Electronic Journal of Polish Agricultural Universities (EJPAU) founded by all Polish Agriculture Universities presents original papers and review articles relevant to all aspects of agricultural sciences. It is target for persons working both in science and industry,regulatory agencies or teaching in agricultural sector. Covered by IFIS Publishing (Food Science and Technology Abstracts), ELSEVIER Science - Food Science and Technology Program, CAS USA (Chemical Abstracts), CABI Publishing UK and ALPSP (Association of Learned and Professional Society Publisher - full membership). Presented in the Master List of Thomson ISI.
Volume 10
Issue 2
Environmental Development
Available Online: http://www.ejpau.media.pl/volume10/issue2/art-01.html


Marcin Zieliński, Mirosław Krzemieniewski
Department of Environment Protection Engineering, University of Warmia and Mazury in Olsztyn, Poland



This paper shows the results of the research on the effect of microwave radiation on the changes of nitric compounds in a reactor with a biofilm. The bioreactor was placed inside a microwave tube, in which it was exposed to radiation. Municipal wastewater were supplied to the bioreactor from the retention tank, to which they returned having flowed through the reactor’s packing. The whole mechanism worked in a periodic system at the 24-hour retention of the wastes supply.

The research made use of specific properties of microwave heating, which involved selective heating of only substances marked by suitable dielectric properties. Owing to the effect obtained by means of a right construction of the reactor and the synchronization of the microwave generator and the volumetric pump, microwave energy was directed mainly to the biofilm.

The results achieved show the influence of microwave radiation on the course of nitric changes in the biofilm. In a control reactor, which was not exposed to radiation, the changes of nitric compounds ended up in oxidizing ammonia nitrogen, and nitrogen losses stemmed from the biomass synthesis exclusively. However the input of microwaves resulted in anoxic conditions in the biofilm and the reductive changes of nitrates and nitrites. It cannot be stated unequivocally to what extent the nitrogen losses occurred as a result of heterotrophic denitrification and to what due to the ANAMMOX process. Nevertheless nitrogen removal in the reactor exposed to radiation reached 55% whereas in the control reactor – below 9%.

Key words: nitrogen compounds, microwave, nitrification, denitrification, ANAMMOX.


The interaction of electromagnetic radiation with substances is dependent on the amount of energy carried by quanta or, in the wave context, the frequency of electromagnetic wave. At the quantum energy above 1 x 102 eV (radiation x, radiation gamma) it is possible to separate an electron from an atom and cause its ionization. Lower magnitudes of microwave radiation from the UV range are sufficient to weaken and rip molecular bonds. Quanta of the radiation from the visible light range and infrared radiation posses energy that is only able to activate electrons on the outer electron shells. This can make molecules turn into excited states, due to which they are capable of forming particular chemical bonds.

Microwaves are part of electromagnetic spectrum with the wavelength range from 1 mm to 1 m and the frequency range from 300 MHz to 300 GHz respectively. The energy carried by quanta of microwave radiation is too little to be absorbed by electrons; it does not lead to their excitation, much less to the separation of an atom. The interaction of microwave radiation with molecules does not cause alterations in their structure. Electromagnetic field of microwave frequency affects substances depending on their internal structure, precisely on their dielectric properties, that is their ability to electric conduction. Materials that are marked by high conductance and low capacity (capacity retention) (e.g. metals) have a high dielectric losses rate. When a dielectric losses rate is very high a microwave penetration depth approaches zero. Materials with such a dielectric environment should be treated as reflective microwaves (reflectors). Materials with a low dielectric losses rate have a high penetration depth (plastics). As a result very little energy is absorbed in the material and such a substance is permeable to microwaves. Microwaves deliver energy most effectively to substances whose dielectric environment is set between these boundaries. Water is an example of such a substance. It is accepted that as a result of microwave radiation the vibration of dipolar molecules primarily contributes to the temperature rise of the substance. The migration of ions has also such an influence but not as significant as the former one [6, 11] Microwave energy is diffused in a form of heat coming from the internal resistance of rotation, which means that dipolar molecules in movement, as a result of friction, dissipate energy that they have obtained from microwaves. This causes the temperature rises of the substance.

