Resuscitation of inactive ammonia-oxidizing archaea and complete nitrifiers by extracellular electrons from a heterotrophic bacterium, Bacillus amyloliquefaciens

Resuscitation of inactive ammonia-oxidizing archaea and complete nitrifiers by extracellular electrons from a heterotrophic bacterium, Bacillus amyloliquefaciens | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Resuscitation of inactive ammonia-oxidizing archaea and complete nitrifiers by extracellular electrons from a heterotrophic bacterium, Bacillus amyloliquefaciens Shanghua Wu, Yuzhu Dong, Haonan Fan, Ye Deng, Xuliang Zhuang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-76082/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Nitrification in the soil is dominated by ammonia-oxidizing archaea (AOA), ammonia-oxidizing bacteria (AOB) and the newly discovered complete nitrifiers, which are members of complex communities. Complicated relations have been found in microbial communities, but the relations between heterotrophic bacteria and autotrophic ammonia oxidizers (AOM) are unclear, and experimental models are lacking. Here, we constructed a microcosm based on DNA stable isotope probing that was established between a heterotrophic bacterium, Bacillus amyloliquefaciens (BA), and the soil autotrophic AOM. Results: The inoculation of BA changed the activities of three indigenous nitrifiers. The originally marginal AOA and complete nitrifiers were active in the nitrification process as well as the AOB, which led to mitigation of N 2 O and an increase in NO. The network analysis showed that the inoculated BA was indirectly and positively linked to AOA through a Microbacteriaceae that contains genes encoding proteins responsible for transporting electrons. Further, the microbial fuel cell system indirectly confirmed the potential regulation of extracellular electron transfer (EET) between these two species. Conclusions: With our findings, a useful model to investigate the relations between heterotrophic bacteria and AOM was constructed, and evidence of EET in complex regulations was provided. General Microbiology ammonia-oxidizing bacteria and archaea complete nitrifiers Bacillus amyloliquefaciens extracellular electron transfer nitrous oxide nitric oxide Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background Nitrous oxide (N 2 O) is the third most abundant and long-lived greenhouse gas in the atmosphere. Anthropogenic emissions are the most important sources of global N 2 O production, comprising nearly 30–45% of total emissions, with approximately two-thirds of this derived from agriculture and soil[ 1 ]. Microbial transformations of nitrogenous compounds, especially heterotrophic denitrification and chemolithoautotrophic aerobic nitrification, are the two dominant contributors to N 2 O emissions[ 2 ]. Classically, heterotrophic denitrification has been considered to be the major biological source and sink of N 2 O in the terrestrial nitrogen cycle, but N 2 O emissions from nitrification are thought to be of equal importance to those from denitrification in recent decades. N 2 O results directly from the ammonia-oxidizing activity of traditional nitrifiers and indirectly from the activities of ammonia-oxidizing bacteria, ammonia-oxidizing archaea, and the newly discovered complete nitrifier (complete ammonia oxidation; comammox) Nitrospira inopinat a[ 1 – 9 ]. Nitrifier denitrification may account for up to 100% of N 2 O emissions from ammonia in soils and is more significant than classical denitrification at low oxygen concentrations, especially fluctuating aerobic-anaerobic conditions[ 10 , 11 ]. Given predicted increases in farmland and fertilizer application (EPA reference), agricultural soils are likely to contribute up to 59% of total N 2 O emissions by 2030[ 12 ], increasing the need to explore approaches to manage and reduce N 2 O emissions from agricultural systems[ 13 , 14 ]. In our previous work, a strain of Bacillus amyloliquefaciens (BA) that significantly reduced N 2 O emissions by 35%-50% after inoculation was isolated from rice rhizosphere, and the results suggested that the