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Previous Article | Next Article 
Applied and Environmental Microbiology, February 1999, p. 396-403, Vol. 65, No. 2
Department of Plant-Microorganism Interactions, Netherlands
Institute of Ecology, Center for Terrestrial Ecology, 6666 ZG,
Heteren, The Netherlands1;
Laboratory
for Microbial Ecotechnology, Research Institute for Agricultural
Microbiology, St. Petersburg-Pushkin 8, 189620, Russia2;
Department of Manure
Technology, Institute of Agricultural and Environmental Engineering
(IMAG-DLO), NL-6700 AA, Wageningen, The
Netherlands3;
Department of
Microbiology, Wageningen Agricultural University, 6703 CT, Wageningen,
The Netherlands4; and
Center
for Environmental Biotechnology, University of Tennessee, Knoxville,
Tennessee 37932-25755
Received 18 September 1998/Accepted 9 November 1998
Although the practice of composting animal wastes for use as
biofertilizers has increased in recent years, little is known about the
microorganisms responsible for the nitrogen transformations which occur
in compost and during the composting process. Ammonia is the principle
available nitrogenous compound in composting material, and the
conversion of this compound to nitrite in the environment by
chemolithotrophic ammonia-oxidizing bacteria is an essential step in
nitrogen cycling. Therefore, the distribution of ammonia-oxidizing
members of the Nitrogen is one of the most
important elements in plant nutrition, and the high crop yields
obtained by modern agriculture have been greatly facilitated by the
application of nitrogen-rich fertilizers. Traditionally, chemical
fertilizers have been the major source of nitrogen amendment in
agricultural systems, but recently the use of biofertilizers, derived
from animal excretia, has become increasingly important. Manure
production has increased considerably during recent years, and in some
countries animal production is limited by the manure disposal
regulations (6, 18). As manure contains a large proportion
of the nutrients ingested by an animal, it has the potential to be a
valuable source of nutrients for plants when it is recycled and used as
a biofertilizer. Although manure may be treated as waste in regions
where the levels of animal production are high, it can also be regarded
as a valuable resource and used either directly or after processing. In
addition to providing nutrients for agricultural crops and helping
alleviate the problem of excess animal waste, biofertilizers can also
improve soil fertility and suppress pathogenic microflora, and the
effects can last for up to several seasons (2, 53).
Composting is one of the oldest and simplest methods of organic waste
stabilization (34). The major goal of composting is to
provide a stable product that is high in nutrients which are easily
accessible by plants. Due to the rapid hydrolysis of urea and the
deamination of unincorporated peptides, ammonia (or ammonium in its
undissociated form) is the most important nitrogenous compound available in composting materials (18). This form of
nitrogen is often not taken up by plants very readily and can act as a substrate for nitrification. Nitrification in the environment can lead
to pollution of groundwater due leaching of nitrate (3) or
can be coupled with denitrification, which results in a loss of
nitrogen from the system or the production the greenhouse gas nitrous
oxide via incomplete denitrification (32). Moreover, when
nitrogen is applied in the form of ammonia, there is potential loss due
to ammonia volatilization, which may give rise to eutrophication or
acidification of both surface water and groundwater (9, 17, 27,
48). Strict controls on the amounts of nutrients that are spread
on agricultural land require an ability to track deposited nitrogen,
which makes stabilization of animal manure products through composting
an essential step in the use of these products as biofertilizers.
Although workers have examined nitrogen balance during composting
(22, 30), this parameter has not been described well yet.
Inbar et al. (18) qualitatively characterized nitrification as a normal process in compost that depends on the composting conditions used. Nodar et al. (28) reported the presence of "very few ammonium oxidizers and nitrite oxidizers" belonging to
undetermined genera in a poultry dung-pine sawdust mixture, and the
number of nitrifiers decreased after prolonged storage of poultry
slurry (29). Thus, although nitrifiers have been recognized
as potentially important organisms in composts and composting
materials, their species composition, distribution, and activity have
not been assessed yet.
The lack of data concerning ammonia oxidizers in compost may be due to
the difficulties encountered in studying this specialized group of
organisms by conventional culture-based techniques. Ammonia oxidizers
have low maximum growth rates and produce low biomass yields, and
pure-culture isolation is extremely time-consuming and unrepresentative
(36).
