FEMS MicrobiologyLetters68 (1990)53-60
Publishedby Elsevier
53
FEMSLE03919
Denitrifying population dynamic and evolution of nitrogen gas
during a composting process
Sergio Casella a n d A n n a Rutili
lstituto Microbiologia Agraria. Universita" di Pisa Centro di Studio per la Microbiologia del Suolo, CNR, Pisa, Italy
Received27 October1989
Revisionreceivedl0 November1989
Accepted 15 November1989
Key words: Denitrification; Composting; Nitrogen gas evolution
1. SUMMARY
Little information exists about nitrogen losses
through microbial activity during treatment of
solid urban waste (SUW) by processes such as
composting. In the present study, in addition to
evaluating the pattern of nitrogen losses by denitrification at different stages of the process, a
comparison between the method of Pochon and
Tardienx, and an improved gas chromatographic
method for estimating denitrifyingpopulations was
undertaken. Though the MPN (Most Probable
Number) enumerations were higher using the colorimetric method than the gas chromatographic
one, the patterns of the two graphs showing numhers of denitrifiers during composting were the
same. The highest numbers were revealed immediately after loading the reactor (107-10S/g
d.w.), lower numbers of denitrifiers were found in
the second sampling corresponding to the thermophific phase (103-104/g d.w.). These numbers increased gradually as the waste material stabilized
Corres~
to: Dr. Sergjo Casella, Isfituto Microbiologia
Agtaria, Univcrsita+di Pisa,Via del Borgheuo,80, 56100Pisa,
Italy.
(10th to 123rd day of composting) to again reach
values of 10~-10s/g d.w.
2. INTRODUCTION
Denitrification is a component of nitrogen cycling in soils and waters of most terrestrial
ecosystems. Bacteria are the only organisms having the physiological capacity for denitrification, a
process defined as the reduction of ionic to gaseous nitrogenous oxides and N 2 []]. Most of the
denitrifying bacteria in soil are chemoheterotrophs
[2]. The amount of water-soluble or readilydecomposable organic matter, the major substrate
for heterotrophic microbial respiration, seems
largely to control denitrification in soil [3].
There have been numerous studies on the influence of the type and amount of organic matter
on reduction of NO~- when the energy sources for
this reduction derive from native soil organic
matter, plant debris, model compounds or
wastewaters [4-8]. Little is known, however, about
nitrogen losses through denitrification in the treatment of solid urban waste (SUW), by processes
such as composting. The importance of nitrogen
0378-1097/90/$03.50© 1990 Federationof EuropeanMicrobiologicalSocieties
losses as a consequence of denitrification are
therefore not yet known.
Causes and amounts of nitrogen lost during
composting may vary greatly according to the type
of waste, the system used and the operational
parameters of the process [9]. Enumeration of
denitrifying bacteria indicates the denitrifying
capacity of the populations inhabiting composted
organic waste, and its variation at different stages
of composting. As a consequence it will be possible to estimate the potential for nitrogen losses
dependent on these populations during the process. In the later stage, denitrifying bacterial enumeration can be useful in order to evaluate the
effect of compost addition to various kind of soil.
Tiedje [1] reviewed methods developed for enumerating denitrifier population. In the present
study we compare the results of two different
methods to determine the relative ability of each
to quantitate denitrifying populations.
One method follows the disappearance of N O ;
with the diphenylamine (DA) colorimetric test as
evidence of denitrification [10]. In the second
method, accumulation of N20 in enrichment cultures behind an acetylene block is used as confirmation of denitrification [11].
3. MATERIALS AND METHODS
3.1. Materials and analysis
The biodegradable organic fraction of SUW
was sampled during one of several composting
trials performed at the Institute of Microbiologia
Agraria, Universita di Pisa, between the springs of
1985 and 1986. Chemical-physical characteristics
of the starting material and of the compost obtained have been previously described [12]. Denitrifier populations were followed through all
stages of the process. Samples were taken at days
0, 6,10,17, 31 (end of stabilization stage) and 123
(later curing stage), At each sampling time, enumerations were obtained in triplicate.
3.2. Preparation of samples, culture conditions and
media
Ten-gram (wet-weight) samples were dispersed
in 90 ml of sterile distilled water by blending for 2
min. Tenfold dilution series from 10 -I to 10 -~
were prepared in sterile distilled water for each
replicate. The culture medium was as described by
Pochon and Tardieux [10], but containing 3 mM
KNO3; pH after autoclaving was 7.2. Each culture
tube (14.5 ml) containing 5 ml of culture medium,
was inoculated with 0.5 ml each of the appropriate
serial dilution, sealed (Subbaseal), and incubated
after flushing for 1 rain with Helium gas through a
sterile 0.22 lam filter, in the dark at 28°C. Five
tubes were employed per dilution. Enumeration
was carried out after incubation of 14 and 21
days.