mitigation was mostly due to the inhibition of nitrification[ 12 ]. However, the detailed process of inhibition and relations of organisms behind the regulation of BA have not been studied, especially those between heterotrophic BA and three groups of autotrophic AOM. AOA amoA genes are abundant and widespread in soils, frequently outnumbering AOB amoA genes[ 15 ], and comammox bacteria are also abundant in some soils[ 16 , 17 ]. The relative abundance and dominance of these three types of ammonia-oxidizing microorganisms (AOM) are affected by abiotic factors, including pH, temperature and moisture content[ 18 ], and by several nitrification inhibitors. AOM may also be influenced by interactions with other microorganisms, e.g., through cross-feeding and quorum sensing[ 19 , 20 ], and by direct or indirect electron transfer between species. The latter is rarely studied and can be investigated using microbial fuel cells (MFCs). Geobacter sulfurreducens and Shewanella oneidensis are the two most commonly used model strains in MFCs because of their good efficiency in transferring electrons; in MFCs, protons are the only thing allowed to pass through the membrane between the anode and cathode chambers and complete the circuit[ 21 ]. In this study, we constructed microcosms to investigate the influence of the heterotrophic bacterium BA on nitrification and three different types of nitrifiers. We also constructed MFCs to study the possible extracellular electron transfer between BA and AOA. Together, these results suggest a complex regulation strategy in which heterotrophic bacteria affect autotrophic organisms, and extracellular electron transfer may be a possible link between the two types of microbes. Methods Soil microcosms and stable isotope probing (SIP) The influence of BA on nitrification was investigated in soil microcosms consisting of 10 g of sieved acidic soil (mesh size of 3.35 mm) from Southwest China used in our previous work[ 12 ]. Microcosms were adjusted to 60% maximum water-holding capacity in 120-ml serum bottles, sealed with rubber stoppers and aluminium caps and incubated at 30 °C in the dark for 4 weeks. The headspace was sampled and adjusted to 5% (vol/vol) CO 2 by injection through the rubber septum, and each bottle was flushed for 5 min weekly with synthetic air (20% O 2 , 80% N 2 ) between sampling and injection. For every week, 100 µg urea-N g − 1 dry weight soil was added by liquid to each bottle. Three treatments were established, each in triplicate microcosms. In Treatment 12C-B, the headspace contained 12 C-CO 2, and active BA was inoculated. The inoculum was grown in liquid batch culture to stationary phase in M9 Minimal Medium ( 13 C-glucose was used in treatments of 13 C-CO 2 to avoid additional 12 C into the microcosms) at 30 °C and 180 rpm for 28 h. Cells were separated by centrifugation at 8000 rpm and washed three times with sterilized water to remove the remaining culture medium. The final cell suspension was adjusted to 3 × 10 8 CFU ml − 1 , and 1 ml of this suspension was added weekly by injection through the rubber septum into each microcosm for 3 consecutive weeks. Treatment 13C-B was identical to Treatment 12C-B, except that the headspace contained 13 C-CO 2 . Treatment 13C-BS was identical to Treatment 13C-B but was inoculated with B. amyloliquefaciens sterilized by autoclaving as a control. Soil chemical analysis Soil pH was determined in water at a ratio of 1:2.5 (w/v) using a pH meter (Accumet Excel XL 60, Fisher Scientific, Singapore). The NO 3 − and NH 4 + concentrations were determined after extraction in 2 M KCl (soil:solution ratio of 1:10 (w/v)) and measured using a continuous flow analyser (SAN++, Skalar, Breda, Holland). Instantaneous CO 2 , NO and N 2 O measurements N 2 O and CO 2 concentrations in the headspace were measured using an Agilent gas chromatograph (Agilent Technologies Inc., Wilmington, DE) equipped with a micro-TCD and a flame ionization detector, respectively. NO concentration was measured using a NO-NO 2 -NO x chemiluminescent analyser (42i Thermo Environmental Instruments Inc., USA)[ 22 ]. DNA extraction and SIP