The Effect of Biofertilizer on The Diversity of N 2 O Reducing Bacteria in Paddy Fields of Sukabumi , Indonesia

Some of the methanotrophic bacteria and N 2 O reducing bacteria have been proven to be able to support the plant growth and increase the productivity of paddy. However effect of the methanotrophic and N 2 O reducing bacteria application as a biofertilizer to indigenous N 2 O reducing bacteria is still not well known yet. The aim of this study was to analyze the diversity of N 2 O reducing bacteria in lowland paddy soil based on a nosZ gene. Soil samples were taken from lowland paddy soils in Pelabuhan Ratu Sukabumi, West Java, Indonesia. There were two treatments for the paddy field soil, ie. biofertilizer-treated field 20% fertilizer (50 kg/ha) with the addition of biofertilizer and 100% fertilizer. PCR amplification of nosZ gene was successfully conducted using nosZF and nosZR primer pair. Denaturing Gradient Gel Electrophoresis (DGGE) process was conducted at 150 V for 5.5h. There were three differences nosZ bands were sequenced. The phylogenetic analysis showed that they were close to uncultured bacteria. Microbial diversity in the biofertilizer-treated field was higher than that of in the 100% fertilizer-treated field. The biofertilizer treatment has higher in microbial diversity than that of applied non-biofertilizer paddy fields. This research might have impact in the application of biofertilizers due to the emission of N 2 O as a green house gas from paddy fields farming activity. The biofertilizer has great potential application in sustainable environmental friendly agriculture systems.

One of metagenomic approach which allows analyzing the diversity of microbial communities, especially denitrifier communities in paddy soil samples is Denaturing Gradient Gel Electrophoresis (DGGE) methods.The principle of this technique is a separation of same length DNA fragments that have different sequences of base pairs.The DGGE technique has been successfully used to determine the complex microbial community diversity (Muyzer & Smalla, 1998).The study on the diversity of denitrifying bacteria encoding nosZ gene in the paddy fields using the metagenomic approach in Indonesia is very limited.Therefore, this research aim was to determine the bacterial diversity of denitrifiers in paddy fields, based on the nosZ gene through metagenomic analysis approach.These research is expected becoming a part of mitigation leading to alleviate N 2 O emission from paddy fields, so there are need study about the diversity of denitrifier community which able to reduce N 2 O into N 2 due to nitrogen anorganic fertilizer applications.These research also expected becoming basic information in order to make biofertilizer contained denitrifier which able to reduce N 2 O emission from paddy fields.

METHODS Biofertilizer composition and soil sampling
Biofertilizer used was containing methanotrophic bacteria Methylocystis rosea BGM 1, Methylocystis parvus BGM 3, Methylococcus capulatus BGM 9, Methylobacter sp.SKM 14, and N 2 O reducing bacteria Ochrobactrum anthropi BL2 (Table 1).Paddy cultivar used was IR64, which mostly applied by farmers in Java.The experiment was conducted in Citarik, Pelabuhan Ratu Sukabumi West Java, Indonesia.There were two treatments of biofertilizer or fertilizer in the experiment i.e. 20% dose of fertilizer (50 kg/ha fertilizer with biofertilizer) and 100% chemical fertilizer.The standard dose of fertilizer was according to Ministry of Agriculture Regulation (Permentan, 2007).Soil sampling was performed three times i.e. at 0 days after planting (DAP), 60 DAP and 120 DAP.The soil sample was taken randomly from 5 different location of paddy field and then mixed together become one part in order to get the DNA extract.

DNA extraction and quantification
Soil DNA extraction was conducted using the Soil DNA Extraction Kit (TIANGEN, Beijing, CN), following the protocol as suggested by the producer.Total DNA genome was extracted