3.3. Enumeration of denitrifying populations: first
method
This method follows NO~- disappearance in
enrichment cultures, determined by the DA colorimetric test, as evidence of denitrification as described by Pochon and Tardieux [10]. After 14
and 21 days of incubation, from 0.1 to 1.0 ml of
each culture tube medium was withdrawn by gastight syringe (disposable tuberculin type) and
tested for NO~- content by adding about 25 mg
urea, a few drops of H2SO4 (to eliminate N20)
and dropwise up to 5 drops of DA reagent (prepared by dissolving 0.2 g of diphenylamine in 100
mi of conc sulfuric acid). A blue coloration indicated the presence of NO~- and was scored negatively for denitrification; a colorless reaction was
scored positively. For each dilution, the number of
positive among the five tubes was determined and
the concentration of the viable organisms was
estimated by referral to standard McCready tables
[131.
3.4. Enumeration of denitrifying populations: second
method
With this method, gas chromatographic detection of N20 in the presence of CzH2 confirmed
denitrification [11]. After 14 and 21 days of incubation, 0,1 ml of sterile 150 mM KNO 3, equivalent to 0.21 mg of NO~'-N, was aseptically added
to each culture tube with a gas-tight syringe to
stimulate N20 production. Then 50 /~1 C2H 2, a
headspace concentration of 0.5% v/v, was aseptically added by injection through a 0.22/~m filter
plus syringe assembly. Tubes were again in-
cubated in the dark at 28°C until N20 production
was clearly detectable (at least 10 nmol N20 per
0.1 ml gas samples).
3.5. N20 assay
Gas samples of 0.1 ml were analyzed for N20
with a Dani-3800 gas chromatograph equipped
with a Ni electron capture detector and 3-m length
of Poropak Q 80/100 mesh column with a 2 mm
inner diameter. Temperature of analysis were
200°C for the detector and 120°C the injector
port. Oven temperature was 60°C. The carried
gas was helium at a flow rate of 30 cm/min. A
standard curve produced from a series of N20
dilutions. Tubes exhibiting detectable N20 production were considered positive. For each dilution, the number of positive among the five replicates was determined and concentrations of the
viable organisms estimated with the aid of a
standard McCready table [13].
02 concentrations were determined for the same
gas samples analyzed for N20 through the same
gas chromatographic equipment.
4. RESULTS AND DISCUSSION
The population fluctuations shown in the two
graphs in Fig. 1 can be explained by the different
ecological conditions arising in the material dur-
10 Log
no.cell$/gd.w.
• •
4
,
0
÷
~
20
4-
40
60
80
T~ne (cloys)
D,Atest
100
120
140
--4"- G.C. test
Fig. l . Enumeration o f denitrifying bacteria d u r i n g the cornp o s t i n g process (iD
L D A test method,
I
•
acetylene blockage method). T h e values represent the means:t:
S D for three rclpficates.
ing the transformation of the biodegradable
'~rganic fraction into compost. A heterogenous
h~ roflora capable of metabolizing soluble organic
m=tter (simple sugars, proteins, fats) initially develops rapidly [9]. During this phase oxygen is
rapidly consumed; and, if not replaced, conditions
of partial anaerobiosis arise, slowing down biooxidation. In the present composting process the
biodegradable organic fraction was anaerobic at
the time of loading the reactor due to an absence
of aeration during transport from the selection
plant to the composting site. Low oxygenation
and high amounts of easily degradable organic
matter encourage the development of heterotrophic organisms which are able to ferment or
carry out anaerobic respiration. These condition
explain the presence of denitrifiers in greater number in the first sample than in those that followed.
After loading the reactor and initiating aeration, exergonic biooxidation rapidly recovers and
the temperature rises to values typical of the thermophilic phase in the first stage of the process
[12]. Strong selectivity towards mesophilic microflora during the early stage may explain the sudden decline in the number of denitrifiers in sample
from day 3, which was within the full thermophilic
stage. Most denitrifying microorganisms are not
thermophilic; only a few species of Bacillus denitrify and form spores. The only denitrifiers identified in self-heating materials at thermophilic
temperatures (48-69°C) are members of the genera Pseudomonas and Bacillus [14]. Moreover,
Keeney et al. [15] reported that at temperatures
> 50°C chemodenitrification may dominate over
biological denitrification.
