Denitrification in Methanogenic Reactors

Proceedings of the 9th World Congress, Anaerobic Digestion 2001, Antwerpen, Belgium, September 2-6. Part 2, pp. 227-229

Denitrification in methanogenic reactors : state of art N. Bernet*, J. C. Akunna**, J. P. Delgenès* and R. Moletta* * Institut National de la Recherche Agronomique (INRA), Laboratoire de Biotechnologie de l'Environnement, Avenue des Etangs, F-11100 Narbonne, France. e-mail : [email protected]. ** Urban Water Technology Centre, School Science and Engineering University of Abertay, Dundee DD1 1HG, Scotland, UK. e-mail : [email protected].

ABSTRACT This paper presents the major findings into the interactions, activities and nature of the microbial populations associated with a combined denitrification/methane-production system. The paper will also report on the studies on factors affecting the performance of such systems. Some of the major findings include the following: - Anaerobic sludges not acclimatised to nitrates or nitrites have the ability to denitrify as well as produce methane. - Denitrification is not the only nitrate reduction pathway in anaerobic systems. Dissimilatory nitrate reduction to ammonia (DNRA) is also possible in the presence of fermentable carbon and at high C/N-NO3 ratio. - Nitrogen oxides induce inhibition of methanogenic activity up to a complete reduction of these compounds. This inhibition and interactions between methanogenic and denitrifying populations have been studied and there consequence on the process development are discussed.

KEYWORDS Anaerobic digestion, Denitrification, Nitrogen removal, C/N ratio, INTRODUCTION During anaerobic digestion of wastewater with high nitrogenous compound s content, organic nitrogen is mainly reduced to ammonia which is not further degraded in anaerobic conditions. A post-treatment may be necessary to remove ammonia before discharge, biological nitrogen treatment processes (nitrificationdenitrification) being the most widely used process. In the case of wastewater with a relatively low COD/TKN ratio following anaerobic treatment (a common occurrence in high rate anaerobic systems), the organic carbon remaining in the digested effluent may be insufficient to achieve complete denitrification and the addition of an external carbon source is then required. An alternative process configuration, in which denitrification takes place in the methanogenic reactor, has been first proposed about ten years ago (Kuroda et al., 1988; Hanaki and Polprasert, 1989; Akunna et al., 1992; Garuti et al., 1992). In this configuration, carbon is first used for denitrification and then remaining organic carbon is converted to methane, thus ensuring complete denitrification and organic carbon removal. The digester is coupled to an aerobic nitrification unit also completing the organic carbon removal. NITROGEN OXIDE REDUCTION IN ANAEROBIC DIGESTERS Using a chemostat fed with glucose and nitrate or nitrite, it was shown that the proportion of dissimilatory nitrate reduction to ammonia (DNRA) in anaerobic systems depends on influent wastewater COD/N(NO2+NO3) ratio. DNRA is dominant activity at high COD/N-(NO2+NO3) and decreases as the ratio is

