Alina Mihailova1, Olga Muter1, Silvija Strikauska2, Baiba Limane1, Andrejs Berzins1, Uldis Viesturs1,2,3, Dzidra Zarina1,3

1University of Latvia, Institute of Microbiology and Biotechnology, 4 Kronvalda blvd., Riga LV-1586, Latvia, olga.muter@inbox.lv2Latvia University of Agriculture, 2 Liela str., Jelgava LV-3001, Latvia, 3 Institute of Wood Chemistry, 27 Dzerbenes str., Riga LV-1006, Latvia

Comparison of various amendments on the growth of the targeted bacteria association and ammonia biodegradation

I n t r o d u c t i o n

M a t e r i a l s a n d m e t h o d s

R e s u l t s a n d d i s c u s s i o n

C o n c l u s i o n s Acknowledgements

6

6,5

7

7,5

8

8,5

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14

media with different additives

pH

Control

Association(after 14days)

-90

-80

-70

-60

-50

-40

-30

-20

-10

0

10

20

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14

media with different additives

Eh,

mV

0

10

20

30

40

50

60

70

80

90

100

Non

-dai

ry c

attle

Dai

ry c

attle

She

ep

Pig

s

Hor

ses

Pou

ltry

Nitr

ogen

, kg/

year

0

2000

4000

6000

8000

10000

12000

14000

1991

1993

1995

1997

1999

2001

2003

2005

2007

Years

NH

3, k

tonn

es/y

ear

Dairy cattle Non-dairy cattlePigs Sheep Horses Poultry

The recent concern is the volatilization of gases from animal facilities with the major emission being nitrogen in the form of ammonia. Ammonia emissions in the atmosphere lead to the formation of small airborne particles with potential effects on human health. Biofiltration is used to remove odours and various volatile organic and inorganic compounds in contaminated off-gas streams. Currently, there is a growing interest in the applications of biofiltration techniques in a variety of settings.

In our study, the current tendency in ammonia concentrations emitted from agricultural sector in Latvia was analized. Besides, biotechnological solutions for the reduce of ammonia emission were discussed. Biofiltration is closely connected with a search for the optimal conditions for microbial biodegrading activity. This study was also focused on the testing of various amendments in the medium during cultivation of bacteria association with the aim to determine the factors, which influence the physiological state of the association and therefore could improve its further use as inoculum for ammonia biodegradation. Optimization of nitrification and denitrification processes with the use of bacteria association could improve the biofilter efficiency.

Keywords: ammonia, nitrification, biofiltration, amendments, cell adherence.

Calculation of ammonia emission in agricultural sector. Ammonia emission was calculated according to EMEP/CORINAR (EEA, 2005). Emission factor is based on estimation of emission rates of a given pollutant for a given source, relative to units of activity. The basic equation applies variables, including an averaged emission factor and activity data, i.e. animal numbers.Microorganisms and growth conditions. In our work, the ammonia-biodegrading association (PNNS, i.e. Pseudomonas spp., Nitrobacter app., Nitrosomonas spp., Sarcina spp.) previously isolated from the biological activated sludge of the fish factory wastewater treatment plant, was used. Two mineral medium were used for cultivation. Medium A, g/l: (NH4)2SO4 – 2.5; K2HPO4 – 1.0; NaCl – 2.0; MgSO4 x 7 H2O – 0.5; FeSO4 x 7 H2O – 0.001; CaCO3 – 10 g. Medium B, g/l: (NH4)2SO4 – 2.5; Na2HPO4 x 12 H2O – 38.0; KH2PO4 – 0.7; NaHCO3 – 0.5; MgSO4 x 7 H2O – 0.1; FeSO4 – 0.0081; CaCl2 – 0.0139 g. Amendments were used as follows: cabbage leaf extract (CLE) (samples A1 and B2), molasses (A3 and B4), yeast extract (A5 and B6), glucose (A7 and B8), fructose (A9 and B10), as well as a mixture of all mentioned amendments in proportionally, i.e. 5-fold, reduced concentrations (A11 and B12). The samples A13 and B14 did not contain any amendment. Glucose and fructose were added to medium in concentration 2.5 g/l. Other amendments were used in concentrations to achieve approximately the same level of reducing sugars in medium. Cultivation of the PNNS association in the liquid A and B medium was performed in 15ml glass tubes containing 10 ml liquid medium at +30 C with agitation at 180 rpm in the dark during 14 days. Concentration of inoculum in the samples at the beginning of experiment was 2.0 x 106 CFU/ml.Analytical procedures. Total nitrogen was determined according to ISO 5983-2:2005. Concentration of NH3 and NO2- were determined colorimetrically with Nessler and Griss reagents, correspondingly. pH and Redox potential were measured by electrode (Hanna pH213). All chemicals used in these experiments were analytical grade. For scanning electron microscopy the samples were fixed in glutaraldehyde solution and dried at +30C for about 2 h. Dried samples were coated with gold in an Eiko Engineering Ion Coater IB-3 and observed in a JEOL scanning microscope JSM T-200 at an acceleration voltage 30 kV.