A fall in temperature (on about the tenth day in
the present trial) indicates the end of the thermophilic phase and the onset of the mesophilic stage
(second stage in the composting process). This
temperature drop reflects the beginning of the
transformation of complex long-chain compounds
(cellulose and lignin), particularly by eumycetes
and actinomycetes [16]. The more easily available
organic fractions which, in soil, seem to affect
denitrification to a considerable degree, are exhausted in this phase. In soil studies denitrification activity appears closely related to extractable
organic glucose [17]. As a result, the addition of
simple sugars, particularly glucose, causes a related growth and activity of denitrifiers [7]. These
results may indicate the reason for the slow recovery in growth of denitrifiers after the thermophilic phase in this compost pile. The slight decrease in numbers of denitrifiers detected in the
final sampling (123rd day of composting) can also
be ascribed to the humification of the material
occurring at this stage.
Another factor controlling denitrifier growth
may be microbial competition for N O f - N present
in the substrate. In soil N O f - N limits the increasing denitrifying biomass when below 1 mg/kg soil
[18]. The organic fraction of SUW usually contains higher amounts of this element (roughly 150
mg/kg of dry matter of the organic fraction loaded
into the reactor). However, since there is a much
higher proportion of heterotrophic microorganisms in SUW than in the soil, NO~--N or,
more generally, inorganic nitrogen, may still be a
limiting factor.
The enumeration of denitrifiers during composting is vital to understanding the cycling of
nitrogen in such a process. However, counting is
infrequently attempted because of uncertain methodology. Our results show that using the second
MPN method may provide a reasonable estimate
of denitrifier populations, for example, when using
the first MPN method, tubes with no trace of
NO 3 were evaluated as positive for denitrification. The first method, however, does not discriminate between denitrification or dissimilatory
and assimilatory reduction of NO 3 to NH 4. This
is true even if N O r is detected, as that ion is a
common intermediate of both pathways.
Fig. 1 shows that MPN enumerations were
higher using the first than the second MPN
method, though the overall patterns of the two
graphs showing numbers of denitrifiers during
composting were the same,
In the first samples taken (immediately after
loading the reactor) high numbers of denitrifiers
were revealed by both methods with several corresponding values. In the second sampling (thermophilic phase), lower numbers of denitrifiers were
found, particularly with the second MPN method.
These numbers increased gradually as the waste
material stabilized, but in all samples the popula-
tion was smaller when detected by the second
method.
Results of the DA colorimetric test were not
always unequivocal. In theory, the presence of
NO 3 should be revealed by a blue coloration,
whereas colorless samples should indicate the absence of this ion. In practice, even when extra care
was taken in carrying out the assay, an unexpected
green color frequently developed, making the test
difficult to evaluate. These cultures were scored
negatively.
Results of the DA test incubation for 14 and 21
days were frequently different (Table 1). After 14
days some of the higher dilution cultures (10 -3 ,
10 -6, 10 -7) showed the presence of N O r . These
were scored negatively. After 21 days, the same
tubes were colorless at: 10 -5 , 10 -6 (day 31), 10 -4 ,
10 -5 (day 10), 10 -3, 10 -4 (day 2) and at least up
to the 10 -~ dilution level for all other samples.
Determination after 28 days of incubation (data
not shown) gave the same results as those of the
21-day determinations.
The results of the N20-assays after 14 and 21
days were also different (Fig. 2). The differences,
however, were not related to the presence or absence of N20 (Table 1) but in the amount of N20
produced. After 14 days highest amounts of N,O
were found in the lowest dilution levels (Fig. 2).
After 21 days of incubation the results were different in that increasing amounts of N20 were found
at the highest dilution levels. There was an increase from 10 - I to 10-5; we then noted a slight
decrease in extinction range dilution tubes (10 -5 ,
10 -6, 10 -7 and 10 -8, day 0). Interestingly, N20
was found after 14 days in some of the samples
requiring a full 21-day incubation to become positive using the DA test (Table 1).
In the low dilution levels most of the N20 was
produced within a few hours; whereas, in the most
diluted samples (particularly 10 -6 to 10-8),
evolution of N20 was still occurring after incubation for 24 h (Fig. 2). In all samples, however, and
with either incubation period (14 or 21 days), N20
was clearly detectable after incubation for 5 h. For
this reason, even if the mean N20 production
rates were different (Table 2), tubes were scored at
this incubation time. Table 2 also shows that the
amount of N20 found in each tube (at all dilution
N20
D.A,
24
N20
D.A.