reduced (Akunna et al., 1992). Methane production decreases as the ratio decreases, mainly because more organic carbon is consumed during denitrification with small amounts remaining for methane production. Methane production commences when there is no N-oxides in the system. Batch reactors have been shown to favour denitrification and methanogenesis compared to continuous flow conditions, because both processes (denitrification and methanogenesis) can be separated in time (Akunna et al., 1994a) in the former. The nature of the carbon source is also an important parameter: in the presence of fermentative substrates such as glucose or glycerol, DNRA is shown to be the main N-oxides reduction pathway in batch cultures. In the presence of acetic and lactic acids, denitrification is the major biochemical process during nitrate and nitrite reduction. (Akunna et al., 1993). Thus, it appears that fermentative micro-organisms are responsible for DNRA. The proportion of nitrate converted to ammonia depends on the rate of denitrification (with both the fermentable organic substrates and the simpler by-products of fermentation in the medium) and the amount of nitrate compared with the amount of carbon compounds undergoing acidogenesis. When the carbon compounds in medium are only in the form of alcohols or organic acids, DNRA does not occur and the only nitrate reduction pathway is denitrification. The implication of this finding in the design and operation of a combined denitrification/methane-production system is that to reduce DNRA, nitrate can be recycled from the nitrification system not with the raw influent, (when the wastewater to be treated contains fermentable substrates), but added separately away from the zones dominated by acidogenesis. EFFECT OF NITROGEN OXIDES ON METHANOGENIC BACTERIA Nitrate and its reduced forms produced during denitrification, nitrite and nitrous oxide, were studied for their influence on methane production from acetate by the methanogenic strain Methanosarcina mazei (Clarens et al., 1998). While 0.5 mg N-NO2.l-1 and 0.8 % nitrous oxide in the gas phase completely suppressed methane production, 1 g N-NO3.l-1 resulted in only 83 % inhibition. Co-culture experiments showed that M. mazei, growing in the presence of nitrate, produced methane from acetate until the denitrifying bacterium Pseudomonas stutzeri was inoculated and denitrification began. This suggests that methanogenesis by M. mazei is inhibited by denitrification activity, including reduced nitrogen forms produced, rather than by nitrate itself or competition for acetate between denitrifying and methanogenic bacteria. The effect of nitrate addition on the anaerobic digestion of an industrial sulphate rich wastewater was also investigated in batch cultures (Percheron et al., 1999). A lag phase, probably caused by a high initial sulphide content, preceded denitrification. During this lag phase, methane production was not affected by nitrate concentrations as high as 500 mg NO3-N.l-1. Methane production stopped as soon as denitrification started. Concurrently, an increase of the redox potential and a transient nitrite production were observed. These physical and chemical changes in the system might have caused the inhibition of methanogenesis CONSEQUENCES ON THE PROCESS DEVELOPMENT Because nitrogen oxides have been found to inhibit (reversibly) methanogenic bacteria, denitrification and methanogenesis should be separated in the reactor, either spatially (plug-flow or particulate biofilm reactors) or in time (batch reactors such as SBR). In the first case, the creation of macro- and/or micro- environments within the system should occur so that the different bacteria involved in the reactions could grow in zones within the reactor favourable to their metabolic activities. This configuration has been carried out using an upflow biofilter fed with a synthetic wastewater containing glucose as a carbon source (Akunna et al., 1994b). In the second case, a system coupling two sequencing batch reactors, anaerobic and aerobic, for the treatment of piggery wastewater, methane production followed denitrification in the anaerobic reactor. Overall performances of TOC and TKN removal of respectively 81 to 91% and 85 to 91% were achieved The process ensured an enhanced organic carbon consumption in the anaerobic system since methane production started after organic carbon utilisation for denitrification. This configuration was found to bring