Fig.1. A global emissions inventory for ammonia compiled for the main known sources. The estimated global emission for 1990 was about 54 x 109 kg N/year (Bouwman et al., 1997).

Evaluation of ammonia emitted from animal husbandry sector in LatviaThe volatilization of gases from animal facilities with the major nitrogen emission in the form of ammonia is known as a serious environmental problem worldwide. The global ammonia emissions are summarized in the Fig.1. About of 21 % from the total ammonia emission is resulted from livestock and poultry (Bouwman et al., 1997). Recently various approaches are used to perform environmental models on a macroeconomic level to estimate agricultural contribution to climate change, acidification and other ecological consequences.

Analysis of statistical data on ammonia emission from animal husbandry sector in Latvia showed the same tendency as in Europe as a whole, i.e. reduction in animal numbers. Thus, in 2007 the numbers of dairy cattle, non-dairy cattle, pigs, sheep, horses and poultry in Latvia was 2.8-fold; 2.2-fold; 3,8-fold; 4,3-fold; 1,7-fold, and 2,9-fold decreased, correspondingly, as compared to 1990 (Central Statistical Bureau of Latvia, 2008). To estimate the changes in ammonia emission caused by reduction in the number of animals, the calculation on ammonia emission by one animal was performed. The calculated results are shown in table 1.

Annual emissions of nitrogen and ammonia were calculated for different animals and shown in Fig.2 and Fig.3. As shown in Fig.3, during last 10 years the theoretically calculated amount of ammonia emitted from livestock and poultry in Latvia did not changed noticeably. The exception is non-dairy cattle, which number in Latvia was increased for this period almost twice (Fig.3).

The data on dynamics of ammonia emission from farms should be taken into consideration for biofilter construction.

Dairy cattle

Non-dairy cattle

Pigs Sheep Horses Poultry

NH3,kg/year 20,3 6,1 18,3 5,5 4,6 1,4 1,1 0,3 10,3 2,9 0,4 0,1

Fig. 2. Annual emission of nitrogen generated by one animal.

Fig. 3. Annual changes in ammonia emission generated by domestic animals in Latvia.

Principles for a biofiltration system to reduce ammonia emission in the farms

Air purification in animal houses is one of the most significant technological solutions, which can noticeably reduce ammonia emission.Our effort was focused on the development of a biofiltration method ensuring a high ammonia concentration and a limited oxygen environment. Biofiltration process was realized in modified solid-state fermentation system. The investigations were made at different ammonia concentrations in inlet gas and packing loads. The biodegradation of volatile compounds was investigated in one and two stage systems with inert carrier material and bacteria association. A one-stage biofiltration system with the ammonia load 0.41 g/m3h ensured the biological elimination capacity 0.33 g/m3h due to the nitrification processes. A two-stage system with the ammonia load 0.78 g/m3h ensured increased total removal efficiency up to 0.69 g/m3h as the result of the denitrification process.For further investigations the pilot scale system for air biofiltration was suggested (Fig.8).

Work was supported by the Latvian Council of Science, projects 05.1484, 04.1100, 04.1076, 06.0031.1. Aloizijs Patmalnieks and Lidija Saulite are gratefully acknowledged for SEM. Authors thank Dr. Ritvars Sudars for fruitful discussions.

Effect of medium composition on the growth of the bacteria association and ammonia biodegradation

The main objective of this study was to determine the factors, which influence the physiological state of the association and, therefore, could improve a further use of this association as inoculum for ammonia biodegradation in the biofiltration process. Two different salt compositions (buffered and non-buffered), as well as organic amendments (glucose, fructose, molasses, cabbage leaf extract (CLE), yeast extract) were tested.

Growth of bacteria association in medium with different amendments

Fig. 6. Changes of pH (A) and RedOx potential (B) in culture medium after 14-days cultivation of the PNNS association. (Description of the samples see in Materials and methods)

Fig. 7. Nitrogen content in liquid medium with different amendments. (Description of the samples see in Materials and methods)

A. Relationship between total nitrogen and N-NH4+ in liquid medium before inoculation of the PNNS association.

Analysis of dynamics in ammonia emission from livestock and poultry based on Latvia farms during last decades was performed and showed the tendency of reduction in animal numbers. Among amendments tested in this study during cultivation of the PNNS association, only a cabbage leaf extract demonstrated a sufficient decrease of the total nitrogen in medium during 14-days cultivation, i.e. from 0.5 g/l to 0.1 g/l. This effect should be studied detailed in future.Formation of colonies onto the glass tube surface upon the growth of the PNNS association could serve as a tool for further experiments on cell immobilization onto the carrier in biofilter for ammonia biodegradation. Nitrite formation was detected in the samples with the whole association and with Nitrobacter spp. and Nitrosomonas spp. Cultivation of Pseudomonas spp. and Sarcina spp. alone did not provide the effect mentioned above (results not shown). These facts indicate to the important role of the whole association.The pilot scale system for air biofiltration was suggested.