gradient fractionation DNA was extracted from 0.5 g of dry soil using the FastDNA SPIN kit for soil (MP Biomedicals, Santa Ana, CA, USA) according to the manufacturer’s instructions. The extracted DNA was dissolved in 80 µL of sterile, deionized, nuclease-free water and divided into two parts: one part was stored at 4 °C prior to quantitative PCR and the other was stored at -20 °C for further use. The quantity and purity of soil DNA were determined by a Nanodrop ND-1000 UV-Vis Spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). SIP fractionation was performed as described previously[ 15 , 23 ]. For each sample, 3.5 µg of soil DNA was mixed with CsCl (buoyant density of 1.725 g ml − 1 ) before ultracentrifugation at 44,100 r.p.m. (~ 177,000 gav ) in a Vti 65.2 rotor (Beckman Coulter, cat. no. 362754) at 20 °C for 44 h with vacuum and maximum acceleration and without braking (requiring an additional 2 h to stop). DNA fractionation was carried out by displacing the gradient medium with sterile water from the top of the ultracentrifuge tube using an NE-1000 single syringe pump (New Era Pump Systems Inc., Farmingdale, NY, USA) with a precisely controlled flow rate of 0.375 ml min − 1 . Up to 15 DNA gradient fractions were generated with equal volumes of approximately 380 ml, and a 65-ml aliquot of each fraction was used for refractive index measurement using an AR200 digital hand-held refractometer (Reichert, Inc., Buffalo, NY, USA). The fractionated DNA was purified and dissolved in 30 µL of sterile, deionized, nuclease-free water[ 24 ]. Quantitative PCR Functional marker genes (bacterial and archaeal amoA ) and amoA of Nitrospira clade A were quantified using Premix Ex Taq (TaKaRa, Japan) and gene-specific primers. The primers used for Nitrospira clade A were coma-244f/coma-659r. Details of plasmid standards, gene-specific qPCR primers, reaction mixtures and thermal programmes are described in previous studies[ 17 , 25 ]. Each sample was quantified in duplicate using the CFX Connect™ Real-Time PCR Detection System (Bio-Rad laboratories). The algorithm that was used to calculate the efficiency (E) and threshold cycle (CT) was based on the kinetics of each individual reaction. BA abundance was determined using Q-PCR based on the method reported by Yong et al. [ 26 ]. Primers targeting the pgsB gene were used for Q-PCR: pgsB726-f (5’-TGGCGCCATGAGA-ATCCT-3’), pgsB791-r (5’-GCAAAGCCGTTTACGAAATGA-3’) and pgsB-probe (5’-FAM-CCGCTGCTCAGCACGAAGGAGC-TAMRA-3’). Thermal cycling consisted of an initial denaturation at 95 °C for 30 s, followed by 40 cycles of denaturation at 95 °C for 5 s and annealing and extension at 60 °C for 34 s. High-throughput sequencing The heavy fractions (considered as the active species including ammonia oxidizers and their mostly closely linked species incorporating 13 C into their genomes during the DNA-SIP incubation) of Treatment 13C-B were sent for 16S rRNA sequencing of both bacteria and archaea. To construct a network to investigate the niches of AOA and AOB, we used universal primers for bacteria and archaea. The V3-V4 region of the bacterial 16S rRNA gene was amplified using the primers 341F (5'-CCTAYGGGRBGCASCAG − 3') and 806R (5'-GGACTACNNGGGTATCTAAT − 3')[ 27 ]. The V4 region of the archaeal 16S rRNA gene was amplified using the primers 524F10extF (5'-TGYCAGCCGCCGCGGTAA-3') and Arch958RmodR (5'-YCCGGCGTTGAVTCCAATT-3')[ 28 ]. Fragments from each soil sample were sequenced using MiSEq. Following gene amplification, 3 µl of the PCR product was used for agarose gel electrophoresis (1%) to confirm the results of amplification. PCRs of 4 replicates for each sample preparation were combined and quantified with PicoGreen. From each sample, 200 ng of the PCR product was collected and pooled with other samples for one sequencing run. The pooled mixture was purified with a QIAquick Gel Extraction Kit (QIAGEN Sciences, Germantown, MD, USA) and re-quantified with PicoGreen. The purified mixture was then diluted and loaded as described in the MiSeq Reagent Kit Preparation Guide (Illumina, San Diego, CA, USA). Construction of a microbial fuel cell (MFC) system A