By Sergei KALYUZHNY, Dr. Sc. (Chem.), Deputy Head of the Chemical Enzymology Chair of the Chemical Department of Moscow State University named after M. V. Lomonosov

Nitrogen compounds to be found in the sewage of many production units pose a serious threat to the environment. The laws of the Russian Federation stipulate that enterprises shall remove these toxic agents well in advance. In their efforts to improve the traditional purification system and remove a number of flaws typical of it, a group of Russian and foreign researchers joined their efforts to producea new biological process called DEAMOX in 2006.

Nitrogen compounds in the sewage of food-manufacturing enterprises, which are largely represented by ammonium and organic substances that are easily transformed into ammonium in the course of processing by organic substances, accumulate, with their high concentrations formed as a result. The discharge of this kind of waste into rivers and lakes leads to undesirable consequences. First, oxygen contained in it is involved in the biological ammonium oxidation process, and its quantity is reduced. And ammonia formed as a result serves as a strong poison for fish. Secondly, the ground waters located nearby are polluted. Thirdly, the polluted water reservoirs undergo the process of silting, for, along with phosphorus, nitrogen is an important source of plant nutrition. Is that problem actually topical for this country? Take a few figures, by way of example. As a result of starch processing nitrogen content totals 204 - 1,042 mg/1, and the respective figure for yeast manufacture is 250 - 1,200 mg/1. The figures of such pollution for liqueur-and-vodka and alcohol plants are 700 - 850 and 1,300 - 1,450 mg/1, respec-

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Biological nitrogen cycle.

tively. Given that the maximum admissible concentration (MAC) of ammonium (N-NH₄) for a typical Russian water canal is as a rule 30 mg/1, of nitrite (N-NO₂) - 1, and of nitrate (N-NO₃) - 50 mg/1, it is only too obvious that the situation is quite grave. That is why it is necessary for the enterprises to process in advance their sewage discharged into the city sewerage system. More often than not the MAC of various nitrogen forms in that sewage is close to exceptionally strict standards for disposal into water reservoirs used for fish farming: for (N-NH₄⁺), - 0.4 mg/1, for (N-NO₂) - 0.02, for (N-NO₃) - 9.1 mg/1.

FROM CALCULATIONS TO EXPERIMENT

So, the enterprises shall remove nitrogen compounds well in advance in order to rule out eutrophication* of natural water reservoirs, for, as a result, they will be unsuitable for bathing, angling, and so on. Various physical, chemical and biological water-processing methods are used depending on the required degree of water purification. Ionic exchange and reverse osmosis are used in this case in order to produce drinking water. Ammonia blowing into its gaseous phase, chemical denitrification**, chemical precipitation, with struvite (MgNH₄PO₄) formed as a result, for instance, and also nitrogen assimilation for biomass growth are among methods used to process sewage. The method of microbiological nitrification-denitrification and of ammonium anaerobic oxidation may serve both ends, however in practice precisely this method is used for sewage purification. We shall discuss this method in detail now and refer to it hereinafter as "nitrification***-denitrification." That would be quite logical, for the chemical version of this method, interesting as it is from a theoretical viewpoint, is of no practical importance at present.

Let's, first, describe the biological cycle of nitrogen and, next, set about discussing the traditional method of nitrogen removal. Nitrogen finds its way to the biosphere mainly through its fixation in molecular form by certain bacteria, with ammonium formed as a result and, next, assimilated by other microorganisms, water-plants and other plants. Ammonium is released in the process of decomposition of the biomass accumulated by them and oxidized as a result of the effect of autotrophic bacteria, such as, for instance, Nitrosomonas and Nitrobacter, being transformed, first, into nitrite and, next, into nitrate (the nitrification process).

Since the removed sewage components contain nitrogen largely in a reduced form (ammonium), they should be subjected to nitrification. In the generally accepted purification methods this stage is usually interconnected with denitrification where nitrate acts as an oxidizer of the organic substance. Not only anoxic conditions**** but also a donor of electrons are necessary for its effective

* Eutrophication means the process of enrichment of rivers and lakes with biogenous elements accompanied by increased water productivity. - Ed.

** Denitrification - the process of transformation of nitrate and other oxidized forms into molecular nitrogen as a result of the effect of various hemoorganotrophic and phototrophic bacteria in the absence of oxygen. - Ed.

*** Nitrification-transformation by the so-called nitrifying bacteria of the ammonium salts of the soil and water reservoirs into nitrates assimilated by plants. The process is of primary importance to nitrogen turnover in the biosphere. - Ed.

**** Anoxic conditions-anaerobic conditions but with nitrate acting as electron acceptor (instead of oxygen). - Auth.

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Diagram of DEAMOX process.

Concentrations of components of nitrogenous pollutants, mg N/1: A - at the inlet to the purification system; B - at the outlet.

conversion into molecular nitrogen. And methanol or organic pollutants often act as such.

The said interconnection of processes and their optimal combination are essential for the successful solution of the problem. However, the traditional microbiological method for the removal of nitrogen pollutants proves not practicable enough due to a great amount of energy consumed for keeping the required oxygen concentration in the medium for nitrification and to the need to find an easily bio-dissoluble donor of electrons for the denitrification process.