The ammonia-oxidizing bacteria comprise the following two monophyletic
lineages within the class Proteobacteria, based on 16S rRNA
gene sequences: the genus Nitrosococcus in the Denaturing gradient gel electrophoresis (DGGE) is a powerful tool for
analyzing microbial communities. It has been used to separate mixed PCR
products after amplification of 16S ribosomal DNA (rDNA) fragments
(25), and this technique has been adapted to the study of
ammonia-oxidizing bacteria (19). DGGE band patterns can be
characterized by hybridization with specific oligonucleotides that
target internal sites, as demonstrated for broad taxonomic groups
within the domain Bacteria (46) and the seven
recognized sequence clusters within the
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Molecular Analysis of Ammonia-Oxidizing Bacteria of the
Subdivision of the Class Proteobacteria in Compost and
Composted Materials
ABSTRACT
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Abstract
Introduction
Materials and methods
Results and discussion
References
subdivision of the class Proteobacteria
in a variety of composting materials was assessed by amplifying 16S
ribosomal DNA (rDNA) and 16S rRNA by PCR and reverse transcriptase PCR
(RT-PCR), respectively. The PCR and RT-PCR products were separated by
denaturing gradient gel electrophoresis (DGGE) and were identified by
hybridization with a hierarchical set of oligonucleotide probes
designed to detect ammonia oxidizer-like sequence clusters in the
genera Nitrosospira and Nitrosomonas. Ammonia
oxidizer-like 16S rDNA was detected in almost all of the materials
tested, including industrial and experimental composts, manure, and
commercial biofertilizers. A comparison of the DGGE and hybridization
results after specific PCR and RT-PCR suggested that not all of the
different ammonia oxidizer groups detected in compost are equally
active. amoA, the gene encoding the active-site-containing subunit of ammonia monooxygenase, was also targeted by PCR, and template concentrations were estimated by competitive PCR. Detection of
ammonia-oxidizing bacteria in the composts tested suggested that such
materials may not be biologically inert with respect to nitrification
and that the fate of nitrogen during composting and compost storage may
be affected by the presence of these organisms.
INTRODUCTION
Top
Abstract
Introduction
Materials and methods
Results and discussion
References
subgroup and the genera Nitrosomonas and Nitrosospira
(containing the former genera Nitrosovibrio and
Nitrosolobus) in the subgroup (15, 47, 54,
55). Whereas it is thought that the genus
Nitrosococcus is restricted to marine habitats
(52), the -subgroup proteobacterial ammonia oxidizers
appear to occur in a broad range of environments (4). The
monophyletic nature of the -subgroup proteobacterial ammonia
oxidizers has facilitated the design of PCR primers and oligonucleotide
probes that target the 16S rRNA gene in this group at different
taxonomic levels, and the use of these primers and probes has led to
recent progress in the analysis of ammonia oxidizer populations
(16, 24, 43, 50).
-subgroup proteobacterial
ammonia oxidizers (Fig. 1) (42,
43). The structural gene for the active subunit of ammonia
monooxygenase, amoA, has also recently been the target of
PCR-based studies (17), and competitive PCR can be used to
estimate the concentration of a target sequence in a given sample
(44).
View larger version (24K):
[in a new window]
FIG. 1.
Schematic phylogenetic tree for the -subgroup
proteobacterial ammonia oxidizers. The construction of the tree was
based on a distance matrix and neighbor-joining analysis, as described
by Stephen et al. (42). The height of each triangle
represents the number of sequences in the sequence cluster used for
tree construction, and the length of each triangle is proportional to
the sequence diversity within the cluster. The cluster designations, as
proposed by Stephen et al. (42), are indicated inside the
triangles. AEM-3 is a sequence that was recovered from marine sediment
and does not fall into any of the sequence clusters described
(23). The group labeled "other -proteobacteria" was
included as an outgroup and represents only a small sample of the
available -subgroup proteobacterial 16S rDNA sequences.
