Stratification of activity and bacterial community structure in biofilms grown on membranes transferring oxygen

Abstract

Previous studies have shown that membrane-aerated biofilm (MAB) reactors can simultaneously remove carbonaceous and nitrogenous pollutants from wastewater in a single reactor. Oxygen is provided to MABs through gas-permeable membranes such that the region nearest the membrane is rich in oxygen but low in organic carbon, whereas the outer region of the biofilm is void of oxygen but rich in organic carbon. In this study, MABs were grown under similar conditions but at two different fluid velocities (2 and 14 cm s(-1)) across the biofilm. MABs were analyzed for changes in biomass density, respiratory activity, and bacterial community structure as functions of biofilm depth. Biomass density was generally highest near the membrane and declined with distance from the membrane. Respiratory activity exhibited a hump-shaped profile, with the highest activity occurring in the middle of the biofilm. Community analysis by PCR cloning and PCR-denaturing gradient gel electrophoresis of 16S rRNA genes demonstrated substantial stratification of the community structure across the biofilm. Population profiles were also generated by competitive quantitative PCR of gene fragments specific for ammonia-oxidizing bacteria (AOB) (amoA) and denitrifying bacteria (nirK and nirS). At a flow velocity of 14 cm s(-1), AOB were found only near the membrane, whereas denitrifying bacteria proliferated in the anoxic outer regions of the biofilm. In contrast, at a flow velocity of 2 cm s(-1), AOB were either not detected or detected at a concentration near the detection limit. This study suggests that, under the appropriate conditions, both AOB and denitrifying bacteria can coexist within an MAB.

FIG. 1.

Hypothetical structure of an MAB treating wastewater.

FIG. 2.

MAB thickness as a function of time for biofilms grown at a fluid velocity of 14 cm s⁻¹. •, air-fed biofilm; ○, oxygen-fed biofilm. Error bars represent the standard deviations of the means (n = 9).

FIG. 3.

Dissolved oxygen profiles of air-fed (a) and oxygen-fed (b) biofilms grown at a fluid velocity of 14 cm s⁻¹. Different profiles represent data collected on different days.

FIG. 4.

Protein concentration profiles within MABs grown at fluid velocities of 2 cm s⁻¹ (a) and 14 cm s⁻¹ (b). •, air-fed biofilm; ○, oxygen-fed biofilm. Error bars represent the standard deviations of the means (n = 3).

FIG. 5.

Profiles of respiratory activity as measured by the reduction of INT to INT-formazan within MABs grown at fluid velocities of 2 cm s⁻¹ (a) and 14 cm s⁻¹ (b). •, air-fed biofilm; ○, oxygen-fed biofilm.

FIG. 6.

DGGE of PCR-amplified 16S rRNA gene fragments of an oxygen-fed biofilm grown at a fluid velocity of 2 cm s⁻¹. Individual gel lanes are identified by a number indicating the distance (in millimeters) from the membrane where the sample was collected. Letters and arrows identify specific bands that were excised from the gel and sequenced. The results of the nucleotide sequence analysis for these PCR-DGGE bands are shown in Table 2.

FIG. 7.

Population profiles of ammonia-oxidizing (amoA) and denitrifying (nirS and nirK) bacterial populations as determined by competitive quantitative PCR within air-fed (a) and oxygen-fed (b) biofilms grown at a fluid velocity of 14 cm s⁻¹. •, nirS; ○, nirK; ▴, amoA.