MFC system was constructed to assess possible extracellular electron transfer between heterotrophic BA and autotrophic AOM. Two-bottle MFC reactors (total volume of each bottle was 100 mL) were constructed as described by McAnulty in 2017[ 21 ]. The anode side was the soil used in this experiment, and the cathode side was subjected to 3 different treatments: BA, sterile BA as the negative control and Shewanella oneidensis MR-1 as the positive control. The MFC systems were incubated at 30 °C in the dark for 14 days. Data analysis After assigning each sequence to the appropriate sample according to its barcode and allowing up to two mismatches, a total of 30,000 reads from both ends were obtained as a partitioned run for each sample. All the OTUs belonging to ammonia-oxidizers were picked up from the bacterial and archaeal OTU table after classification. Network analysis was performed and visualized using Cytoscape (Version 3.72). All statistical analyses were carried out in the R platform (3.6.2, http://www.r-project.org ). The Shapiro–Wilk test and Bartlett test were used to check whether the data conformed to normality and homoscedasticity, respectively. The post hoc Tukey HSD test was used to check if the relationship between two sets of data was statistically significant. Results Inhibition of nitrification by BA The influence of BA on nitrification was assessed by measuring differences in NH 4 + , NO 3 − , CO 2 , NO and N 2 O concentrations during incubation of microcosms (Fig. 1 ). The increases in NO 3 − , NO and N 2 O concentrations in Treatments 12C-B and 13C-B were significantly less than those in Treatment 13C-BS. The nitrate concentration in Treatments 13C-B and 12C-B was significantly (P < 0.05) lower than that in 13C-BS after incubation for 28 days. The nitric oxide concentration in 13C-B and 12C-B was significantly (P < 0.05) higher than that in 13C-BS. The N 2 O concentration in 13C-B and 12C-B was significantly (P < 0.05) higher than that in 13C-BS. In contrast, the CO 2 concentration decreased after inoculation of microcosms with active BA. The CO 2 concentration in 13C-B and 12C-B was significantly (P < 0.05) higher than that in 13C-BS. These factors all lead to the potential inhibition of the nitrification process by BA. However, NH 4 + in Treatments 13C-B and 12C-B was also less than that in Treatment 13C-BS. The ammonium concentration in Treatments 13C-B and 12C-B was significantly (P < 0.01) lower than that in Treatment 13C-BS. DNA-SIP To identify which ammonia oxidizers incorporated 13 CO 2 during incubation, both archaeal and bacterial amoA genes were quantified in DNA from different fractions (Fig. 2 a and b). The quantitative abundance of AOB was greatest around a buoyant density of ~ 1.71–1.72 g ml − 1 in Treatment 12C-B and ~ 1.73–1.75 g ml − 1 in Treatments 13C-B and 13C-BS. In contrast, the archaeal abundance was greatest at ~ 1.71–1.72 g ml − 1 in 12C-B and 13C-BS and ~ 1.73–1.75 g ml − 1 in 13C-B. This indicates that bacterial amoA was labelled in Treatment 13C-BS, while in Treatment 13C-B and 12C-B, both ammonia-oxidizing archaea and bacteria were labelled after incubation for 28 days. Sequencing of 16S rRNA genes in the heavy fractions indicated that neither the AOA nor AOB were the most abundant species in the 13 C-labelled 16S rRNA-based communities. Within Thaumarchaeota, 93.6% of the 13 C-labelled AOA community fell within the Soil Crenarchaeotic Group and 1.5% within South African Gold Mine Gp 1. Within the bacteria, 80% of the AOB and NOB sequences fell within the Nitrospira group, while few traditional AOB, e.g., Nitrosococcus , or NOB, e.g., Nitrobacter , were observed in labelled DNA (Fig. 2 c and d). Activity of comammox after stimulation by BA Given the relatively high proportion of Nitrospira 16S rRNA sequences in 13 C-labelled DNA, specific amo A primers for comammox were quantified in DNA from heavy fractions. This analysis indicated that comammox, especially Nitrospira clade A, was active in our BA-induced systems (Fig. 3 ). The relative abundance of Nitrospira clade A was greatest around a buoyant density of ~ 1.71–1.72 g ml − 1 