In organisms that contain a lot of water it is impossible to distinguish the influence of microwaves explicitly due to the high absorbance of water molecules [11] There are two mechanisms which can lead to energy absorption by a protein molecule. The first mechanism involves a direct interaction with the microwave field, as a result of which protein molecules with the dipole moment begin to rotate. The other mechanism consists in the absorption of microwave energy by a solution, and then the acceptance of the thermal energy resultant by a protein. Proteins have a particular distribution of charges – an isoelectric point. They contain polar chains which may be rotationally excited by microwave energy. Also the water molecules in the hydrated layer of proteins can be brought into rotation by electromagnetic energy [10].

In the presented study the research on the use of microwave electromagnetic radiation as a factor allowing of creating thermal conditions, and thus the course of nitric changes in the reactor with the biofilm has been revealed. Microwave heating has a volumetric quality, and at the same time it is marked by selectivity [6] With a suitable packing selection and an appropriate governing of the system it is possible to lead energy directly to the biofilm, which should affect the activity of the biofilm and the course of biochemical changes.

The aim of the presented research was to determine the influence of microwave radiation on the changes of nitric compounds in the reactor with the biofilm.


The research was carried out by means of a trickling filter with the biofilm, which was placed inside the tube and exposed to microwave radiation. The cover and the packing of the reactor were made of material permeable to microwaves. The active volume of the reactor was V= 145 cm3 with the right surface of the filling s = 202 m2·m-3; the theoretical active surface of the reactor was F=0.029 m2.

A magnetron, from which radiation was transmitted through the waveguide to the reactor tube, was the source of microwave radiation. The generator, whose total power was 800W, generated microwave radiation of 2.45 GHz frequency at 52% efficiency. The magnetron emitted radiation with a constant efficiency, and the amount of supplied energy was regulated by the length of its work and break time. The work of the microwave generator was synchronized of the wastewater supplied to the bioreactor. Every time the reactor was to be turned on the inflow of wastewaters was cut off. As a result microwave energy was mainly absorbed by the biofilm rather than the wastewaters being treated. After each radiation phase the volumetric pump dosing wastewaters was turned on again and the whole system resumed to a treatment phase.

As a result of the microwave radiation the temperature inside the biological reactor rose. The highest temperature of the biofilm was observed directly after the working phase of the microwave generator had finished. Then the temperature dropped during the treatment phase (Tab. 1). The ambient temperature as well as that of the control reactor remained constant at 21.5°C.

Table 1. The temperature inside the bioreactor dependent on the amount of the radiation supplied and the size of the hydraulic loading

The parameters of the system

The amount of the energy supplied

The temperature after the end of the radiation phase [°C]

The temperature after the end of the treatment phase [°C]

Hydraulic loading
Sewage pumping
Retention tank – reactor

0.05 m/h
3.5 times per day

0.15 m/h
10.5 times per day


1.3 Ws





2.5 Ws





5.0 Ws




This research made use of the municipal wastewaters, which were taken directly from the municipal collector once every 24 hours at a fixed time. The average value COD of the wastewaters was 220 mg O2 · l-1, at a standard deviation δ = 21.1. The observed proportion of BOD5 to COD was 0.7 on average. The average amount of total nitrogen remained at the level 45 mg NTNT · L-1, whereas the proportion of total nitrogen to BOD5 – at the level 0.26.