Our goal was to study the distribution and community composition of
-subgroup proteobacterial ammonia oxidizers in different types of
compost and composting materials in order to determine to what extent
these organisms might be responsible for nitrogen transformations in
these substrates. The method that was most suitable for community
analysis of multiple samples was separation of specifically amplified
16S rDNA fragments by DGGE, followed by membrane transfer and
hybridization with specific oligonucleotide probes (43). 16S
rRNA was simultaneously targeted by reverse transcriptase PCR (RT-PCR)
to determine which of the ammonia oxidizer populations detected were
most active in the composting materials tested (11, 35, 51).
PCR amplification of the amoA gene helped confirm that
ammonia-oxidizing bacteria were present, and competitive PCR was used
to estimate the number of target molecules present per gram of material
in the various samples. A most-probable-number (MPN) analysis was also
performed with some samples in order to compare culture-dependent
detection and molecular detection of ammonia-oxidizing bacteria in
manure and compost.
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MATERIALS AND METHODS |
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Abstract
Introduction
Materials and methods
Results and discussion
References
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Sampling procedures.
The chemical compositions of the
samples used are shown in Table 1. The
samples were obtained between October 1996 and January 1997 at the
locations described below, and they were transported at 4°C and kept
at this temperature until they were used. The samples were processed
within 1 week of collection. All of the samples except the activated
sludge sample were homogenized prior to DNA isolation with a model PT
laboratory sample divider (Retsch, Haan, Germany) adapted with a
3-mm-grid sieve. The activated sludge sample was thoroughly mixed prior
to DNA isolation.
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Industrial compost used for mushroom cultivation. Samples were obtained from an industrial composting plant designed to produce substrate for cultivation of the edible mushroom Agaricus bisporus (Lange) Imbach. At this plant the process is fully automated and has a throughput of approximately 10,000 metric tons of compost per week. Straw-rich horse manure, livestock manure slurries, and wheat straw are the raw materials, and gypsum and water are added to the composting mixture during the initial stages of the process. The composting process takes approximately 2 weeks. During the first week the temperature is kept within the range from 65 to 75°C, after which a 1% inoculum consisting of the end product is added to the mixture, and the compost is allowed to mature for an additional 1 week at the ambient temperature. Samples were obtained from five different stages of the process, as follows: at the beginning of the composting process (t=0 sample), after 4 days of composting (t=4 sample), after 6 days of composting (t=6 sample), after the end product was added to the 6-day-old compost (6d+ sample), and at the end of the 2-week process (end sample).
Experimental pig manure and chicken manure composts. Pig and chicken manure composts were obtained from the Wageningen Agricultural University Department of Environmental Technology (Wageningen, The Netherlands) and the All-Russia Institute of Agriculture (Moscow, Russia). The starting material for the compost obtained from the Wageningen Agricultural University was pig manure mixed with straw. This mixture was allowed to mature and stabilize for 6 months in small, dense composting piles, and the temperature was not monitored. Samples were taken simultaneously after 6 months of composting from both the top (top sample) and bottom (bot sample) of a composting pile.
The Moscow sample (aer sample) was obtained from an experimental compost production plant in which chicken manure slurry and peat were used. The chicken manure slurry was premixed with peat, and the moisture content was adjusted to 70%. The aerobic composting process took place in specially fitted 10-m3 containers which had floor slots that allowed air to be supplied to the composting mixture. The compost was aerated six times each day by forcing air through the floor slots for 5 min. The composting process was conducted at 65 to 75°C, and the total treatment time was 7 days. The compost was stored for 5 months prior to sampling.Chicken manure and pig manure samples. The pig manure sample (pig man sample) was obtained from the solid fraction below the stall slats at a pig farm in Wageningen, The Netherlands. The manure was mixed with straw and then stored for 1 week prior to sampling.