in Treatments 12C-B and 13C-BS and at ~ 1.73–1.75 g ml − 1 in Treatment 13C-B. Moreover, we amplified clade B at first but did not obtain any positive results. Network of the microcosms In order to elucidate the niche of BA after inoculation into the microcosms, we constructed a network of microorganisms, including archaea and bacteria (Fig. 4 ). The network showed that both nitrifiers and BA were not heavily connected to other organisms because of their small size in the whole community. However, BA and Thaumarchaeota were linked indirectly by Microbacteriaceae , and the edge indicated positive relations between both BA- Microbacteriaceae and Microbacteriaceae -Thaumarchaeota. AOA in the MFC system To further investigate whether AOA provides positive feedback to electron transfer from the outside, we constructed a MFC system, and the abundance of AOA was quantified by Q-PCR. The abundance of AOA in the presence of BA was significantly higher than that in Treatment SBA (Fig. 5 ), but it was not significantly higher between Shewanella and SBA. The abundance of amoA in Treatment C was 2.62 × 10 5 g − 1 dry weight soil, which was much lower than that in the other three treatments with inoculation of both active and sterile microbes. The abundance of AOA in Treatment BA was 6.65 × 10 5 g − 1 dry weight soil, which was significantly higher than that in the presence of Shewanella and Sterile BA (6.08 × 10 5 (P < 0.05) and 5.71 × 10 5 (P < 0.05), respectively). Discussion This study suggests that the three autotrophic AOM, AOA, AOB and comammox bacteria Nitrospira clade A, were active in ammonia oxidation. The system involved a heterotrophic bacterium (HB), BA, which suggested a special relation between the HB and autotrophic AOM in the soil to be confirmed in the future. This provides compelling evidence that the regulation of the extraneous HB on the nitrification process involved the enhancement of oxidation of NH 2 OH to NO through AOA and Nitrospira clade A and the inhibition of the production of N 2 O through AOB, leading to a significant increase in NO and mitigation of N 2 O in the nitrification process. It also provides evidence that the potential regulating strategy is extracellular electron transfer between HB and AOM, especially AOA. Previous studies have suggested a complex relation between heterotrophic bacteria and autotrophic AOM, including competition for NH 4 + and cooperation where organic carbon is low[ 29 – 31 ]. There are different ways microbes interact, e.g., cross-feeding, co-metabolism, cell-to-cell communication[ 20 , 29 , 32 ] and interspecies extracellular electron transfer (IEET)[ 33 ]. In our constructed microcosms, we found the co-activity of AOA, AOB and the newly discovered comammox in the ammonia-oxidizing process. After BA was inoculated into the microcosms, it successfully resuscitated the inactive ammonia-oxidizing archaea and complete nitrifier Nitrospira in the soil, which was previously dominated by ammonia-oxidizing bacteria[ 15 , 34 ]. Nitrification is a two-step process:[ 14 ] ammonia (NH 3 ) is oxidized via hydroxylamine (NH 2 OH) to nitrite (NO 2 − ) and, subsequently, nitrite is oxidized to nitrate (NO 3 − ). Within these two steps, two processes contribute to N 2 O emissions. The first is aerobic N 2 O formation from the abiotic reaction of the intermediates NH 2 OH, NO and NO 2 − , termed chemodenitrification. NH 3 is first oxidized to NH 2 OH by the enzyme ammonia monooxygenase (AMO), which belongs to the superfamily of copper-dependent membrane-bound monooxygenases[ 35 ]. Subsequently, NH 2 OH is oxidized to NO by hydroxylamine dehydrogenase (HAO) under oxic and anoxic conditions[ 1 , 4 , 7 ]. NH 2 OH released in this process may be oxidized to N 2 O by oxidants, including Fe 3+ and MnO 2 , while NO 2 − and NO may be reduced by reductants such as Fe 2+ , Cu 2+ or humic substances[ 36 ]. The second is nitrifier-denitrification, an enzymatic process in which NO 2 − is reduced to N 2 O via NO; reduction of NO to N 2 O is catalysed by two classes of cytochrome c nitric oxide reductases (NORs). Chemodenitrification is associated with