123
.
.
+ + --. . . .
. . . . .
+++++
.
+++++
+++++
+++++
+++++
+++++
+++++
+++++
+++++
+++++
+++++
+++++
+++++
.
+++++
+++++
14 ~
l0 -4
+++++
+++++
+++++
+++++
+++++
+++++
+++++
+++++
++++
+++++
+++-+++
+++++
++ ++- - -
+++++
. . . . .
. . .
++ . . . . . . . . .
+++++
+++++
21 ~
++
+++++
+++++
+++++
+++-++
++
++
+++++
++ . . .
+ . . . .
~ . . . .
+++++
+++++
21 c
A l l t h e s a m p l e s a t 10 - 9 d i l u t i o n level resulted as . . . . .
c incubation time in days.
All t h e s a m p l e s a t 10 - t d i l u t i o n level resulted as + + + + +
a s a m p l i n g t i m e i n days.
b D . A . : c o l o d m e t t i © M . P . N , m e t h o d ; N 2 0 ; m o d i f i e d M . P . N . m e t h o d b y g,c. N 2 0 d e t e c t i o n .
~ negative tube).
+++++
+++++
+++++
+++++
+++++
+++++
.+ .+ .+ .+ .+ . . . . . .+++++
+++++
. . . . . . . . . .++.+.++.+.++. +. . . .
+++++
+++++
+++++
+ . . .
+++++
+++++
+ . . . .
+ . . . .
+++++
+++++
+++++
14 ~
14 c
+++++
10 - 3
21 c
10-2
Dilution levels
(+ = positive tube, -
N20
D.A.
31
N20
D.A.
17
N20
D.A.
6
10
D.A.
0
N20
D,A.
N20
test b
days a
.
.
.
.
.
.
.
.
+++
+ + - - + + - - -
+++--
+ + - - . . . . .
.
.
+
+ . . . .
14 c
10 - 5
.
.
.
.
.
.
.
+++++
++ . .
+++--
++ . .
+++++
. .
+++
+
.
+++++
+++++
21 c
.
.
+
C n m n ~ 6 s o n o f r e s u l t s b e t w e e n f i r s t a n d s e c o n d M P N m e t h o d f o r d i f f e r e n t d i l u t i o n l e v e l s a f t e r 14 a n d 21 d a y s i n c u b a t i o n
Table I
.
.
.
.
.
.
.
.
.
.
.
. . . . .
. . .
+ + - - -
.
. . . . .
iii?!
.
.
+++-.
.
.
.
.
.
.
.
.
.
.
+
. .
++
.
+++++
.
.
++
. .
.
+
.
?iii!+
.
.
+++---
.
.
.
.
+++++
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
. . . . .
. . . . .
?iiii
.
.
~ . . . .
14 c
. . . . .
10 -7
14 c
21 ¢
10-6
.
.
.
.
.
.
.
.
.
+++++
+++++
.
.
.
.
.
:::::
.. .
.
~++++
21 ¢
.
.
.
.
.
.
.
.
.
.
.
.
:::::
.
. . . . . . . . . .
. . . . . . .
. . . . . . . . . .
. . . . . . . . . .
:::::
. .. .. . . . . . . . . . . . . . . . . .
.
21 ~
. . . . . . . . . .
14 c
IO - 8
_
Sarn:
l
14 Days
t
21 Days
~4
i
0
2
1-
7,{:
2
1i
i
24
5
15
~4
T{me t hour's )
Fig. 2. Total amount of N20 evolved at different ~'~!ution levels
of each sampling after 5,15, 24 h incubation at 14 an 21 days.
Numbers on the lines represent the dilution level.
levels and after either 14 or 21 days), was greater
than the 6.4/~mol that would have been detectable
in the gaseous phase if the NO~- had been added
before the N20 assay had been completely reduced to N20 (this quantity was calculated
according to the Bunsen absorption coefficient
[19D.
The amount O 2 in the tubes after 14 days
decreased from atmospheric levels to about 0.5~
of the total in the lowest dilution levels (10-I,
10 -2, 10 -3, 10 -4, 10-s), and to about 1.5~ in the
highest (10-6,10 -7, I0 -z, 10 -9 ) (data not shown).
After 21 days of incubation the O 2 concentration
decreased to between 0.17 and 0.5~ for all dilution levels, with a few exceptions (10 -7, 10-9).