The research was carried out in the periodic system. Once every 24 hours the whole bulk of the wastewaters in the retention tank was exchanged. The wastewaters were conveyed from the retention tank to the biological reactor, and afterwards they came back to the retention tank (Fig. 1). At the hydraulic loading q=0.05 m3 · m-2 · h-1 and q=0.15 m3· m-2 · h-1 the whole bulk of wastewaters flowed trough the bioreactor n=3.5 times and n=10.5 times every 24 hours respectively. The loading of the reactor right surface with organic pollutant load expressed in a form of COD remained at the level A“COD = 8.6g COD · m-2 · d-1, whereas the total nitrogen load was A’TNT =1.6 g N · m-2 · d-1.

Fig. 1. A: A research system diagram 1) bioreactor; 2) microwave tube; 3) retention tank; 4) wastewaters supply to the reactor 5) microwave generator; 6) the exchange of the wastewaters in the retention tank
B: A diagram of the research system work phases

For comparative purposes another research was conducted by means of a reactor that was not exposed to microwave radiation, but which operated in identical hydraulic conditions and with the same pollutant load.

In order to determine the characteristic features of the changes of nitric compounds occurring in the reactors, the content of total nitrogen, organic nitrogen, ammonium nitrogen, nitrate nitrogen and nitrite nitrogen in the raw and treated sewage was analyzed once every 24 hours. The research for each experimental series was conducted for 45 days.

The results obtained allowed to calculate average values, standard deviation and standard error for every factor being analyzed. In the case the value of the standard error was below 10% it was accepted that the results were marked by low changeability, and the calculated average value was reliable. The average values obtained in this way set the basis for determining the ultimate results.


The exposure of the biofilm to microwave radiation considerably affected the nitrogen removal efficiency in the sewage. Owing to the radiation supply nitrogen was removed not only by biomass synthesis but also as a result of reduction of oxidized forms. The maximum efficiency of nitrogen removal reached 55% in the case the hydraulic loading of the reactor q = 0.15 m3 · m-2 · h-1. This effect was obtained at the amount of the radiation supplied 10 Ws (Fig. 2). In the comparative reactor, in the identical technological conditions, the efficiency of the process was not higher than 8.6%. Microwave radiation made nitric removal operate mainly due to the reduction of oxidized forms rather than biomass synthesis (Fig. 2). At the maximum nitrogen removal of 24.75 (55%) only 4.73 mg · d-1 was used for the synthesis. In the case of the comparative reactor the nitrogen removal efficiency was almost in 100% due to biomass synthesis. The observation of the changes of nitric compounds at the lowest amount of radiation 5.1 Ws and the hydraulic loading of the reactor q = 0.05 m · h-1 contributed to the statement that the amount of oxidized nitrogen 22.55 mg N · d-1 was almost identical as in the case of the control reactor 24.44 mg N · d-1 (Fig. 3). In addition, due to microwave radiation 10.67 mg N · d-1 was reduced, whereas in the control reactor NOx reduction was not observed. Thus the higher degree of the sewage exchanged was the more nitrogen was oxidized. What is more in the system modified by the lowest amount of radiation 5.1 Ws the nitrification rate reached 34.43 mg N · d-1 (76.5% of total nitrogen load), but in the control system only 25.11 mg N · d-1 (53% of total nitrogen load).

Fig. 2. Participation of reduction and biomass synthesis in nitrogen removal

The results obtained show that microwave radiation generated anoxic spheres in the biofilm, as a result of which nitrification and the reduction of oxygen forms occurred simultaneously. Microwave energy causes volumetric heating of substances. If one takes into account that the biofilm was fairly thin as compared to the microwave penetration depth one can assume that heating occurred steadily in the whole cross-section. When higher temperature was applied, the amount of oxygen in external, more active layers of the biofilm, decreased more rapidly, which led to the generation of anoxic spheres. The relationship between the generation of anoxic spheres and the temperature of the biofilm is confirmed by the research by Hao at al. [5], who claimed that temperature drop leads to the growth of oxygen penetration depth.