INTRODUCTION
Application of inorganic nitrogen fertilizer by farmers over years tends to increase in order to improve soil fertility and agricultural production.The excessive application of N fertilizers likes NPK and urea may have negative impacts to the environment.This application lead to the increasing of N 2 O (nitrous oxide) emission from the nitrification and denitrification processes.The agricultural landfield is become the largest source of N 2 O emissions to the atmosphere (Smith et al., 2010).N 2 O has potential effect of greenhouse gas 310 times higher than CO 2 (Forster, 2007).The concentration of N 2 O in the atmosphere has been increased by 20% since pre-industrial era (Montzka et al., 2011).N 2 O emissions are influenced by nitrogen fertilizer application rate.The number of applied nitrogen fertilizer are correlates with the increasing of N 2 O emissions (MacKenzie et al., 1998).Submerged paddy fields system provides anaerobic or oxygen limited condition supports denitrification process (Chen et al., 2012).
Denitrification is gradual reactions to reduce nitrate into N 2 gas.Nitrate and nitrite are used by denitrifiers as an electron acceptor in a microoxic or anoxic environment (Knowles,1982).There are four important enzymes in the denitrification process, which are nitrate reductase which convert NO 3 into NO 2 -, nitrite reductase which convert NO 2 to NO, nitric oxide reductase which convert NO to N 2 O, and nitrous oxide reductase which convert N 2 O into N 2 .Uncomplete denitrification process generates the formation of N 2 O gas as the end product of nitrate reduction (Canfield et al., 2010).
The alteration of N 2 O into N 2 need nitrous oxide reductase enzyme encoded by nosZ gene.Nitrous oxide reductase is the key enzyme in the N 2 O reduction in the denitrification process (Jung et al., 2013).This enzyme can be used to mitigate N 2 O emissions because the enzyme is able to catalyze N 2 O reduction into N 2 (Orellana et al., 2014).The reduction of N 2 O into N 2 in the terrestrial environments is done by denitrifiers using nitrous oxide reductase enzymes (NosZ) (Zumft & Kroneck, 2007).The potential of microbial communities to reduce N 2 O into N 2 can be predicted by determination of nosZ genes abundance using metagenomic analysis (Henry et al., 2006).Analysis of the microbial diversity in the ecosystem samples is important to improve the knowledge of metabolisms, biodiversity, genetic resources and understanding the function and roles of the microbes in the environments (Fakruddin & Mannan, 2013).then quantified using Nanodrop 2000 spectrophotometer (Thermo Scientific, Wilmington, USA).

Amplification of nosZ gene
The extracted DNA was amplified by PCR T1-Thermocycler (Biometra, Goettingen, DE).Amplification was performed using primer pairs of nosZ-1126F genes with GC clamps at the 5 'end and nosZR.The sequences of nosZ-1126F (: 5 'CCG CCG CGC CGC GGC GCG GCGGCG GGG GGGGGGG-GGGCTBGGGCCRTT-GCA GCA CGG-3') and the sequence of the reverse nosZR was 5'-GAAGCGRTCCTTSGA-RAACTTG-3' (Throback et al., 2004).The PCR mix of 25μL was prepared containing 2μL primers with a concentration of 10 pmol, 12.5 μL of KAPA 2G Robust Hotstart Readymix 2× (KAPA Biosystems, USA), 4 μL of DNA template (~100 ng) and 6.5 μL of Nuclease-free water (NFW).The PCR conditions were used based on modification of the Touchdown PCR system with conditions of initial denaturation at 94 °C for 2 min denaturation at 94 °C for 1 min, annealing at 60 °C for 45 sec, elongation at 72 °C for 1 min, for 10 first cycles annealing temperatures was lowered 0.5°C per cycle, and to the continuing 25 cycles of PCR the start at 94 °C for 1 min, and annealing 53 °C for 45 sec, elongation 72 °C for 4 min.The expected size of PCR product is about ~500 bp.The PCR products were run on 1% agarose gel electrophoresis.Gel staining using ethidium bromide 0.1%, and then visualized using the G:BOX Gel Documentation (Syngene, Frederick, USA).
The DGGE process was carried out at 60 °C, voltage of 150 V for 5.5 h Polyacrylamide gel was stained with ethidium bromide (EtBr) of 0.1% for 15 min and visualized by using G: BOX Gel Documentation (Syngene, Frederick, USA).The images were then analyzed using G: BOX and CLIQS 1D software (Total Lab, GB).The DNA bands were then cut and put in 100 mL sterile nuclease-free water then eluted and stored at 4 °C overnight for further analysis.
Abundance based on OTU (Operational Taxonomic Unit) obtained from each treatment OTU, which represent abundance of bacterial community determined based on the band volume of DGGE polyacrylamide gel which had analysed by CLIQS 1D software followed by determination of OTU by PAST 3 software

Reamplification of nosZ gene amplicon
A total of ~50 ng of the eluted DNA was amplified by using primer pairs nosZF and nosZR and without GC clamps.Mastermix PCR conditions were set up similar to the previous experiment.The DNA amplicons were sent to a laboratory providing sequencing services.