ammonia oxidation by ammonia-oxidizing bacteria (AOB), ammonia-oxidizing archaea (AOA) and comammox, while nitrifier denitrification has only been reported in AOB[ 1 , 3 , 4 , 8 , 37 ], and all genome-sequenced AOA and comammox lack NOR[ 1 , 5 , 38 , 39 ]. The inoculation of BA and the change in AOM status in situ obviously changed the rate of nitrification. The consumption of CO 2 and production of N 2 O and NO 3 − were lower in Treatment 13C-B compared to Treatment 13C-BS, indicating that BA inhibited the nitrification process[ 40 – 43 ]. However, NH 4 + remained in the soil, and the higher emissions of NO in Treatment 13C-B after inoculation did not support the inhibition of nitrification. The concentrations of NH 4 + in Treatment 13C-B were less than those in Treatment 13C-BS, and the concentration of NO was slightly higher in 13C-B, which led to an enhancement of the ammonia-oxidizing process. The analysis of ammonia-oxidizing microorganisms in this system helped us to resolve this problem. Our soil used in this work was agricultural soils collected from a typical acidic soil area in China, in which AOB play a dominant role, as previous works have suggested[ 34 , 44 ]. The results of SIP in Treatment 13C-BS confirmed the dominance of AOB in our experimental soils. However, the results also suggested that both AOA and Nitrospira clade A become other active organisms that oxidize NH 3 to NH 2 OH in Treatment 13C-B. This indicated that the initially inactive and feeble AOA in agricultural soils was resuscitated by the inoculation of BA and began to be active in function with AOB in situ . The labelled AOA in Treatment 13C-B mainly belong to Soil Crenarchaeotic Group, which indicates their dominant function in the ammonia-oxidizing process. After that, the AOB in Treatment 13C-B were sequenced, and we found that only a few AOB were classified, including Nitrosospira and Nitrosococcus . We wondered if any other species participated in this process, especially the newly isolated comammox. Interestingly, the results of Q-PCR of comammox showed that Nitrospira clade A was labelled in the heavy fractions. Taken together, after the inoculation of BA into the acidic soils, the whole ammonia-oxidizing process was redefined; instead of the sole activity of AOB as usual, both AOA and comammox were more active and made use of NH 3 . The results further revealed the abnormal increase in the production of NO in Treatment 13C-B. As we have mentioned before, NO is an important intermediate in the nitrification process both in biotic and abiotic pathways[ 1 , 4 , 8 , 45 ]. However, in almost all the AOA and comammox genomes sequenced to date, no canonical nitric oxide reductases (NORs) have been detected, despite the wide presence of a nitrite reductase gene ( nirK ) in AOA. Although cytochrome P450 and other enzymes possibly involved in the production of N 2 O and acting as NOR may be detected in the future, it can only take action in some AOA in the presence of excess nitrite[ 1 , 8 , 46 ]. After the inoculation of BA and the recovery of AOA and comammox in the soil, more NO was produced in the microcosms (Fig. 6 a). The results and relations between the inoculated BA and ammonia-oxidizing microorganisms indicate a complex competence and cooperation among these species. BA is a widely isolated heterotrophic bacterium that holds a good NH 4 + affinity in oligotrophic environments, and the inoculation of BA consumes NH 4 + in soil, which makes it more competitive for nitrification by AOM[ 47 ]. This in turn provides opportunities for AOA and comammox, whose affinity for ammonium is better than AOB[ 1 , 38 , 39 , 47 , 48 ], and makes the co-function of AOM in soils possible[ 49 ]. In addition, the inoculation of BA into the soils changed the original niche in situ , and new relations between BA and the ammonia-oxidizers were constructed. The network showed that BA and the AOA Thaumarchaeota are at the edge of the network, and they are indirectly and positively connected by a Microbacteriaceae . Both BA and Microbacteriaceae contain genes encoding multiheme cytochrome c , which is good for the