ENVIRONMENTAL ENGINEERING

ENVIRONMENTAL
ENGINEERING
May 22-23, 2008
The 7th International Conference
Faculty of Environmental Engineering
Vilnius Gediminas Technical University
Saulėtekio ave 11, LT-10223 Vilnius, Lithuania
Phone: +370 5 2745090; Fax.: +370 5 2744731; e-mail: ap2008@ap.vgtu.lt
COMPARISON OF VARIOUS AMENDMENTS ON THE GROWTH OF THE
TARGETED BACTERIA ASSOCIATION AND AMMONIA BIODEGRADATION
Alina Mihailova1, Olga Muter1, Silvija Strikauska2, Baiba Limane1, Andrejs Berzins1,
Uldis Viesturs1,2,3, Dzidra Zarina1,3
1
University of Latvia, Institute of Microbiology and Biotechnology, Kronvalda boulvd., 4, Riga LV-1586, Latvia,
2
Latvia University of Agriculture, Liela str., 2, Jelgava LV-3001, Latvia
3
Institute of Wood Chemistry, 27 Dzerbenes str., Riga LV-1006, Latvia
E-mail: alina.mihailova@inbox.lv,
olga.muter@inbox.lv,
Silvija.Strikauska@llu.lv,
andrejs54@inbox.lv,
lumbi@lanet.lv,
dz2333@inbox.lv
Abstract. The effect of medium composition on the growth of the PNNS (i.e. Pseudomonas spp., Nitrobacter
spp., Nitrosomonas spp., Sarcina spp.) association was studied. The main objective of this study was to
determine the factors, which influence the physiological state of the association and, therefore, could improve
further use of this association as inoculum for ammonia biodegradation. Two different salt compositions
(buffered and non-buffered), as well as organic amendments (glucose, fructose, molasses, cabbage leaf extract
(CLE), yeast extract) were tested. Among amendments tested in this study, only CLE 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. 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. 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.
Keywords: ammonia, nitrification, biofiltration, PNNS association, amendments, cell adherence.
Abbreviations: CLE – cabbage leaf extract ; CFU – colony forming units; PNNS – Pseudomonas spp.,
Nitrobacter spp., Nitrosomonas spp., Sarcina spp. ; SEM – scanning electron microscopy.
1.
aspects of ammonia biofiltration process, which
involves physical, chemical and biological
interactions [5].
In the process of biological ammonia oxidation
either bacteria of a single species or bacterial
associations can be used. As growth rates of nitrifying
bacteria are extremely low, the acclimation time to
reach steady state sometimes takes 1-2 months [3].
It has been reported that in mixed cultures
nitrification reactions frequently are observed to
proceed more rapidly in the presence of heterotrophic
microorganisms than in pure culture [6]. Organic
metabolites formed by the chemosynthetic bacteria
could readily account for any extensive development
of heterotrophs in inorganic nitrification media.
Isotopic labelling studies have proved that different
Introduction
At present, ammonia emission and subsequent
wet and dry deposition are a significant waste
management problem facing animal husbandry and
agriculture. The traditional treatment of ammonia is
based on physical and/ or chemical processes [1].
Biofiltration is an emerging technology [2], and in
comparison with traditional methods of air pollution
control, it offers a number of advantages for the
treatment of low concentrations of polluted air
streams [3]. Biological ammonia removal occurs by
the process of its nitrification to nitrate. Nitrate can
subsequently be denitrified to nitrogen. Both
autotrophic and heterotrophic nitrification can take
place [4]. There are many uncertain and variable
218
organic compounds are able to incorporate into cells
of autotrophic bacteria. These compounds include
acetate, pyruvate, [7, 8, 9, 10], a-ketoglutarate and
succinate [8], amino acids [8, 11, 12], fructose [13].