Fig. 4. Formation of the colonies on the glass tube surface after 14-days cultivation of the PNNS association in liquid B medium amended with molasses (sample B4).

Description of the samples see in Materials and methods)

Fig. 5. SEM micrograph of the colony grown on the glass tube surface after 14-days cultivation of the PNNS association in liquid B medium amended with molasses (sample B4).

Table 1. Annual ammonia emission generated by one animal.

A

B B. Changes of the concentration of the total nitrogen in liquid medium after 14-days cultivation of the PNNS association.Description of the samples see in Materials and methods.3, 10, 12 samples – total nitrogen in medium after cultivation – not determined.

Fig.8. Technological scheme of biofiltration system.

1 – scrubber; 2 – bioreactor; 3 – ventilator; 4 – circulation pump; 5 – sedimentation tank.

- contaminated air; - purified air; - water-dust mixture; - purified circulation water.

Two different salt compositions used in this experiment, i.e. A and B medium, showed a similar effect for bacteria growth, however in medium B the growth was slightly higher. Development of the PNNS association during 14-days cultivation was monitored via OD540 measurement, nevertheless the results on culture turbidity was not used in this paper because of heterogeneity of growing culture. Turbidity in the samples was enhanced already in 2 days after beginning of the experiment. In turn, the maximum turbidity was detected to 7-10 days of cultivation. Afterwards, samples get less turbid due to formation of flakes and slimy fraction. In the samples A13 and B14, i.e. with medium A and B, but without any amendments, bacteria growth was not detected during 14-days cultivation.

Formation of colonies onto the glass tube surface upon the growth of the PNNS association could serve as a tool for further experiments on cell immobilization in biofilter for ammonia biodegradation (Fig.4, 5). The use of the whole association was shown more effective in terms of its application for ammonia biodegradation, as compared to single bacteria species of this association.

Changes of N-NH4+ and total nitrogen during growth of the PNNS

association

Addition of different amendments to the basal salt medium can noticeably change the nitrogen content and, therefore, influence the development of bacteria association and ammonium biodegradation. As shown in Figure 7A, initial concentration of ammonium in all samples was similar and varied in the range of 0.32 - 0.57 g N/l. In turn, amount of the total nitrogen in medium significantly varied in dependence on amendment added to medium. Thus, addition of yeast extract resulted in an increase of the total nitrogen in medium from 0.45 g/l to 1.95 g/l (average data) (Fig.7A).

Changes in the content of the total nitrogen were detected after 14-days cultivation. Thus, the total nitrogen concentration was significantly decreased in the samples 1 and 2, i.e. amended with CLE (Fig.7B). This fact requires more detailed study in order to distinguish the processes of nitrification, denitrification and incorporation of nitrogen-containing compounds into cell biomass.

The ability to carry out both heterotrophic nitrification and denitrification is characteristic to some heterotrophic species as Alcaligenes, Pseudomonas. The PNNS association contains Pseudomonas spp., which theoretically can provide, under certain conditions, nitrification and denitrification processes. This study is supposed to be continued in future.

Changes of pH and Eh during growth of the PNNS association

The pH level of culture media, as well as its redox potential plays a crucial role in bacteria metabolism, and particularly, in nitrification and denitrification processes. At the beginning of cultivation the pH level in all samples was ranged from 7.3 to 8.0. Exception was the samples, containing medium A and yeast extract, i.e. samples 5 and 11, where the pH level was 5.5 and 6.7, correspondingly. As shown in Figure 6A, the pH level of culture medium was changed after 14-days cultivation. Mostly it attributes to the medium A, which is, due to its salt composition, not buffered. As it is known, equilibrium between gaseous and hydroxyl forms of N-NH4+ is dependent on the pH level. At pH below 7.5-8.0 volatilization of ammonia is insignificant.

Redox potential in tested samples at the beginning of cultivation varied in the range of -12 -60 mV, except the samples with medium A amended with yeast extract, where the Eh level achieved +78 and +13 mV, correspondingly. After 14-days cultivation, redox potential in the samples was ranged in diapasone from -67 mV to +10 mV (Fig.6B). Kemp et al. reported that nitrification rate was zero at redox levels of –200 mV and significant rates were observed in sediments when redox values were between –100 and 0.00 mV. Wießner et al. reported that the ammonia removal processes were found to be firmly established, including for moderately reduced redox conditions with high efficiencies for Eh>−50 mV.

1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

1. excreta from domestic animals;

2. excreta from wild animals;

3. synthetic fertilizers;

4. oceans;

5. biomass burning;

6. crops;

7. human population and pets;

8. soils under natural vegetation;

9. industrial processes;

10. fossil fuels.

0

0,5

1

1,5

2

2,5

1 2 3 4 5 6 7 8 9 10 11 12 13 14

media with different additives

N,

g/l

Control (withoutinoculum)

PNNS association

B

0,0

0,5

1,0

1,5

2,0

2,5

3,0

3,5

1 2 3 4 5 6 7 8 9 10 11 12 13 14

media with different additives

N,

g/l

N

NH4A

N-NH4+