The material from which the chicken bedding sample (chi bed sample) was obtained had been in place for 3 weeks before sample was obtained. The chicken dropping sample (chi man sample) was collected 2 weeks after the previous waste collection, and the fresh chicken manure sample (chi fre sample) was collected just after deposition by laying hens and therefore had minimal contact with the outside environment prior to collection. These three samples were obtained from a commercial farm in Heesch, The Netherlands, which was used for chicken egg production.Dry bioorganic fertilizers. Commercial dry bioorganic fertilizer samples were obtained from St. Petersburg, Russia; these samples were products of treatment of agricultural animal excretia. Bamil (BAM) is a fertilizer derived from activated sludge and primary settler sediments obtained from purification plants used to process pig farm slurry. The activated sludge and sediment are mixed at a 1:1 ratio and dried at 70 to 80°C for approximately 1 h in order to obtain a moisture content of 10%. ECUD (ECU) is a product derived from anaerobic fermentation of chicken slurry. Chicken manure (moisture content, 75%) is diluted with water to a moisture content of 85% and loaded into anaerobic tanks, where manure fermentation takes place at 40°C continuously for 7 to 8 days. The solid fraction obtained is subsequently dried at 70 to 80°C to reduce its moisture content to 10%. BIOGUM (BIO) is obtained in a similar fashion but after aerobic treatment of chicken slurry.
Activated sludge. As chemolithotrophic ammonia-oxidizing bacteria are known to occur in high numbers in aerobic activated sludge, a calf slurry-fed activated sludge sample (AcS sample) was used as a positive control in this study (50). The AcS sample, which consisted of two 1-liter samples that were subsequently pooled, was obtained from the manure treatment plant of the Gelderland Organization for Manure Treatment in Ede, The Netherlands.
Enumeration of ammonia-oxidizing bacteria and characterization of
highest positive MPN dilutions.
Ammonia-oxidizing bacteria were
enumerated by using the MPN method of Verhagen and Laanbroek
(49), as modified by Bodelier et al. (5). The
medium used was the medium described by Schmidt and Belser
(39), and positive cultures and cell counts were determined
by the methods described by Verhagen and Laanbroek (49).
One-half of the dilutions were examined after 8 weeks of incubation at
28°C, and the other half were examined after 20 weeks of incubation.
MPN analyses were performed with only six samples (Table
2). Nitrification activity was determined by the presence of nitrate and/or nitrite (>0.1 mM
NO3
plus NO2), as
detected with a model Traacs 800 autoanalyzer (Technicon Instruments
Corp., Tarrytown, N.Y.). Culture medium (1 µl) from each highest
positive dilution was used directly as a template for ammonia
oxidizer-specific PCRs. For the analysis of recovered 16S rDNA, we used
the PCR, DGGE, and hybridization conditions described below for compost
samples.
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Nucleic acid extraction, PCR, RT-PCR, and competitive PCR.
DNAs were extracted from all of the samples by using the method
described by Stephen et al. (42), as modified by Kowalchuk et al. (19). Direct specific amplification of -subgroup
proteobacterial ammonia oxidizer 16S rDNA was performed by using
primers CTO189f-GC and CTO654r and the conditions described by
Kowalchuk et al. (19). The PCR products were separated by
standard agarose gel electrophoresis (1.5% agarose, 0.5× TBE [1×
TBE is 0.04 M Tris base plus 0.02 M acetic acid plus 1.0 mM EDTA, pH
7.5]) and were visualized by the ethidium bromide fluorescence method.
For the nested PCR we first performed a eubacterium-specific PCR with
primers pA and pH (8) and then performed a second PCR with
primers CTO189f-GC and CTO654r. The first PCR was performed by using
approximately 50 ng of template DNA and Tbr polymerase (Dynazyme;
Finnzymes, Iploo, Finland) as recommended by the manufacturer, and the
following thermocycling program was used: one cycle consisting of 2 min at 94°C; 30 cycles consisting of 30 s at 94°C, 60 s at
55°C, and 75 s at 72°C (with the time increasing 1 s/cycle);
and one cycle consisting of 5 min at 72°C. The reaction volume was of
25 µl. The PCR products (25 µl each) were examined by
electrophoresis in a 0.5× TBE-1% low-melting-point agarose gel
(Boehringer, Mannheim, Germany), followed by ethidium bromide staining.
For all of the samples, the product of the expected size (1.5 kb) was
excised from the gel (100 mg of gel material). The gel fragment was
melted by heating it for 5 min at 65°C, and 1 µl of the resulting
preparation was used as the template in the second PCR performed with
primers CTO189f-GC and CTO654r as described above.