transport of electrons and facilitates ammonia oxidation, especially AOA[ 50 – 52 ]. To date, no cytochrome c has been found in AOA compared to its wide expression in AOB and comammox[ 53 – 55 ]. Previous studies have confirmed the influence of IEET on anaerobic methane oxidizing microbes[ 33 ], which is similar to ammonia-oxidizing microbes. Therefore, we constructed a MFC system to investigate the potential IEET between BA and AOA. Although the MFC systems were carried out for only 14 days, the abundance of AOA in Treatment BA was significantly higher than that in the control. This indicated that the electrons transferred from BA were as high as the typical Shewanella at the cathode side, and the electrons may play a positive role in the abundance of AOA in the soil. Of course, this is only preliminary evidence for the IEET between these two species, but the difference between the 3 treatments did provide evidence that AOA reply positively to the electron transferred from other organisms. Altogether, this suggests that BA is regulated by all autotrophic AOM in acidic soils via the competence of NH 4 + (competence resource) and the facilitation of electron transport (energy supplying), a hypothesis that warrants further experimental elucidation (Fig. 6 b). Conclusions This is a novel discovery of the resuscitation of AOA and newly found complete nitrifiers under the originally dominant AOB in the ammonia-oxidizing process. The results were detected based on the complex interactions between microbes. In our work, after the inoculation of BA, the whole nitrification in soil was changed, the ammonia-oxidizing process was inhibited; in addition, the emission of N 2 O was much lower, and the production of NO increased owing to the lack of NOR in AOA and comammox. We provide a possible explanation for the special relationship between BA and AOM; both the competence of ammonium with AOB and the facilitation of electron transport with AOA may have led to these results. However, far more research needs to be performed, not only to testify and confirm the relations and electron transfer between these microorganisms but also to determine if some other microbes are involved in this process. Additionally, the mechanisms by which BA regulates the ammonia-oxidizing process at metabolic levels should be elucidated. Declarations Ethics approval and consent to participate Not applicable Consent for publication Not applicable Availability of data and materials Sequences data of 16S rRNA high-throughput sequencing are available in NCBI Sequence Read Archive repository database (www.ncbi.nlm.nih.gov/sra) under accession number SRR12587975 to SRR12588174. Competing interests The authors declare that they have no competing interests Funding This research was supported by the National Natural Science Foundation of China (Nos. 41907273, 31670507 and 91951108). Authors' contributions S.W. conducted the experiments with the help of Y.D. and F.H. S.W. and Y.D. analyzed the data with the help of Y.D.. S.W. wrote the manuscript. S.W. and X.Z. initiated the project and directed the research. Acknowledgements The authors would like to thank Professor James I. Prosser for his general help in revising the manuscript and provide strains for further study. Author information Affiliations Key Laboratory of Environmental Biotechnology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China Shanghua Wu, Yuzhu Dong, Haonan Fan, Ye Deng & Xuliang Zhuang College of Resources and Environment, University of Chinese Academy of Sciences, Beijing 100049, China Shanghua Wu, Yuzhu Dong, Haonan Fan, Ye Deng & Xuliang Zhuang References Kits KD, Jung MY, Vierheilig J, Pjevac P, Sedlacek CJ, Liu S, et al. Low yield and abiotic origin of N 2 O formed by the complete nitrifier Nitrospira inopinata . Nat Commun. 2019;10(1):1836; doi: 10.1038/s41467-019-09790-x. Kuypers MMM, Marchant HK, Kartal B. 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Wang B, Qin W, Ren Y, Zhou X, Jung MY, Han P, et al. Expansion of Thaumarchaeota habitat range is correlated with horizontal transfer of ATPase operons. ISME J. 2019;13(12):3067-79; doi: 10.1038/s41396-019-0493-x. Xu HJ, Wang XH, Li H, Yao HY, Su JQ, Zhu YG. Biochar impacts soil microbial community composition and nitrogen cycling in an acidic soil planted with rape. Environ Sci Technol. 