Phylogenetic Analysis
The sequences of nosZ gene amplicons were analyzed using ChromasPro (Technelysium, AU) for assembly and trimming process.The sequences were then compared to the database in GeneBank by using BLAST-N (Basic Local Alignment Sequence Tools for nucleotide) (blast.ncbi.nlm.nih.gov).Phylogenetic analysis was performed by using MEGA 6.0 software (Tamura et al., 2013) and the phylogenetic tree was constructed using neighbor-joining method with 1000x bootstrap according to the Bayesian Information Criterion (BIC) score.

DGGE profiles of nosZ gene diversity
The DGGE profiles of the samples show variety DNA bands indicated the differences of nosZ gene diversity.There were five thick bands  (Hapsari, 2008;Astuti, 2009) BGM3 Paddy field in Bogor Methylocystis parvus (Hapsari, 2008;Astuti, 2009) BGM 9 Paddy field in Bogor Methylococcus capsulatus (Hapsari, 2008;Astuti, 2009) SKM14 Paddy field in Sukabumi Methylobacter sp.(Hapsari, 2008;Astuti, 2009) BL2 Paddy field in Bogor Ochrobactrum anthropi (Setyaningsih, 2010) of nosZ gene amplicon that always appeared in all samples of DGGE.DGGE profiles of the samples showed variety DNA bands indicating the differences of nosZ gene diversity.Samples of biofertilizer application showed more diverse nosZ gene diversity indicating the diversity of N 2 O reducing bacteria.Interpretation analysis trough CLIQS 1D program showed the increasing number of bands from the beginning day of planting (DAP) up to 120 DAP (Figure 1).According to OTU obtained from each treatment showed that there were different type of nosZ gene abundance as shown in OTU diagram from the K0 to P120 treatment (Figure 2).The OTU of 0 DAP showed that P0 treatment had higher abundance and diversity compared with K0, at the 60 DAP showed that the abundance of K60 are higher than P60, but the diversity are lower in K60.In the 120 DAP as a last treatment showed that those high abundance in K120 but low in diversity compared with P120 that lower in abundance but higher in diversity.The most dominan OTU appeared in 11, 12, 2, 1, 6, 7 compared with other different bands which appeared in different percentage (Table 2).
Clustering analysis showed that diversity of N 2 O reducing bacterial community during the vegetative phase of paddy was different with that of the generative phase.The diversity of N 2 O reducing bacteria on 0 DAP of both the biofertilizer and 100% fertilizer treatment was very close resemblance, similar to the N 2 O reducing bacteria diversity on 120 DAP of the both treatments.
There are three nosZ gene within the microbial genomes were successfully amplified using primers nosZ from seven chosen band derived from six paddy field soil samples.The BLAST-N of the three succesfully sequenced bands indicated that the nucleotide sequences had a close similarity with the nosZ gene sequence of uncultured bacteria (Table 3).These results were confirmed by the results of BLAST-X that the amino acid sequences were similar with that of nitrous oxide reductase enzyme (Table 4).nosZ gene diversity of N 2 O reducing bacteria.
The thickness of the bands showed the abundance of microbes, the thicker and clearer bands mean more abundant species, because the band in the gel will be formed when cell density of the species has at least 10 6 cells/g soil (Gelsomino et al., 1999).The abundance of bacterial community which represent from visualization of band volume and the OTU result showed the fluctuation in each treatment (Figure 1).The abundance and diversity of bacterial commu-   (Sylvia, 2005).The biofertilizer containing i.e. methanotroph bacteria and nitrate reducing bacterium Ochrobactrum anthropi were able to increase the growth of paddy plant.Bacterial isolates used has been shown to promote the growth of paddy due to their ability to fix N 2 (Pingak, 2013;Bintarti, 2014).Application of the biofertilizers that affecting microbial communities was found in the paddy field, denitrifying bacteria encoding nosZ gene.
The temporal shift of microbial communi-ty in the rhizosphere could be caused by organic molecules released from roots due to growth phase changes of paddy plant (Aulakh et al., 2001).Root exudates of photosynthate compounds can provide nutrients to increase the microbial populations (Bai et al., 2000).Paddy plants require nitrogen during vegetative phase until tillering and panicle formation stage (Dobermann & White, 1999).
The surface soil condition more oxic at the end of planting due to decreasing of irrigation, but deep inside the soil is still anoxic.The increasing diversity of bacterial communities is probably because of at the end phase of growing paddy, the content of nitrogen-based nutrient in the soil is still abundant and partially absorbed by root plants which made increasing of bacterial encoding nosZ genes population due to denitrification process.In the other hand, application of this biofertilizer also decreasing levels of N 2 O emissions from initial 24.51 to 6.99 mol/day/ha at the end of the treat-ment which conducted at Sukabumi paddy fields soil (Sukmawati, 2016).This research showed the correlation between high bacterial diversity encoding nosZ with decreasing N 2 O emissions from paddy fields.In the other hand, this biofertilizer composition also made high fluctuation diversity of pmoA gene encoding methane monooxygenase enzyme which consumes methane especially in vegetative phase of paddy growth (Sutanto, 2014).However in nifH gene diversity, both of those experiments in Sukabumi paddy field were no fluctuation of diversity and tend to be low in diversity (Hadianta, 2014).Both of those experiment were conducted in Sukabumi paddy field.
The diversity of bacteria in the control treatment was high expressed, however it did not to determine to affect reduction of N 2 O emission because the gene was not actively expressed.Whether the nosZ genes in the field actively expressed analyzed based on presence of mRNA of the genes can be done (Perez et al., 2014).
From the shift of bacterial diversity of each sample, it can be assumed that the bacterial communities diversity at the vegetative phase is different from the diversity at the generative phase (Figure 2).Based on the phylogenetic tree analysis, there were found that three DGGE bands were close with uncultured bacteria (Figure 3).This result also supported with BLAST-X analysis result which proves that the amino acid sequences were similar with that of nitrous oxide reductase enzyme.Based on BLAST-P result with amino acid sequences proved that protein domain was related with Tat conserved protein domain.The twin-arginine translocation (Tat) protein export system is present in the cytoplasmic membranes of most bacteria and archaea and has the highly unusual property of transporting fully folded proteins (Palmer & Berks, 2012).Tat in nosZ gene has function in the functional respiratory system of N 2 O in the periplasmic compartment, Tat translocon is used to transport apoenzyme from cytoplasm to the periplasm.