DGGE, blotting, and hybridization analysis.
The PCR products
examined by DGGE included the products obtained after specific nested
PCR and RT-PCR, as described above. The fragment used spanned 465 bp of
the 16S rRNA gene and included a 36-bp GC clamp (41)
introduced during the PCR. DGGE was performed with a D-Gene system
(Bio-Rad Laboratories, Hercules, Calif.) by using the protocol of
Muyzer et al. (26) as adapted for analysis of -subgroup
proteobacterial ammonia oxidizers (19). DNA fragments with
known ammonia oxidizer sequence cluster affinities (Fig. 1)
(42) were also electrophoresed on DGGE gels (38 to 50%
denaturant) as controls for subsequent hybridization analyses (data not
shown). The DNA was stained with ethidium bromide and rinsed twice in deionized water prior to UV transillumination.
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RESULTS AND DISCUSSION |
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Abstract
Introduction
Materials and methods
Results and discussion
References
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Recovery of ammonia oxidizer DNA by PCR and RT-PCR.
The PCR
and RT-PCR results obtained for the 15 compost and manure samples and
one activated sludge sample tested are summarized in Table 2. Direct
amplification with the -subgroup proteobacterial ammonia
oxidizer-specific primers yielded PCR product for only six of the
compost or manure samples. Although some samples contained significant
amounts of humic substances, PCR inhibition was not observed, and all
samples yielded products of the expected size with the
eubacterium-specific primers pA and pH (8; results not shown). Addition of 5 × 103 N. europaea cells g1 to sterilized sand and addition of
104 cells g1 to the sterilized bot compost
sample resulted in positive PCR signals. The latter level of
inoculation was sufficient to produce positive PCR signals in samples
which were originally negative.
1 for sterilized sand and 103 cells
g1 for the sterilized bot sample.
rRNA was extracted from all of the samples analyzed, as confirmed by
RT-PCR performed with F968 and R1401, although the signals obtained
with the three dry biofertilizers were much weaker than all of the
other signals (results not shown). RT-PCRs performed with only primers
CTO189f-GC and CTO654r yielded detectable products with the t=0, top,
aer, and AcS samples (Table 2). Unfortunately, the fragment produced by
amplification with primers F968 and R1401 did not contain the region
spanned by primers CTO189f-GC and CTO654r and could not be used as a
template in a nested PCR analysis. RT-PCRs performed with primers pA
and pH also yielded unsatisfactory results, perhaps due to the length
of the fragment, so that no nested RT-PCR analysis could be carried
out. The minimum numbers of log-phase N. europaea cells
required to produce positive RT-PCR signals with the sterilized sand
and bot samples were 103 and 3 × 103
cells g of soil1, respectively. Addition of 3 × 103 log-phase N. europaea cells g of
soil1 to samples which were negative for the RT-PCR
performed with primers CTO189f-GC and CTO654r yielded detectable RT-PCR signals.
Table 2 also shows the results obtained when the PCR targeted the
amoA gene, and competitive PCR performed with a deletion construct, p428-NAB_8_23 (44), allowed us to estimate the
target concentrations in the samples analyzed. The minimum numbers of linear amoA deletion fragments per reaction mixture which
resulted in positive PCR signals were 10 when the fragments were added to water or DNA extracted from sterilized sand and 20 when the fragments were added to DNA extracted from the sterilized bot sample;
5 × 102 and 103 N. europaea
cells g of soil1 were the minimum numbers of cells which
resulted in positive amoA PCR results for the sterilized
sand and bot samples, respectively. It is not yet known how efficiently
the amoA primers recognize all of the lineages within the
-subgroup proteobacterial ammonia oxidizer clade (37).
Incomplete coverage of all of the -subgroup proteobacterial ammonia
oxidizer targets by the amoA primers might explain why this
assay had an intermediate level of sensitivity compared to the direct
and nested 16S rDNA PCR assays (Table 2) despite the fact that it was
as sensitive as the nested 16 rDNA PCR assay when controls were used.
