Microbial ecology of intermittently aerated reactors treating swine wastewater molecular approaches for identifying key nitrogen-removing bacteria /

by Mota, Cesar.

MOTA, CESAR. Microbial ecology of intermittently aerated reactors treating swine wastewater: molecular approaches for identifying key nitrogen-removing bacteria. (Under the direction of Dr. Francis L. de los Reyes III). Swine farms produce large amounts of manure/wastewater that need to be treated before final disposal. Swine wastewater has traditionally been treated using anaerobic lagoons that do not remove nutrients effectively, and the treated effluent that is sprayed on cropland usually has high contents of nitrogen and phosphorus. Excess land application of animal waste can cause contamination of ground and surface waters, and may lead to eutrophication, ultimately resulting in depletion of dissolved oxygen, fish kills, and changes in water color, odor, and taste, making water unsuitable for consumption. Therefore, there is currently great demand for more effective swine wastewater treatment technologies. One promising alternative for treatment of livestock waste consists of anaerobic digestion followed by intermittent-aeration for removal of nitrogen through nitrification and denitrification. As nitrification and denitrification are biological processes, better understanding of the ecology of the microorganisms involved and their response to key operating conditions may ultimately lead to improved performance of nitrogen removal systems. The objective of this study was to investigate the microbial ecology of nitrogen-removing bioreactors treating swine wastewater and the effects of key operating conditions on nitrogen removal efficiency. Laboratory-scale intermittently-aerated reactors were fed anaerobically digested swine wastewater with ammonia concentrations up to 175 mg NH3-N/L and operated with different aeration to non-aeration (ANA) time ratios. Changes in the fractions of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) were monitored using 16S rRNA- and amoA-based molecular approaches. Nitrosomonas/Nitrosococcus mobilis were the dominant AOB and Nitrospira were the dominant NOB in all reactors. Nitrosomonas sp. Nm107 was detected in all reactors, regardless of the reactor’s performance. Close relatives of Nitrosomonas europaea, Nitrosomonas sp. ENI-11, and Nitrosospira multiformis were occasionally detected in all reactors. NOB were more sensitive than AOB to long anoxic periods, resulting in nitrite accumulation and lower total NOB rRNA levels. Anoxic periods of 4 h resulted in partial nitrification, followed by denitrification via nitrite, suggesting that efficient nitrogen removal can be achieved at lower operational costs due to savings related to lower oxygen and organic matter system requirements. The difficulty in identifying active denitrifying bacteria motivated the conception of an innovative approach for identifying bacteria based on functional genes and subsequent sorting of labeled cells for proper phylogenetic identification through the use of 16S rRNA fingerprinting techniques. mRNA fluorescent in situ hybridization (FISH) was performed using tyramide signal amplification (TSA) and horse radish peroxidase (HRP)-labeled oligonucleotide probes targeting transcripts of nirS, the gene that codes cytochromecontaining nitrite reductase, an important enzyme that catalyzes the reduction of nitrite to nitric oxide. For the first time, the simultaneous in situ detection of all three groups of bacteria involved in nitrogen removal from wastewater (denitrifying, ammonia-oxidizing, and nitrite-oxidizing bacteria) was possible using the method developed in this research. Our results revealed close spatial relations among all three groups of bacteria targeted. A number of bacterial colonies hybridized with both nirS mRNA and the 16S RNA of ammonia oxidizing bacteria, suggesting that members of AOB might possess and express nirS genes in addition to the already known nirK genes present in some AOB such as Nitrosomonas europaea. Labeled nitrite reducers were sorted from the background microbial community using flow cytometry (FCM) for subsequent phylogenetic analysis based on 16S rRNA genes. Results suggest that the dominant in situ nitrite reducers were closely related to Acidovorax BSB421. The molecular approach developed in this research has great potential to unravel the longstanding question of which environmental processes are attributed to which microorganisms in natural and engineered habitats.