Our estimates of populations in organic waste
material are consistent with the soil studies of
Volz [20], showing that nitrate reducers were more
prevalent than denitrifiers. The first MPN method
does not, therefore, seem suitable for enumeration
of denitrifying bacteria. Interpretation of the colorimetric test is not always straightforward due to
the different colors which sometimes developed.
Moreover, several samples scored as negative at 14
days come up positive after 21. Thus, even if
nitrate reducers were present, they were undetected because they were incapable of reducing
considerable amounts of nitrate. Changes in results of the DA test carried out at different incubation were previously described [21].
The enzymes for N-oxide reduction are induced
under low 02 conditions [22,23]. in the present
study all the tubes were incubated at 02 levels low
enough to permit the expression of denitrification
enzymes (below 16.5~ air saturation [22]). Test
cultures with the highest 02 concentrations yielded
the lowest concentrations of N20. This was more
a reflection of low bacterial number than of
elevated 02 levels. Extending the period of incubation of these tubes by one week permitted the
total amount of N,O produced to reach nearly
identical quantities for all dilution levels. More
importantly, however, the negatively-scored samples became positive after this additional period of
incubation; this is the observation that confirms
the validity of this test, as compared with the
uncertainty of the DA method.
The biological production of N20 typically observed in denitrifier cultures [24] and among natural soil flora [25], is not limited to this group of
microorganisms. Other possible sources of N20
are nitrate respiration by non-denitrifying NO~"
reducers [26], nitrification [2"/,28], and chemo-denitrification [29]. N20 production seems however
to be ascribable to these processes only under very
specific environmental or cultural conditions
[16,30]. Therefore, media and culture conditions,
in particular O 2 concentration in the tubes, are
selective towards denitrifying activity, N20 can be
considered mainly to result from this process.
Uncertainties concerning the evaluation of
gaseous nitrogen losses from the soil in general,
and from compost, particularly during the process,
suggest that monitoring the bacterial population
involved in the transformations of nitrogen can be
Table 2
Evolution of N20 at different dilution levels of each sampling after 5 h incubation, at 14 and 21 days
Time a
Sampling
0
6
10
17
24
31
123
Dilution levels
incub.
10 - I
10 -2
10 -3
10 -4
10 -5
10 -6
10 -7
10 - s
14
21
14
21
14
21
14
21
14
21
14
21
14
21
6.8+0.2
3.0+0.5
5.9+0.1
2.9_+0.8
6.0+0.2
3.4+0.2
7.5_+0.3
3.0+0.1
7.0+0.3
3.4_+0.1
7.4+0.3
3.3_+0.1
6.6_+0.3
3.0+0.2
4.0+0.1
3.3+0.8
3.7+0.1
3.3_+0.1
3.9+0.1
3.0_+1.0
4.6_+0.1
3.6+0.1
4.0-+0.2
3.6_+0.2
4.8+0.2
3.4_+0.2
4.0+0.2
3.4+0.1
1.9+0.1
3.3+0.1
1.4+0.1
4.8+1.3
1.3_+0.1
4.5_+0.2
i.4_+0.1
4.8_+0.1
1.6_+0.1
4.7_+0.2
1.7-+0.1
5.0_+0.3
1.6_+0.1
4.8_+0.3
1.4+0.1
5.2-+0.3
1.3+0.2
5.9_+0.1
0.8-1-0.1
5.6_+0.1
0.7+0.1
5.6+0.3
0.01
5.6+0.1
1.9_+0.1
3.4+0.2
i.9+0.1
3.2_+0.2
1.9-+0.1
4.0-+0.1
2.0_+0.1
3.4_+0.2
1.9_+0.1
3.9+0.1
- - - - -
i.1 +0.1
6.2_+0.2
1.3_+0.1
5.7_+0.4
1.1 _+0.1
6.3+0.5
0.8_+0.1
5.9_+0.3
The rates represent the means + SD for five replicates.
a sampling and incubation time are expressed in days.
All samples at 10-gdilution level resulted as - - All the values are expressed as/~mol N-N20 tube- t. h - i.
of great interest for better understanding
zation of compost.
the utili-
ACKNOWLEDGEMENTS
W e w o u l d l i k e t o t h a n k M a r i n a B o n f a n t i for
essential assistance in manuscript preparation, M.
De Bertoldi, G. Picci, W.J. Payne and J.M. Lynch
for critical reading of the manuscript.
This work was supported by National Research
Council, Italy.
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