It is demonstrated that the activity of heterotrophic denitrifying bacteria is to less extent dependent on temperature than nitrifying bacteria. Kadlec and Reddy [9] claim that the optimum temperature for denitrification oscillates between 35-40°C. According to the researchers in the temperature range 15-35°C the temperature rate theta for denitrification is 1.07. In the author’s research the temperature in the reactor, whose maximum denitrification efficiency (the amount of radiation 2.5 Ws, q = m3 · m-2 · h-1) was not higher than 25.3°C after the generator phase had ended, and during the treatment phase was not lower than 22.8°C. In the comparative reactor, at the temperature 18°C, denitrification occurred in a very limited way. Thus it ought to be assumed that if temperature factor in the temperature range 15-35°C does not affect the activity of denitrifying bacteria significantly, the generation of anoxic spheres inside the biofilm, mainly due to the quicker oxygen consumption in the external layers, was of crucial significance. However the rise of denitrificants activity by means of temperature appeared to be to much less extent significant.

The likely occurrence of simultaneous nitrification and denitrification in the attched biomass has been widely reviewed in literature [2, 7, 13, 16, 17] show that these processes can occur simultaneously if there are both nitrificants and denitrificants in the biofilm, and the amount of oxygen is high enough to oxidize organic compounds and cause nitrification, and low enough to ensure denitrification. The oxidization of organic compounds and ammonium nitrogen generates anoxic spheres in the more internal layers of the biofilm [1, 7]. Puznava at al. [12] proved that the concentration of dissolved oxygen at the level 0.5-3.0 mgO2 · L-1 prevents the biofilm from being completely penetrated by oxygen and denitrification in the internal layers of the biofilm is likely to occur. In the bed filled with polystyrene globules denitrification obtained by Puznava at al. [12] was 71% with the simultaneous nitrification of 96-98%. The authors research indicated the growth of denitrification efficiency with the rise of the degree of sewage exchange between retention tank and bioreactor. For instance, at the amount of radiation 10.0 Ws and the hydraulic load q = 0.05 m3 · m-2 · h-1 (whole sewage flowed between retention tank and bioreactor 3.5 time per day) the amount of reduced nitrogen was 14.4 mg N · d-1 (Fig. 3), whereas when the hydraulic load was q = 0.15 m3 · m-2 · h-1 (10.5 time per day) the amount of reduced nitrogen reached 20.03 mg N · d-1, and in the control system it was not higher than 0.3 mg N · d-1 (Fig. 4). Presumably higher frequency of the sewage exchange in the retention tank made substrates be more susceptible to biochemical changes in the biofilm. The sewage that returned to the retention tank contained organic compounds as well as nitrates and nitrites. Thanks to this, denitrification in the anoxic spheres of the biofilm was allowed to occur. The residual participation of denitrification 0.7% in nitrogen removal in the case of comparative reactor let us assume that the anoxic spheres were generated mainly due to microwave radiation rather than technological parameters connected with the reactor loading with the pollutant load.

Fig. 3. Changes of nitrogen compounds in depends on microwave amount in 0.05 m3 · m-2 · h-1 hydraulic load

Fig. 4. Changes of nitrogen compounds in depends on microwave amount in 0.15 m3 · m-2 · h-1 hydraulic load