Krummel and Harms reported that addition of
formate, acetate, pyruvate, glucose, or peptone had a
negligible effect on NO2 – formation in two
Nitrosomonas strains [9]. Other research shows that
addition of some amino acids to N. europaea cells
was reported to increase production of NO2 – and
production of protein [12]. Hommes et al. [13]
reported that Nitrosomonas europea cultures
containing mannose, glucose, glycerol, mannitol,
citrate or acetate show little or no growth.
Thus, the effect of organic amendments on
growth and activity of nitrifying bacteria is
discussible.
In the present study, an autotrophic ammoniabiodegrading association (PNNS) previously isolated
from the biological activated sludge of the fish factory
wastewater treatment plant, was used [14, 15]. This
study was mostly focused on the testing of various
amendments in the medium during association growth
with the aim to determine the factors, which influence
the physiological state of the association and therefore
could improve further use of this association as
inoculum for ammonia biodegradation. Experiments
were performed with batch cultures.
2.
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.
3.
Materials and methods
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 [modified from 14]. 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 [modified from
16]. 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. CLE was
prepared as described in [15]. 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.
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
219
Results and discussion
Growth of PNNS 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 is not used
in this paper because of heterogeneity of growing
culture. For this reason it is quite difficult to
distinguish the phases of culture growth. 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.
Besides, formation of colonies on the surface of
glass tubes was detected. These colonies became
visible already after 48h of incubation as the small
points with further growth in size (Fig.1). The
colonies were distributed on the bottom of tubes, in
the middle part, as well on the upper side of the
cultivation medium. Size of the colonies ranged from
3-4 mm in diameter to smaller (most probably, there
were also invisible micro-colonies on the surface,
thus, the smaller diameter of the colonies is not
indicated here). Colonies differed by their
morphology. One kind of colonies is presented on the
Figure 2.
1 cm
Fig 1. 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).
2,5
N-NH4+
N, g/l
2
1,5
1
0,5
I
0
1
2
3
4
5
6
7
8
9
10 11 12 13 14
Samples with different media content
Control (without inoculum)
PNNS association
2,5
2
N, g/l
I. The center of the colony.
1,5
1
0,5
0
1
II
2
3
4
5
6
7
8
9
10 11 12 13 14
Samples with different content
Fig 3. Nitrogen content in liquid medium with different
amendments
I - Relationship between total nitrogen and NNH4+ in liquid medium before inoculation of the
PNNS association.
II - 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.
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.3II). This fact requires more detailed study in order to
distinguish
the
processes
of
nitrification,
denitrification and incorporation of nitrogencontaining compounds into cell biomass.
The ability to carry out both heterotrophic
nitrification and denitrification is characteristic to
some heterotrophic species as Alcaligenes [17, 18,
19], Pseudomonas [20, 21, 22]. 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.
II. The periphery of the colony.
Fig 2. 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).
Colonies consisted mostly of the rounded and
slightly elongated cells with the cell length 0.7 – 1.3
µm (Fig.2-I, 2-II). The most active formation of the
colonies adhered on the glass surface, was detected in
the samples amended with glucose and molasses. It is
important to note, that cultivation of single strains of
the PNNS association under the same conditions did
not result in colonies formation on the tube surface.
Changes of the concentration of 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 3A, 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.3-I).
220
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 4-I, 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. At pH of 9.3 the ratio between
ammonia and ammonium ions is 1:1 and the losses
via volatilization are significant [23, 24].
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.4-II).
Kemp et al. [25] 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 [26]. 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 [27].
-40
Description of the samples see in Materials and
methods.
Nitrite formation during growth of the PNNS
association. Nitrite formation in medium during
cultivation was monitored qualitatively. Among all
tested variants, the presence of nitrites during all the
period of cultivation was detected in the samples 3, 6,
and 11, i.e. with molasses, yeast extract and a mixture
of all tested amendments added to cultivation
medium.
