater treatment plant whereby nitrifying biofilms with different thicknesses (50 or 400 µm) were grown in a single reactor, and thus subjected to identical external conditions. The 50 and 400 µm biofilm communities were significantly different. This beta-diversity between biofilms of different thickness was primarily caused by deterministic factors. Turnover (species replacement) contributed more than nestedness (species loss) to the beta-diversity, i.e. the 50 µm communities were not simply a subset of the 400 µm communities. Moreover, the two communities differed in the composition of nitrogen-transforming bacteria and in nitrogen transformation rates. The study illustrates that biofilm thickness alone is a key driver for community composition and ecosystem function, which has implications for biotechnological applications and for our general understanding of biofilm ecology.

Figure 1

Biofilm structure shown by EPS staining of cryosections. The biofilm-water interface is the upper side. (a) Z400 biofilm. (b) Z50 biofilm. Scale bar: 100 µm. (c): Z400 (up) and Z50 (down) biofilm carriers; a ruler in cm is shown for size comparison.

Figure 2

(a) Richness (⁰D), diversity (¹D) and evenness (¹D/⁰D) for the Z50 and Z400 biofilms. (b) PCoA based on the Sørensen index (β_sor).

Figure 3

(a) Standardized effect size for the Sørensen index (β_sor); dashed lines indicate SES values of +2 and −2. (b) β_sor, β_sne (dissimilarity due to nestedness) and β_sim (turnover) values; the sum of β_sim and β_sne is β_sor. (c) Beta diversity ratio. Values were estimated for pairwise comparisons among Z400 replicates (n = 10), Z50 replicates (n = 10) and between the two groups.

Figure 4

(a) Relative read abundance of nitrifiers and anammox bacteria in Z50 and Z400. (b) Relative read abundance multiplied by total solids (TS) measurements for each carrier type. (c) Biovolume fractions of nitrifiers and anammox bacteria, as measured by qFISH.

Figure 5

(a) FISH image of a Z400 biofilm cryosection; the water-biofilm interface is on the top. Green: Nitrosomonas. Red: Nitrospira. Yellow: Nitrotoga. Blue: Brocadia. Grey: SYTO. (b) FISH image of a Z50 biofilm cryosection; the water-biofilm interface is on the top. Green: Nitrosomonas. Red: Nitrospira. Yellow: Nitrotoga. Grey: SYTO. (c) FISH-based population distribution at different biofilm depths in Z400.

Figure 6

DO concentrations profiles in the Z50 and Z400 biofilms. The shaded regions show ranges of DO concentration profiles resulting from different assumption about the fraction of the total dry solids on the carriers that is active bacteria. The dashed horizontal lines show the biofilm-liquid interface.

Figure 7

Potential conversion rates by carrier type during aerobic oxidation of NH4+ (a), aerobic oxidation of NO₂⁻ (b) and anoxic oxidation of NH4 (c) during batch tests. Significant differences between Z50 and Z400 (ANCOVA, p < 0.05) are shown with (*). Red: Z400, Blue: Z50.