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Relative contribution of nirK- and nirS- bacterial denitrifiers as well as fungal denitrifiers to nitrous oxide production from dairy manure compost Koki Maeda, Sakae Toyoda, Laurent Philippot, Shohei Hattori, Keiichi Nakajima, Yumi Ito, and Naohiro Yoshida Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b04017 • Publication Date (Web): 28 Nov 2017 Downloaded from http://pubs.acs.org on November 28, 2017
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Relative contribution of nirK- and nirS- bacterial denitrifiers as well as
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fungal denitrifiers to nitrous oxide production from dairy manure compost
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Koki Maedaa*, Sakae Toyodab, Laurent Philippotc, Shohei Hattorib,
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Keiichi Nakajimaa, Yumi Itoa and Naohiro Yoshidab,d
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a
NARO, Hokkaido Agricultural Research Center, Dairy Research Division, 1 Hitsujigaoka, Sapporo 062-8555, Japan
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b
Department of Chemical Science and Engineering, School of Materials and Chemical
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Technology, Tokyo Instititute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8502,
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Japan.
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c
INRA, UMR 1229, Soil and Environmental Microbiology, 17 rue Sully BP 86510, 21065 Dijon cedex, France
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d
Earth-Life Science Institute, Tokyo Institute of Technology, 2-12-1 Ookayama, Meguro-ku, Tokyo 152-8550, Japan
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＊
18
Tel; +81-29-838-6365
Corresponding author (
[email protected])
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Running title; N2O with unknown isotopic signature from compost
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Current address for KM;
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JIRCAS, Crop, Livestock & Environment Division, 1-1 Ohwashi, Tsukuba, Ibaraki 305-8686,
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Japan
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Abstract
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The relative contribution of fungi, bacteria, nirS and nirK denirifiers to nitrous oxide (N2O)
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emission with unknown isotopic signature from dairy manure compost was examined by
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selective inhibition (SI) technique. Chloramphenicol (CHP), cycloheximide (CYH),
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diethyldithiocarbamate (DDTC) were used to suppress the activity of bacteria, fungi and nirK
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possessing denitrifiers, respectively. Produced N2O were surveyed to isotopocule analysis and its
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15
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abundances were compared by qPCR. Results showed that N2O production was strongly
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inhibited by CHP addition in surface pile samples (82.2%) as well as in nitrite amended core
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samples (98.4%), while CYH addition did not inhibit the N2O production. N2O with unknown
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isotopic signature (SP=15.3~16.2‰), accompanied with δ18O (19.0~26.8‰) values which was
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close to bacterial denitrification, was also suppressed by CHP and DDTC addition (95.3%)
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indicates that nirK-denitrifiers were responsible for this N2O production despite being less
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abundant than nirS-denitrifiers. Altogether, our results suggest that bacteria are important for
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N2O production with different SP values both from compost surface and pile core. However
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further work is required to decipher whether N2O with unknown isotopic signature is mostly due
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to nirK-denitrifiers that are taxonomically different from the SP characterized strains and
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therefore having different SP values rather than being also interwoven with the contribution of
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NO detoxifying pathway and/or of codenitrification.
N site preference (SP) and δ18O values were compared. Bacteria, fungi, nirS and nirK gene
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Keywords: N2O isotopocule, site preference, compost, denitrification
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Introduction
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Livestock manure is a huge source of greenhouse gas (GHG) nitrous oxide (N2O). Global
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N2O emission from livestock manure is estimated as 0.2-0.5 GtCO2 eq/year, taking second place
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of the total emission of livestock sector (up to 10%) with methane (0.2-0.4 GtCO2 eq/year)1.
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Since demand for livestock production is rapidly increasing with growing population especially
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in the developing country2, the mitigation of GHG emission in livestock manure management is
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an urgent issue for sustainable agriculture.
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Composting is one of the most traditional technologies treating animal manure. It is a
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biological decomposition process of organic matters contained in the manure, and various
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microorganisms including bacteria or fungi are involved. It can convert animal manure into
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stable organic fertilizer which can be used with less adverse effects to the environment3.
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Although the cost of the composting is generally higher than direct utilization of raw manure, it
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is considered to be an alternative way to recycle the manure especially for the livestock farm
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with limited lands4.
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In previous studies, we showed that significant N2O emission occurs just after the turning
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of dairy manure compost piles, which is performed to introduce oxygen into the piles to promote
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the degradation of organic matter5. To identify the production mechanisms of the emitted N2O,
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we analyzed the intramolecular distribution of 15N, a stable isotope of N which accounts for
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about 0.375% of natural N atoms, within the N2O molecule (15N site preference; SP value).
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Because the isotope effect is specific to each reaction pathway, SP has different values between
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N2O produced by different microbial processes6 depending on the enrichment of 15N in the
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central N in NNO molecule compared to the peripheral N. Thus, for N2O produced by bacterial
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nitrification or fungal denitrification, SP value ranges from 30 to 40‰7, 8 (In contrast, N2O
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produced by bacterial denitrification exhibits, SP value around 0‰8.