CONCLUSION
There is variation in diversity of bacterial nosZ gene in paddy field soil samples taken from Pelabuhan Ratu, Sukabumi by using DGGE profiles.The majority of nosZ gene in the samples has similarity with uncultured bacteria.The nosZ gene detected diversity is higher in the biofertilizer application paddy fields.Similarly the diversity of the nosZ gene at generative phase is higher than that of at generative phase during paddy cultivation.

Figure 1 .
Figure 1.(A) nosZ gene pattern from DGGE profile with seven (red numbered) chosen band that encised for re-PCR.(B) Gel doc G: BOX photo result and interpretation based visualization on CLIQS 1D program (K0: control 0 DAP P0: treatment 0 DAP (day after plantation), K60: control 60 DAP, P60: treatment 60 DAP, K120: control 120 DAP, P120: treatment 120 DAP).The number of bands represent the variety species of microbial diversity types and OTU (Operational Taxononomic Unit) which obtained from each treatments

Figure 2 .
Figure 2. OTU diagram obtained from each day of treatment (A) 0 DAP, (B) 60 DAP, and (C) 120 DAP showing the curve that represent abundance of bacterial communities

Table 1 .
Reference of isolates which used as biofertilizer

Table 2 .
Rank of the nosZ gene abundance based on OTU from soil sample

Table 3 .
BLAST-N result and from re-PCR band