Enrichment And Characterization Of Anaerobic Ammonia Oxidizing Bacteria From Industrial Sludge Samples Radhika Yadav , Prarthana Nagar, Anjana Desai Department of Microbiology and Biotechnology Centre, Faculty of Science, The M. S. University of Baroda, Vadodara-390002, Gujarat, India. INTRODUCTION Nitrogen removal in domestic and industrial wastewater has been a key concern in protecting our environment. Convensional nitrification-denitrification system is less efficient and is not cost effective due to the requirement of external carbon source and aeration. Consequently greater interest and attention is being given to new biological processes like aerobic denitrification, heterotrophic nitrification and autotrophic denitification.. The Anaerobic Ammonia Oxidation (ANNAMOX) pathway offers an interesting alternative for nitrogen removal but has not been studied extensively for a range of wastewater because they are difficult to enrich. Pure cultures of anammox bacteria have not been obtained so far. Just four annamox genera have been described of which Candidatus Kuenenia, Candidatus Brocadia and Candidatus Annamoxoglobus have been enriched from industrial sludge samples and the fourth genus Candidatus Scalindua has been detected in natural habitats especially in marine sediments and oxygen minimum zones (L. Niftrik et al., 2008). The process involves oxidation of ammonium to N2 with nitrite as electron acceptor under strictly anoxic conditions NH4+ + NO2- N2 + 2H2O. Further enrichment and characterization of annamox bacteria will help in increasing our knowledge about the process, and they can be used in efficient removal of nitrogen from wastewater. MATERIALS AND METHODS Industrial sludge samples from Vadodara, Nandesari and Bharuch were spiked with 1% ammonium sulphate, sodium nitrite and sodium carbonate. They were incubated at pH 8 for 15 days. From them 1ml sample was inoculated in 5ml inorganic medium as described by Konrad Egli et al. (2001) in screw capped bottles to maintain anoxic conditions. To check anammox activity in these samples, reduction in ammonia and nitrite were estimated at regular intervals. The effect of hydrazine, a unique intermediate in the process, was observed on ammonia and nitrite removal and gas formation. Anammox bacteria are anaerobic, chemolithoautotrophic microorganisms having a doubling time of around 11 days (Jetten et al., 1999), hence their primary enrichment was carried out for 1 year in the screw capped bottles. For their secondary enrichment special reactors were designed for anaerobic growth of anammox bacteria. A mixture of 80% Argon (99.999% purity) and 20% CO2 (99.998% purity) was passed through the reactors to create anaerobic condition. Removal of ammonia and nitrite was measured. Gas produced in the process was identified by gas chromatography : model Varian 3600 using Helium (99.999%purity) as the carrier gas. Anammox activity under complete anaerobic condition RESULTS AND DISCUSSION Fig 4. Increase in the ammonia removal was observed in N1 and N2 samples. In N2 removal of ammonia was double in the presence of hydrazine as compared to the sample without hydrazine Fig 8. Reduction in ammonia and nitrite is approximately in the ratio of 1:1. Anammox bacteria are known to utilize ammonia and nitrite in the ratio of 1:1 to 1:1.33 (Schmid et al., 2003). Nitrogen formation Fig 1. Significant removal of ammonia was observed in D1, O, N1 and N2 samples Fig 5. Increase in the nitrite removal was observed in N1 and N2 samples. Nitrite removal and gas production increases in the presence of hydrazine for the anammox bacteria. (Schalk et al., 1998) Fig 9. Significant Nitrogen formation was observed in all the samples. Effect of hydrazine on the gas production N1 N1H N2 N2H D1 D1H Fig 2. Significant removal of ammonia was observed in D1, N1 and N2 samples. As these samples showed significant removal of both ammonia and nitrite, further studies were carried out on D1, N1 and N2 samples Fig 6. Gas bubble was formed in the inverted durham’s tube in the presence of hydrazine in N1 and N2 samples whereas significant effect was not observed in case of D1 sample. Fig 9. N2 sample showed maximum hydrazine utilization. CONCLUSION Amongst enriched samples, N1, N2 and D1 samples showed ammonia and nitrite removal from the media in the ratio of 1:1 along with nitrogen formation under complete anaerobic conditions. Hydrazine utilization was also observed in these samples. This implies that anammox bacteria may be present in the samples. 16S rDNA analysis can be carried out of the above samples to further confirm the presence of anammox bacteria in them. Gas outlet Gas inlet Sampling port ACKNOLEDGEMENT The work presented here was sponsored by GNFC, Bharuch, Gujarat. REFERENCES Egli K et al., (2001) Archives of Microbiol. 175: 198-207 Ingo Schmid et al., (2003) FEMS Microbiol Rev 27: 481-492 Jetten M S M et al., (1999) FEMS Microbiol Rev 22: 421-437 Laura van Niftrik et al., (2008) J. Bacteriol. 190: 708-717 Schalk J et al., (1998) FEMS Microbiol Lett 158: 61-67 Fig 7. Inoculation of enriched samples in specially designed flasks for enrichment under complete anaerobic conditions. Here Pseudomonas flurosence CHAO was used as control. P. flurosence was inoculated in Luria Bertani media whereas the others were inoculated in media described by Konrad Egli et al., (2001). Growth was not obtained even after 7 days of incubation in the control suggesting complete anaerobic environment in the flasks. Fig 3. Schematic representation of anaerobic ammonia oxidation carried out by anammox bacteria showing hydrazine N2H4 as an intermediate (L. Niftrick et al., 2004)