DGGE and hybridization analysis. For consistency, DGGE analyses were performed with products obtained with primers CTO189f-GC and CTO654r after the nested PCR analysis. Directly amplified products were compared with the products obtained by nested PCR when possible, and the DGGE band patterns were similar; there were only minor differences in the intensities of some bands (data not shown). In the four cases in which RT-PCR products were obtained with primers CTO189f-GC and CTO654r, the fragments that were recovered were also subjected to DGGE in order to compare the results with results obtained when 16S rDNA was targeted.
DGGE produced bands with 43 to 47% denaturant (Fig. 2), which is consistent with the results of previous DGGE analyses of-subgroup proteobacterial ammonia
oxidizers (19, 44). Double band patterns were obtained in
many cases, as reported previously, due to an ambiguous position in
primer CTO654r (19). After membrane transfer and a
hybridization analysis, performed with the hierarchical set of probes
used for the -subgroup proteobacterial ammonia oxidizers
(44), all of the visible bands could be identified to the
sequence cluster level. Four of the seven sequence clusters proposed by
Stephen et al. (42) (Fig. 1) were detected, although the
four clusters were never detected in one sample. These sequence clusters were Nitrosospira clusters 3 and 4, which have
previously been detected in soil environments, and
Nitrosomonas clusters 6 and 7, which have been observed in
various environments (23, 42, 50).
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-subgroup
proteobacterial ammonia oxidizer-like sequences were detected (Table
2). The aer sample contained Nitrosospira cluster 3 and
Nitrosomonas cluster 6, and both groups produced detectable levels of 16S rRNA when RT-PCRs were performed. This sample was obtained after 5 months of compost storage, and it is not clear if the
sequences detected came from descendants of cells that had survived the
high-temperature phase, perhaps facilitated by microsites within the
peat structure (56), or cells that had subsequently
colonized the compost.
The chi fre sample had minimal contact with the outside environment
prior to collection, and the presence of Nitrosospira cluster 3 cells in this material suggests that some cells may remain
intact even after they pass through a hen's digestive tract. Whether
these cells were viable is not known, and the ammonia oxidizer activity
was below the detection limit of the RT-PCR assay for all of the manure
samples tested. The other PCR-based assays were also negative for the
chicken manure samples (chi fre and chi man samples), suggesting that
the numbers of ammonia oxidizers were probably low. The pig man sample
also contained only Nitrosospira sequences, including both
cluster 3 and cluster 4 Nitrosospira sequences.
Ammonia oxidizer-like 16S rDNA sequences from Nitrosospira
cluster 3 and 4 organisms were detected in the BAM dry fertilizer, and
only Nitrosospira cluster 3 was detected in the ECU and BIO samples. Both the direct 16S rRNA-targeted PCR and the RT-PCR were
negative when BAM and ECU fertilizer samples were examined, whereas the
amoA-directed PCR was positive. These dried fertilizer products were designed to retain their nutrient contents even after
long periods of storage. Whether low levels of ammonia-oxidizing bacteria, such as those detected here, might significantly influence nitrogen transformations in these fertilizers or fertilizer-treated soil is not known.
The AcS sample contained sequences related to Nitrosospira
cluster 3, as well as Nitrosomonas clusters 6 and 7. RT-PCR
revealed a predominance of Nitrosomonas cluster 6 rRNA,
suggesting that this group is chiefly responsible for the autotrophic
ammonia oxidizer activity in the sludge examined. rRNA from members of Nitrosomonas cluster 7, which contains perhaps the most
culturable ammonia oxidizer species (36) (see below), was
not detected. Only a very faint signal, which was detected only after
hybridization, was observed for Nitrosospira cluster 3, suggesting that the relative activity of this group was also very low
in this sample. The activity of ammonia oxidizers in the sludge
product, which is used as an agricultural fertilizer, might also play a
role in postprocessing nitrogen transformation and nitrogenous gas
emission (32).