Various amendments in cultivation medium
could change the equilibrium between single strains
of the bacteria association in different manner. For
example, Clark and Schmidt [28] reported that
contamination of a culture of Nitrosomonas europea
resulted in enhanced nitrite formation as compared
with pure cultures. Growth of N. europaea was
stimulated in mixed cultures with the heterotrophic
isolate to varying degrees, apparently depending on
the age and density of autotrophic inocula. Mixed
culture stimulation of N. europaea was evidenced
only by a shortened lag phase, since post-lag phase
growth of the autotroph was equivalent in both pure
and mixed cultures. The heterotrophic component had
a nutritional requirement for amino acids when grown
on simple media; presumably, in the mixed culture
system, the heterotroph was provided with amino
acids synthesized by the autotroph [28].
Additional experiments with single strains of the
PNNS association demonstrated completely different
dynamics of nitrogen formation upon cultivation
(results not shown). It demonstrates a complexity of
the processes occurred in the cultivation medium
inoculated with different bacteria strains and amended
by different organic components.
Besides, studies on nitrogen conversion under
low-oxygen and anaerobic conditions have shown that
ammonia can be converted to dinitrogen by processes
other than conventional nitrification of ammonia to
nitrate followed by denitrification of nitrate to
dinitrogen gas [29]. Under low-oxygen conditions, the
production of nitrite from ammonia is favoured over
the production of nitrate [30]. The nitrite can then be
denitrified to nitrous oxide and/or dinitrogen without
being converted to nitrate. This process has been
termed “partial nitrification–denitrification”.
Thus, as shown in literature, nitrites are
intermediates in the complex processes of
ammonification, nitrification and denitrification.
Therefore, the presence or absence of the nitrites is in
itself not enough to explain the processes occurred in
cultivation medium.
-60
4.
Control (without inoculum)
PNNS association
I 8,5
pH
8
7,5
7
6,5
6
0 1 2 3 4 5 6 7 8 9 1011 1213 14
Samples with different media content
II20
0
Eh
-20 0 1 2 3 4 5 6 7 8 9 10 1112 1314
Conclusions
-80
Addition of all tested compounds resulted in
noticeable stimulation of the growth of the PNNS
association. The association inoculated into medium
without amendments – did not develop during 14days cultivation. Most probably, addition of organic
-100
Fig 4. Changes of pH (I) and RedOx potential (II) in culture
medium after 14-days cultivation of the PNNS association.
221
compounds provided an active growth of heterotroph
(e.g. Pseudomonas spp.). At the same time, there have
been many reports about a stimulating effect of
different organic additives on nitrification rate and
growth of autotrophic bacteria [6-13].
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.
Among amendments tested in this study, only
CLE 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.
Yeast extract itself (in concentration of about
2%) significantly influenced the main parameters of
the cultivation medium, i.e. increased pH, as well as
decreased Eh. Addition of yeast extract resulted in 4fold increase of the total nitrogen in medium.
Obviously, for future experiments, yeast extract
should be used in lower concentrations.
Summarizing, the results obtained in this study
provide the information, which is necessary for
further development of biofiltration system for
ammonia biodegradation. Optimization of nitrification
and denitrification processes with the use of bacteria
association could sufficiently improve the biofilter
construction. In particular, the process of
simultaneous nitrification-denitrification (SND) i.e.
without alternating anoxic and oxic phases in time or
space has recently elicited significant interest. The
ability to carry out both heterotrophic nitrification and
denitrification was shown for Pseudomonas spp. [2022], and, therefore could be promising for further
optimisation of ammonia biodegradation. The role of
other species of the PNNS association in the presence
of organic compounds is still not clear. However, the
results obtained in this study, indicate to the important
role of the PNNS association as a whole. For instance,
colony formation on a surface was detected only in
the samples with the whole association. 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.
There are many uncertain and variable aspects of
ammonia biofiltration process, which involves
physical, chemical and biological interactions [31,32].
Knowledge of microorganisms’ ecosystem, their
transformations and interrelations could significantly
improve the ammonia biodegradation.
Acknowledgement
This work was supported by the Latvian Council
of Science, i.e. grants 05.1484, 04.1100, 04.1076.
Aloizijs Patmalnieks and Lidija Saulite are gratefully
acknowledged for SEM.
222
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