Stoichiometric and molecular evidence for the enrichment of anaerobic ammonium oxidizing bacteria from wastewater treatment plant sludge samples

Anammox enrichments were readily developed from seven municipal wastewater treatment plants (WWTPs) sludge, but not with methanogenic granular sludge from two agro-industrial WWTPs. Only 50 d was required for the first evidence of anammox activity from a return activated sludge obtained from a WWTP operated for nutrient removal. The molar ratios of nitrite and ammonium consumption of approximately 1.32 as well as nitrate and dinitrogen gas product ratios of approximately 0.095 provided evidence of the anammox reaction. The presence of anammox was confirmed by polymerase chain reaction (PCR) using primer sets (PLA46F and AMX820R) specific for anammox bacteria. The 16S rRNA gene fragment of anammox bacteria was detected in seven enrichment cultures (ECs) with demonstrated anammox activity but not in the original inocula from which the ECs were derived and also not in the two methanogenic sludge samples, which indicates the PCR predicted the anammox activity. Two genera, Brocadia and Kuenenia, were successfully identified as the Planctomycetes occurring in the clone libraries of successful anammox enrichments. Brocadia dominated in cultures that were respiked extensively; whereas Kuenenia predominated in cultures that were less aggressively respiked. These findings indicate that respiking management may play an important role on selecting the genus of anammox bacteria. The batch enrichment results clearly illustrate that anammox can be readily enriched from municipal sludge from a wide variety of process operations at WWTPs.

► Anammox cultures are readily developed from municipal sludge in batch assay.
► Presence of anammox bacteria was confirmed by stoichiometric and molecular evidence.
► Substrate respiking frequency plays an important role on selecting anammox bacteria.
► Brocadia is enriched in intensively respiked cultures.
► Kuenenia is enriched in less aggressively respiked cultures.