Unique effects of predation on heterotrophic and nitrifying membrane-aerated biofilm reactors (MABRs)

The membrane-aerated biofilm reactor (MABR) is an emerging wastewater treatment technology that uses O₂-supplying membranes as a biofilm support.  Because O₂ is supplied from the biofilm base instead of the bulk liquid, MABR biofilms have distinct microbial community structures and behavior.  Past research showed that protozoan predation in MABR biofilms can create a unique void layer at the base of the biofilm. We hypothesized that the void layer could weaken the biofilm and promote sloughing, and investigated this with heterotrophic and nitrifying MABR biofilms.

Biofilms were grown in flat-sheet MABRs (“Base Case”). As a control, a reactor was supplied with cycloheximide in the media to suppress protozoa (“Suppressed Predation”). Each condition was run in triplicate. A rheometer was used to measure biofilm mechanical strength, and MABR flow cells were used to explore detachment. The biofilms were imaged using optical coherence tomography (OCT) (Ganymede, Thorlabs, Germany), and the images were digitally processed to quantify the biofilm thickness and internal void areas. In all tests, the biofilm was first grown to steady-state, as determined by effluent substrate concentrations and biofilm thicknesses.

In the heterotrophic biofilms, predation increased the internal void ratio from 6 ± 7 % to 50 ± 16 %.  The storage modulus was 1,780 ± 1,180 Pa for the Base Case, compared to 9,800 ± 4,290 Pa for Suppressed Predation. Similarly, the loss modulus was 1,580 ± 729 Pa for the Base Case and 363 ± 189 Pa for Suppressed Predation. When subjected to an increased flow, the biofilm loss was 44 ± 24 % for the flow cell with predation, while only 7 ± 9 % for the control.

In the nitrifying biofilms, predation resulted in a greater fraction of internal voids, at 69 ± 6 % for the Base Case vs. 54 ± 5 % for Suppressed Predation. Also, the increased void ratio by predation reduced the biofilm viscosity and elasticity, resulting in greater detachment.

The loss modulus with Base Case and Suppressed Predation was 242 ± 135 Pa and 3640 ± 1860 Pa, respectively. The storage modulus was 1650 ± 853 Pa with Base Case and 23300 ± 11500 Pa with Suppressed Predation. The relative detached area of biofilm with Base Case and Suppressed Predation were 18 ± 12 and 4 ± 5 %, respectively. Thus, the greater detachment for the Base Case was consistent with the weaker mechanical properties.  Predation also decreased the nitrification fluxes and promoted partial nitrification. The selective loss of NOB, as confirmed by fluorescence in-situ hybridization (FISH) and qPCR, may be due to the larger size of AOB clusters, providing greater resistance to predation.

These findings suggest that the effects of protozoa may need to be considered to predict the behavior of heterotrophic and nitrifying MABRs. Also, a better understanding of the microbial ecology of protozoa may lead to more effective MABR operational strategies.