It is reviewed in the literature data that nitrogen removal from the sewage is likely to occur in the biofilm not only by means of biomass synthesis and denitrification. Hao at al. [5] observed the phenomenon of ammonium nitrogen oxidation (ANAMMOX – Anaerobic Ammonium Oxidation) by autotrophic bacteria occurring in the internal layers of the film. As a result of this process ammonium nitrogen and nitrites are transferred to elementary nitrogen with a small amount of newly-generated nitrate nitrogen. The consumption of ammonium and nitrite nitrogen with the production of nitrates remains in the proportion 1:1, 31:0.22 Van De Graaf et al. [15]. At the limited amount of oxygen some electrons that come from NH4 oxidation are not carried onto O2 but NO2-. According to Helmer et al. [8] as for the reactor with the biofilm it is impossible to state clearly what processes are responsible for nitrogen losses. Depending on the concentration of dissolved oxygen in the biofilm ammonium nitrogen can be subjected to oxidation to nitrites in aerobic conditions; whereas in the conditions of limited concentration of oxygen – anaerobic oxidation to molecular nitrogen with nitrites as the acceptors of electrons. Both processes can occur simultaneously in various layers of the film. By means of oxygen concentration in the ambient medium it is possible to control the velocity of the process that turns NH4 into NO2 as well as anaerobic oxidation of ammonium nitrogen (NH4+ and NO2- to N2) in the biofilm. Both processes are balanced at the oxygen concentration 0.7 mg O2 · L-1. The biofilm consists of an external oxygen layer and an internal anoxic one. Bacteria which oxidize NH4+ prefer the external layer, whereas microorganisms capable of anaerobic oxidation of NH4 opt for the internal one. Presumably ANAMMOX might have been partly responsible for the nitrogen removal in the systems exposed to microwave radiation as it was in the author’s research. Such a claim can be supported by relatively high nitrogen losses at the low proportion of C/N. Microbiological studies on ANAMMOX Strous at al. [14] showed that high temperatures are preferable for these microorganisms, and 40± 3°C is agreed to be the optimum. It was stated that ANAMMOX activity follows the Arrhenius’ law within the temperature range 20-40°C. The temperature rise in the biofilm as a result of the microwave radiation might suggest favorable conditions for the bacteria generating this process. On the other hand, however, the involvement of heterotrophic denitrificants, which also prefer similar temperatures, should not be excluded. According to Helmer at al. (2001) ANAMMOX can be governed by Nitrosomonas bacteria. According to many researchers [3, 4] the maximum activity of Nitrosomonas in aerobic conditions is at 35°C, and above this temperature the activity drops rapidly. Taking the temperature optimum for ANAMMOX into consideration one can assume that Nitrosomonas in anoxic conditions would sustain its ability to oxidize ammonia even at higher temperatures.


Microwave radiation affected the course of nitric changes in the reactor with the biofilm. A homogenous change of thermal conditions in the biofilm generated by microwave radiation caused the generation of anoxic spheres in the deeper layers of the biofilm. It is most likely that at higher temperatures the oxygen consumption in the external, more active layers of the biofilm is higher. In such conditions it was possible to reduce and remove nitrogen from the system not only due to biomass synthesis but also as a result of heterotrophic denitrification or ANAMMOX. In the case of reactor with the biofilm it is impossible to determine unequivocally what processes are responsible for nitrogen losses. The reactor worked in the periodic system, therefore, at the initial stages of the process classic heterotrophic denitrification probably played a dominant role in nitrogen losses. During the treatment process the number of available carbon compounds dropped, so did the proportion of C/N in the sewage.

This might have affected the increasing participation of ANAMMOX in nitrogen removal.

Curiously, such noticeable differences in the course of nitric changes between the reactors exposed to microwave radiation and the control ones, emerged at the temperature difference as low as 3-4°C. Taking into consideration the fact that the experiment was carried out at the temperature range 18-28°C, which is favorable to nitric changes, one can assume that microwaves affected the biofilm also in a non-thermal way, which seems to confirm numerous other researches from this field of study.


The study was financed from the resources of the State Committee for Scientific Research of Poland under project No. 3 P04G 038 22.


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Accepted for print: 3.02.2007

Marcin Zieliński
Department of Environment Protection Engineering,
University of Warmia and Mazury in Olsztyn, Poland
Prawochenskiego 1, 10-957 Olsztyn, Poland
Phone: (089) 523 32 57
email: marcin.zielinski@uwm.edu.pl

Mirosław Krzemieniewski
Department of Environment Protection Engineering,
University of Warmia and Mazury in Olsztyn, Poland
Prawochenskiego 1, 10-957 Olsztyn, Poland

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