Cell enumeration by the MPN method. The cell counts determined by the MPN analysis for autotrophic nitrifying bacteria are presented for the six samples tested in Table 2. The cell counts were below the detection limit for the t=6, chi man, and chi bed samples. Moderate numbers of ammonia-oxidizing cells were detected in the t=0 sample, and levels just above the limit of detection were detected in the end sample. The AcS sample contained extremely high numbers of culturable ammonia-oxidizing bacteria (>1011 cells/g of fresh material). Although high culturability for ammonia-oxidizing bacteria in activated sludge was expected (1), the cell numbers were approximately 2 orders of magnitude higher than the cell numbers reported for a different animal waste-fed activated sludge, as determined by DAPI (4',6-diamidino-2-phenylindole) staining and in situ hybridization after disruption of aggregates by Ultraturrax blending (50). It may be that the shaking method used for the MPN analysis more successfully disrupts sludge flocs or causes fewer cells to be damaged than blending. Alternatively, the sludge examined may simply have contained a higher number of ammonia oxidizer cells.
16S rDNA characterization of the highest positive MPN dilutions. A comparison of the ammonia oxidizer 16S rDNA sequences in MPN enrichment cultures (Table 3) with hybridization results obtained after nested PCR performed with directly extracted DNA (Fig. 2) revealed differences in the ammonia oxidizer sequences detected by the two methods. The culture-based method detected proportionately more ammonia oxidizers affiliated with Nitrosospira cluster 3 and Nitrosomonas cluster 7, the two sequence clusters best represented in pure-culture collections. The number of ammonia-oxidizing cells detected in the t=0 and end samples was greater when the longer incubation period was used, suggesting that some cells were either very slow growing or required an extensive period of adaptation to the growth medium. The fact that the proportion of Nitrosospira cluster 4 organisms relative to Nitrosospira cluster 3 organisms increased with a longer incubation time (Table 3) suggests that the members of the former group may require a longer period of time to become active in the culture medium. Although previous studies produced no evidence suggesting that there was preferential amplification of the groups most frequently detected by the direct PCR-based approaches used in this study (19), it is still not possible to conclude to what extent culture (36) or PCR (45) biases contributed to these differences.
Conclusions.
-Subgroup proteobacterial ammonia
oxidizer-like sequences were detected in almost all of the composting
materials analyzed, although the sensitivities of the different PCR
methods used varied. It is not known yet to what extent the
ammonia-oxidizing bacteria detected might affect nitrogen
transformations before, during, and after composting. However, the
detection of nucleic acids from nitrifying bacteria in these materials
may have important implications for both compost processing and
storage, as well as for the use of compost in agriculture. The main
goal of composting is to produce a stable, highly nutritious substrate.
As ammonia oxidation is often the rate-limiting step during the loss of
nitrogen from systems due to denitrification, conditions which are
favorable for the activity of ammonia-oxidizing bacteria in compost may affect the net nitrogen balance. The potential for nitrification has
previously been considered in connection with the construction and
installation of some compost production plants (21, 38). In
contrast to application of mineral fertilizers, application of
biofertilizers and manure inoculates the soil with allochthonic microflora, and results presented here suggest that composts and even
dried bioorganic fertilizers may inoculate soils with nitrifying bacteria. Several studies have examined nitrification in soils after
the soils have been amended with compost (14, 20, 32, 40),
but they have done so mostly within the context of activation of the
indigenous nitrifying community. The ability to identify and track the
presence and activity of specific groups of ammonia-oxidizing bacteria
should aid in our overall understanding of nitrogen balance problems in
the production, storage, and use of a variety of composting materials.
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ACKNOWLEDGMENTS |
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This study was supported by the Netherlands/Russian joint initiative Development of Biotechnological Methods for Manure Treatment Focused on Fertilizer Production, which was financed by the Netherlands Organization for Scientific Research (NWO). G.A.K. was supported by an NWO grant to the Netherlands Graduate School of Functional Ecology. J.R.S. was supported by grant GR3/8911 from the United Kingdom Natural Environment Research Council.
We thank Carol Phillips for help with the hybridization analyses.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Plant-Microorganism Interactions, Netherlands Institute of Ecology, Center for Terrestrial Ecology, Boterhoeksestraat 22, Postbox 40, 6666 ZG Heteren, The Netherlands. Phone: 31 (0) 2647 91314. Fax: 31 (0) 2647 23227. E-mail: gkowal{at}cto.nioo.knaw.nl.
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Abstract
Introduction
Materials and methods
Results and discussion
References
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