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The SP value of N2O produced from the pile surface just after the turnings was around 2.0‰9,
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which is close to the value of pure bacterial denitrifier cultures. On the other hand, NO2-
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amended pile core samples produced N2O with an unknown isotopic signature of 11.4-20.3‰10,
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which does not fall to any known biological production pathways7, 8, 11-14. This phenomenon
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raised the question about the contribution of different groups of N2O producers. In the previous
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study10, addition of acetylene (10% in the headspace), which inhibits N2O reduction by 3
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denitrification but also N2O production by nitrification15 resulted in higher N2O production,
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therefore suggesting a negligible contribution of nitrification. Because SP value of 37‰ are
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reported for fungal denitrification7, its role in N2O emissions from pile core has also been
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hypothesized by our previous work10. Thus, fungi are known to perform denitrification and
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produce N2O as the end product16. In contrast to denitrifying bacteria, which can possess either a
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copper or a cd1 nitrite reductase encoded by nirS or nirK, respectively, denitrifying fungi possess
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only the nirK-type copper reductase12, 17. Contribution of other processes such as nitrification
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(33‰)8 or N2O reduction18, 19 can also be an alternative explanation for this unknown isotopic
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signature.
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Here, our objective was to identify which microorganisms were responsible for N2O
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emissions from both the surface and core pile and to understand why different isotopic signature
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were observed. For this purpose, we used the selective inhibition (SI) technique using
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bactericide and fungicide as previously reported20-23. In addition, diethyldithiocarbamate
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(DDTC) was used to disentangle the relative contribution of nirK- and nirS-possessing
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denitrifiers24.
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Materials and Methods
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1. Composting experiment
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The composting experiment was performed twice at the National Agriculture Research
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Center for the Hokkaido Region (Sapporo City, Hokkaido). The cows were fed orchard grass
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silage and corn silage, oat hay, alfalfa hay, beet pulp and two types of concentrate mixtures to
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meet their digestible energy requirements, as recommended by the Japanese Feeding Standard
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for Dairy Cattle. Lactating Holstein cow excrement and dried grass (Orchard grass; Dactylis
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glomerata) were used in this study to make the compost.
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About 4,000 kg of dairy cow excrement and 400 kg of dried grass were mixed completely by
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a manure spreader. The compost was piled up on a waterproof concrete floor, and turned once
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every two weeks with a front loader and manure spreader. Each pile had a volume of 7.5 m3 with
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pile dimensions of 4 m in diameter and 1.8 m in height at the start of the experiment. The
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temperatures of the compost piles and the ambient air were measured hourly using a Thermo
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Recorder RTW-30S (Espec, Japan). Since it was known in the previous studies that the sampling
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location of the pile can affect the NO2- or NO3- accumulation9, 10, fresh samples were taken from
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each zone (the pile top, surface, and core) at the start and end of the two composting experiments
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and just before each turning. The detail of the sampling point and time are described in Fig. S1.
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2. Batch incubation experiment and N2O measurement
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Nitrous oxide production rates were determined using the acetylene inhibition technique,
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which results in the accumulation of N2O since the N2O reduction is blocked. About 1 kg of
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fresh compost samples was collected at the start and end of the two composting experiments and
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just before each turning. Samples were homogenized and 5 g of fresh subsamples were used.
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For the core samples, no NO2- or NO3- accumulation occurs and no N2O production can be
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obtained when only core samples are incubated10, 1 ml sodium nitrite solution (200 mM) was
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added to induce N2O emissions. Chloramphenicol (CHP), which inhibits protein synthesis of
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bacteria by interfering peptidyl transferase at 50S ribosomal subunit25, 26, and cycloheximide
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(CYH), which binds to E-site of 60S ribosomal subunit and inhibit protein synthesis of
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eukaryotes by blocking translocation step in elongation27, 28, was used to inhibit specific activity
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of bacteria or fungi, respectively. CHP (10 mg g-1 fresh compost), CYH (10 mg g-1 fresh
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compost) and DDTC (45 mg g-1 fresh compost) were mixed with talc (20 mg g-1 fresh compost)
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manually, and the mixtures were stored in 100 ml flasks. The treatments are CHP, CYH,
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CHP+CYH, DDTC, control 1 (no addition), control 2 (talc only). The optimum amount of these
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chemicals was determined in the preliminary experiment (data not shown). Ten percent of the
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headspace gas was replaced to pure acetylene. We did not purge the head space gas to simulate
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the composting, because the compost turnings are generally done to introduce O2 into the piles,
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and thus the headspace gas contained O2 at the beginning of the batch incubations. Each flask
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was capped with a butyl rubber stopper and sealed with aluminium. After the 24 h incubation at
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30°C, 100 µl of headspace gas was sampled from each flask using a gas-tight microsyringe and
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the N2O concentration was analyzed using a gas chromatograph (GC-14B; Shimadzu, Japan)
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equipped with an electron capture detector (ECD), with two replicates for each treatment.