For a long time it was believed that ammonium germ oxidization could occur solely in aerobic conditions. It was only in the 1970s that Engelbert Broda, an Austrian researcher, proved by his thermodynamic calculations that this process could also develop in anaerobic conditions right to the stage of molecular nitrogen formation provided nitrite was used as the electron acceptor. It was also proved that ammonium anaerobic oxidization, or the anammox process*, is even more practicable from the viewpoint of energy consumption. The discovery produced revolutionary changes in methods for the removal of nitrogenous pollutants, for the anammox-reaction-based systems do not need a carbon source, have a low rate of biomass increase and require less energy resources than the standard systems based on nitrification and heterotrophic denitrification.

Nitrite as a far from typical component of sewage serves as the only hitch in this case, since a separate system is required for its generation (for instance, from ammonium). In the past 15 years a number of processes have been offered for obtaining it from ammonium-containing sewage. For instance, manipulations with diluted oxygen concentrations, inhibition or temperature change-controlled washing of nitrite-oxidizing bacteria from the system (the SHARON process, developed by Chris Hellinga of the Technical University of Delft (The Netherlands) and his colleagues in 1998, is the most widely known). It should be recalled that nitrite is a highly toxic and reaction-prone substance generating various nitrogen oxides, or NO_x gases regarded as highly dangerous from the viewpoint of global warming. Moreover, all methods for obtaining it provide for complicated control.

NEW BIOLOGICAL PROCESS

For the solution of the above-mentioned problems the international research group (with the author of these

* The term was coined soon after the successful experiment, staged by Arnold Mullder of Gist-Brocades Co. (The Netherlands) and his colleagues in 1995, who confirmed that their former calculations were accurate. - Auth.

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lines as its member) offered in 2006 a new biological process for the removal of nitrogenous pollutants- DEAMOX. It is based on the anammox reaction under autotrophic denitrifying conditions of anaerobic biofilm, with sulfide used as the donor of electrons for nitrite to be obtained from nitrate.

Three reactors operate in the system developed by us: an anaerobic reactor that mineralizes the initial polluted waters for sulfide and ammonium generation, a nitrifying rector that receives a part of the sewage obtained at the first stage for nitrate production, and a DEAMOX reactor that finally receives the total volume of liquid to be purified, with molecular nitrogen, carbon dioxide and water produced as a result. We conducted an experimental check on the laboratory installation, specially designed for processing highly concentrated nitrogenous sewage produced, for instance, in the manufacture of baker's yeast. In the process optimization period of 410 days we removed over 90 percent of such admixtures with the nitrogen load of over 1,000 mg per reactor per day.

By applying up-to-date methods (in particular, with the sequence of 16S pDNA* with the subsequent phylogenetic** analysis) we studied a unique community of microorganisms formed in the period of the DEAMOX reactor operation and revealed 66 16S-pDNA clones of bacterial population and 51 clones of the Archaean population. Phylotypes typical of anaerobic and nitrifying reactors were naturally represented in the analyzed biomass. Predominating in the bacterial clone library were Proteobacteria and Bacteroidetes that accounted for 32 and 15 percent, respectively. We also identified Planctomycetes, Chloroflexi, Firmicutes, Verrucomicrobia, Lentisphaerae and Spirochaetales. However, we have failed to determine 11 bacterial groups, for the succession of nucleotides close to the currently known microorganisms was not revealed in them. It should be noted that revealed in two of those groups were non-cultivated (unable to grow in existing media in the pure culture) bacterial clones that probably represent new kinds of anammox bacteria.

Minute nitrite concentrations in the processed sewage outgoing from the DEAMOX reactor testify to the fact that sulfide-oxidizing denitrifyers and anammox bacteria (ammonium oxidizers in anaerobic conditions) are responsible for its syntrophic inter-species transfer. In our case the use of this anion is probably realized between identified Thauera sp. or Thiobacillus sp. and new species of anammox bacteria belonging to unidentified groups.

In discussing the practical significance of the DEAMOX-process it would be logical to compare its characteristics with competitive methods, i.e., the SHARON-ANAMMOX combination and the traditional nitrification-denitirification process. There is no need for control over pH values or over the exogenous donor of electrons in our new method. Moreover, it provides for specific rates for the removal of nitrogenous pollutants that exceed the potential of traditional methods 18 times over and that are comparable to those in the SHARON-ANAMMOX process. However, DEAMOX has a number of advantages over the latter. Primarily, it does not involve complicated control over nitrite generation, and low nitrite concentrations in the reactor eliminate the serious risk of contamination for microrganisms and of emission of No_x gases. Finally, denitrifying conditions contribute to biomass granulation (self-immobilization), which makes it possible to use simple reactor designs such as UASB*** reactor.

It should be noted that the process offered by us can be used for the removal of highly concentrated nitrogenous pollutants from the sewage of food production units, stock-breeding farms, filtrates of solid garbage grounds, return waters after anaerobic fermentation of sewage sediment, and so on. Pilot tests of the DEAMOX process are being held at one of the yeast factories in Russia.

All this gives grounds for hope that we have developed an advanced method for the removal of nitrogen compounds in purification of sewage with high concentrations of this component. The author would like to express gratitude to Biotan Systems International Co. (Delft, The Netherlands) for its financial contribution to this work.

Illustrations supplied by the author

* 16S-pDNA - conserved section of DNA that can, just like fingerprints, inform a researcher as to what species a given organism belongs to. - Auth.

** Phylogenetic analysis - in our case identification of microorganisms by means of their detailed comparison with the currently known species, genera, classes, and so on, represented in the library of microorganisms. - Auth.

*** UASB - a reactor in which the ascending How of the initial sewage is pumped through the granulated biomass layer. - Auth.