Transcript Slide 1

Comparison of various amendments on the growth of the targeted
bacteria association and ammonia biodegradation
Alina
1
Mihailova ,
Olga
1
Muter ,
Silvija
2
Strikauska ,
Baiba
1
Limane , Andrejs
1
Berzins ,
Uldis
1,2,3
Viesturs ,
Dzidra
1,3
Zarina
1University
of Latvia, Institute of Microbiology and Biotechnology, 4 Kronvalda blvd., Riga LV-1586, Latvia, [email protected]
2Latvia University of Agriculture, 2 Liela str., Jelgava LV-3001, Latvia,
3 Institute of Wood Chemistry, 27 Dzerbenes str., Riga LV-1006, Latvia
Materials and m ethods
Introduction
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 +30C 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.
Results and discussion
Evaluation of ammonia emitted from animal husbandry sector in Latvia
Effect of medium composition on the growth of the bacteria association and ammonia biodegradation
The 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.
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
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.
The data on dynamics of ammonia emission from farms should be taken into consideration for biofilter
construction.
human population
and pets;
8.
soils under natural
vegetation;
9.
10.
9.
industrial
processes;
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).
10.
fossil fuels.
0
Years
Dairy cattle
Pigs
Horses
Non-dairy cattle
Sheep
Poultry
Fig. 2. Annual emission of
Fig. 3. Annual changes in ammonia
nitrogen generated by one
animal.
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).
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.
A
8,5
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 (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)
7,5
Technological scheme of
biofiltration system.
0
Nitrogen content in liquid medium with different
amendments. (Description of the samples see in Materials
and methods)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
NH4
N-NH4+
2,0
1,5
0,5
0,0
20
10
1
2
3
4
5
6
7
8
9
10
11
12
13
3
14
-20
-30
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.
4
5
6
7
8
9
10 11 12 13 14
media with different additives
B
0
2
A. Relationship between total nitrogen and N-NH4+ in
liquid medium before inoculation of the PNNS association.
0
Eh, mV
3,0
1
media with different additives
-10
N
2,5
Fig. 7.
Association
(after 14
days)
6
B
3,5
1,0
Control
6,5
Fig.8.
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).
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).
Fig. 6.
8
Fig. 5.
Changes of N-NH4+ and total nitrogen during growth of the PNNS
association
A
7
1 – scrubber;
2 – bioreactor;
3 – ventilator;
4 – circulation pump;
5 – sedimentation tank.
Description of the samples
see in Materials and
methods)
pH
7.
2007
8.
2000
10
0
2005
crops;
4000
2003
6.
6000
2001
7.
8000
1999
biomass burning;
10000
1997
5.
6.
12000
80
70
60
50
40
30
20
1995
oceans;
Changes of pH and Eh during growth of the PNNS association
14000
1993
4.
5.
100
90
0,4 0,1
1991
4.
synthetic
fertilizers;
10,3 2,9
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).
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.
Poultry
NH3, ktonnes/year
3.
3.
1,1 0,3
Poultry
excreta from wild
animals;
4,6 1,4
Horses
Horses
2.
Sheep
Pigs
excreta from
domestic animals;
Pigs
Sheep
1.
1.
2.
Non-dairy
cattle
18,3 5,5
Dairy cattle
NH3,kg/year
Dairy
cattle
20,3 6,1
Non-dairy cattle
Annual
ammonia emission
generated by one animal.
Nitrogen, kg/year
Table 1.
Fig. 4.
N, g/l
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).
Control (without
inoculum)
PNNS association
2,5
2
N, g/l
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.
1,5
1
-40
-50
0,5
-60
-70
0
-80
1
-90
2
3
4
5
6
7
8
9 10 11 12 13 14
media with different additives
media with different additives
- contaminated air;
- water-dust mixture;
- purified air;
- purified circulation water.
Conclusions
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.
Acknowledgements
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.