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3. Analysis of N2O isotopocule ratios Ten ml of headspace gas samples were taken at the end of the incubation experiments and
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stored in pre-evacuated vials. Because the headspace gas samples contain 10% acetylene, the
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acetylene was separated through a pre-column (1/8 in. o.d., 1.5 m long s.s. tube packed with
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silica
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chromatograph-isotope ratio mass spectrometer (GC-IRMS) (MAT 252; Thermo Fisher
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Scientific K.K., Yokohama, Japan) system as described elsewhere to measure N2O isotopocule
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ratios11. Site-specific N isotope analysis in N2O was conducted using ion detectors that had been modified
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for the mass analysis of fragment ions of N2O (NO+) containing N atoms in the center positions of N2O
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molecules, whereas the bulk (average) N and oxygen isotope ratios were determined from molecular
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ions29. Pure N2O (purity >99.999%; Showa Denko K.K., Japan) was calibrated with international
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standards (atmospheric N2 and Vienna standard mean ocean water)11 and used as a working standard for
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the isotopocule ratios. The notation of the isotopocule ratios is shown below. The measurement precision
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was typically better than 0.1‰ for δ15Nbulk and δ18O, and better than 0.5‰ for δ15Nα and δ15Nβ.
gel)
and
the
acetylene-removed
samples
were
introduced
into
a
gas
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δ15Ni = (15Risample / 15Rstd – 1) (i = α, β, or bulk)
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δ18O = (18Rsample / 18Rstd – 1)
(1) (2)
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Here, 15Rα and 15Rβ respectively represent the 15N/14N ratios at the center and end sites of the nitrogen
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atoms; 15Rbulk and 18R respectively indicate the average isotope ratios for 15N/14N and 18O/16O. The
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subscripts “sample” and “std” respectively indicate the isotope ratios for the sample and the standard,
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atmospheric N2 for N and Vienna Standard Mean Ocean Water (V-SMOW) for O. We also define the 15N
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site preference (hereinafter SP) as an illustrative parameter of the intramolecular distribution of 15N:
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15
N–site preference (SP) = δ15Nα – δ15Nβ.
(3)
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4. Chemical analysis of the compost
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About 1 kg of fresh compost samples was collected at the start and end of the two composting
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experiments and just before each turning. Samples were homogenized and fresh subsamples
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were used to measure total solids, volatile solids, inorganic-N, pH and electrical conductivity, or
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stored at -20°C for total nitrogen determination. Total solids (TS) were measured after drying the
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samples overnight at 105°C, and dried samples were powdered and used for C/N ratio
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determination. Volatile solids (VS) were measured after the samples were processed at 600°C
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for 1 h. Total N was measured using raw samples by the Kjeldahl method. The C/N ratio was
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determined using a C/N analyzer (vario MAX CNS; Elementar, Germany).
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To measure inorganic-N, pH and electrical conductivity, 5 g of fresh compost was placed into
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a 50 ml polypropylene tube with 40 ml of deionized water, then shaken (200 rpm, 30 minutes)
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and centrifuged (3,000 g, 20 minutes). The supernatant was collected and NH4+, NO2--N and
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NO3--N were measured using ion chromatography (ICS-1600; Dionex, USA); pH and electrical
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conductivity (EC) were determined with calibrated electrodes (Horiba, Japan).
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5. DNA extraction
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DNA extraction from the compost samples was performed using the commercially available
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DNA extraction kit Isofecal (Nippon Gene, Japan). The extraction was done according to the
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manufacturer’s instructions, and the concentrations of DNA samples were measured by
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BioSpec-nano (Shimadzu, Japan). The purified DNA samples were stored at -80°C until further
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analysis.
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6. Quantification of bacterial 16S rRNA gene, denitrification genes, fungal ITS gene and
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bacterial amoA gene
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qPCR was performed using the commercially available kit SYBR Premix Ex Taq II (Takara,
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Japan), with a 20 µl reaction mix that consisted with that contained 40 ng of template DNA. The
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primer pairs for amplifying bacterial 16S rRNA gene, fungal ITS (internal transcribed spacer)
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region, bacterial denitrification genes (nirS and nirK), bacterial amoA gene and their PCR
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conditions are summarized in Table S1. Reactions were carried out in an ABI 7500 real-time
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PCR system (Applied Biosystems, USA). An external standard curve was prepared using serial
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dilutions of a known copy number of the plasmid pGEM-T Easy vector (Promega, USA)
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containing each gene. The insert gene for the 16S rRNA gene and nirS was Paracoccus
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denitrificans (NCIMB 16712), and that for the amoA gene was Nitrosomonas europaea (NBRC
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14298). Plasmids containing the cloned nirK gene (AB441832), ITS region from dairy manure
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compost5 were used for the standard curve for these genes.
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7. Statistical analysis
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The chemical component and N2O SP values were analyzed by ANOVA using the general linear
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model procedure described by SAS30. Tukey’s multiple range comparison tests were used to
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separate the means. A value of P