about a dilution of the raw wastewater in the digester, reducing the concentrations of inhibitory compounds like ammonia (Bernet et al., 2000). A review of other works carrying out similar configurations confirms the feasibility of such a process, using mainly spatial separation in continuous processes (Kuroda et al., 1988; Hanaki and Polprasert, 1989; Garuti et al., 1992; Akunna et al., 1994b; Tilche et al., 1994; Lin and Chen, 1995; Hendriksen and Ahring, 1996a; Chen et al., 1997; Fang and Zhou, 1999, Mosquera-Corral et al., 2001), but also batch processes (Hendriksen and Ahring, 1996b; Bernet et al., 2000, 2001). CONCLUSIONS The process proposed is able to remove organic matter by anaerobic digestion and nitrogen by nitrificationdenitrification. This configuration optimises the utilisation of the organic carbon which is firstly used for denitrification and the excess converted to biogas. This system is adapted to the treatment of high strength wastewaters with a low C/N ratio or containing mainly non fermentable organic substrates. When fermentation is an inevitable occurrence in the anaerobic process, nitrate can be added to the digester at points where acidogenesis is minimal and organic acids are the dominant organic compounds present, for plug flow reactors.To carry out denitrification and anaerobic digestion in the same reactor, these biological processes must be separated, either spatially (plug-flow reactors) or in time (SBR). REFERENCES Akunna J. C., Bizeau C. and Moletta R. (1992). Denitrification in anaerobic digesters : possibilities and influence of wastewater COD/N-NOx ratio. Environ. Technol., 13, 825-836. Akunna J. C., Bizeau C. and Moletta R. (1993). Nitrate and nitrite reductions with anaerobic sludge using various carbon sources glucose, glycerol, acetic acid, lactic acid and methanol. Wat. Res., 27(8), 1303-1312. Akunna J. C., Bizeau C. and Moletta R. (1994a). Nitrate reduction by anaerobic sludge using glucose at various nitrate concentrations - ammonification, denitrification and methanogenic activities. Environ. Techno., 15, 41-49. Akunna J. C. , Bizeau C., Moletta R., Bernet N. and Héduit A. (1994b). Combined organic carbon and complete nitrogen removal using anaerobic and aerobic upflow filters. Wat. Sci. Tech., 30(12), 297-306. Bernet N., Delgenès N., Akunna J. C., Delgenès J. P. and Moletta R. (2000). Combined anaerobic-aerobic SBR for the treatment of piggery wastewater. Wat. Res., 34(2), 611-619. Bernet N., Delgenès N., Delgenès J. P. and Moletta R. (2001). SBR as a relevant technology to combine anaerobic digestion and denitrification in a single reactor. Wat. Sci. Tech. 43(3), 209-214. Chen K. C., Lin Y. F. and Houng J. Y. (1997). Performance of a continuous stirred tank reactor with immobilized denitrifiers and methanogens. Wat. Environ. Res., 69(2), 233-239. Clarens M., Bernet N. Delgenès J. P. and Moletta R. (1998). Effects of nitrogen oxides and denitrification by Pseudomonas stutzeri on acetotrophic methanogenesis by Methanosarcina mazei. FEMS Microbiol. Ecol., 25(3), 271-276. Fang, H. H. P. and Zhou, G. M. (1999) Interactions of methanogens and denitrifiers in degradation of phenols. J. Environ. Eng., 125(1), 57-63. Garuti G., Dohanyos M. and Tilche A. (1992a). Anaerobic-aerobic combined process for the treatment of sewage with nutrient removal : the ANANOX process. Wat. Sci. Tech., 25(7), 383-394. Hanaki K. and Polprasert C. (1989). Contribution of methanogenesis to denitrification with an upflow filter. J. Wat. Poll. Control Fed., 61(9), 1604-1611. Hendriksen H. V. and Ahring B. K. (1996a). Integrated removal of nitrate and carbon in an upflow anaerobic sludge blanket (UASB) reactor : operating performance. Wat. Res., 30(6), 1451-1458. Hendriksen H. V. and Ahring B. K. (1996b). Combined removal of nitrate and carbon in granular sludge : substrate competition and activities. Antonie van Leeuwenhoek, 69(1), 33-39. Kuroda M., Shima H. and Sakakibara Y. (1988). A study on simultaneous treatment of organic matter and nitrate with a biofilm consisting of methane fermentative bacteria and denitrifying bacteria. Proc. of Environ. & Sani. Eng. Research, 24, 231. Lin Y. F. and Chen K. C. (1995). Denitrification and methanogenesis in a co-immobilized mixed culture system. Wat. Res., 29(1), 35-43. Mosquera-Corral A., Sanchez M., Campos J. L., Mendez R., Lema J. M. (2001). Simultaneous methanogenesis and denitrification of pretreated effluents from a fish canning industry. Wat. Res., 35(2), 411-418. Percheron G., Bernet N. and Moletta R. (1999). Interactions between methanogenic and nitrate reducing bacteria during the anaerobic digestion of an industrial sulfate rich wastewater. FEMS Microbiol. Ecol., 29(4), 341-350. Tilche A., Bortone G., Forner G., Indulti M., Stante L. and Tesini O. (1994). Combination of anaerobic digestion and denitrification in a hybrid upflow anaerobic filter integrated in a nutrient removal treatment plant. Wat. Sci. Tech., 30(12), 405414.