2014;48(16):9391-9; doi: 10.1021/es5021058. Yong X, Zhang R, Zhang N, Chen Y, Huang X, Zhao J, et al. Development of a specific real-time PCR assay targeting the poly-gamma-glutamic acid synthesis gene, pgsB , for the quantification of Bacillus amyloliquefaciens in solid-state fermentation. Bioresour Technol. 2013;129:477-84; doi: 10.1016/j.biortech.2012.11.092. Behrendt L, Larkum AW, Trampe E, Norman A, Sorensen SJ, Kuhl M. Microbial diversity of biofilm communities in microniches associated with the didemnid ascidian Lissoclinum patella . ISME J. 2012;6(6):1222-37; doi: 10.1038/ismej.2011.181. 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Nat Commun. 2018;9(1):1517; doi: 10.1038/s41467-018-03861-1. Heil J, Vereecken H, Bruggemann N. A review of chemical reactions of nitrification intermediates and their role in nitrogen cycling and nitrogen trace gas formation in soil. European Journal of Soil Science. 2016;67(1):23-39; doi: 10.1111/ejss.12306. Vajrala N, Martens-Habbena W, Sayavedra-Soto LA, Schauer A, Bottomley PJ, Stahl DA, et al. Hydroxylamine as an intermediate in ammonia oxidation by globally abundant marine archaea. Proc Natl Acad Sci U S A. 2013;110(3):1006-11; doi: 10.1073/pnas.1214272110. Kits KD, Sedlacek CJ, Lebedeva EV, Han P, Bulaev A, Pjevac P, et al. Kinetic analysis of a complete nitrifier reveals an oligotrophic lifestyle. Nature. 2017;549(7671):269-72; doi: 10.1038/nature23679. Palomo A, Pedersen AG, Fowler SJ, Dechesne A, Sicheritz-Ponten T, Smets BF. Comparative genomics sheds light on niche differentiation and the evolutionary history of comammox Nitrospira . ISME J. 2018;12(7):1779-93; doi: 10.1038/s41396-018-0083-3. Di HJ, Cameron KC, Podolyan A, Robinson A. Effect of soil moisture status and a nitrification inhibitor, dicyandiamide, on ammonia oxidizer and denitrifier growth and nitrous oxide emissions in a grassland soil. Soil Biology & Biochemistry. 2014;73:59-68; doi: 10.1016/j.soilbio.2014.02.011. Scheer C, Rowlings DW, Firrel M, Deuter P, Morris S, Grace PR. Impact of nitrification inhibitor (DMPP) on soil nitrous oxide emissions from an intensive broccoli production system in sub-tropical Australia. Soil Biology & Biochemistry. 2014;77:243-51; doi: 10.1016/j.soilbio.2014.07.006. Lam SK, Suter H, Mosier AR, Chen D. Using nitrification inhibitors to mitigate agricultural N2 O emission: a double-edged sword? Glob Chang Biol. 2017;23(2):485-9; doi: 10.1111/gcb.13338. Sun L, Lu Y, Yu F, Kronzucker HJ, Shi W. Biological nitrification inhibition by rice root exudates and its relationship with nitrogen-use efficiency. New Phytol. 2016;212(3):646-56; doi: 10.1111/nph.14057. Jia Z, Conrad R. Bacteria rather than Archaea dominate microbial ammonia oxidation in an agricultural soil. Environ Microbiol. 2009;11(7):1658-71; doi: 10.1111/j.1462-2920.2009.01891.x. Martens-Habbena W, Qin W, Horak RE, Urakawa H, Schauer AJ, Moffett JW, et al. The production of nitric oxide by marine ammonia-oxidizing archaea and inhibition of archaeal ammonia oxidation by a nitric oxide scavenger. Environ Microbiol. 2015;17(7):2261-74; doi: 10.1111/1462-2920.12677. Jung MY, Gwak JH, Rohe L, Giesemann A, Kim JG, Well R, et al. Indications for enzymatic denitrification to N 2 O at low pH in an ammonia-oxidizing archaeon. ISME J. 2019;13(10):2633-8; doi: 10.1038/s41396-019-0460-6. Martens-Habbena W, Berube PM, Urakawa H, de la Torre JR, Stahl DA. Ammonia oxidation kinetics determine niche separation of nitrifying Archaea and Bacteria. Nature. 2009;461(7266):976-9; doi: 10.1038/nature08465. Daims H, Lucker S, Wagner M. A New Perspective on Microbes Formerly Known as Nitrite-Oxidizing Bacteria. Trends Microbiol. 2016;24(9):699-712; doi: 10.1016/j.tim.2016.05.004. Straka LL, Meinhardt KA, Bollmann A, Stahl DA, Winkler MH. Affinity informs environmental cooperation between ammonia-oxidizing archaea (AOA) and anaerobic ammonia-oxidizing (Anammox) bacteria. ISME J. 2019;13(8):1997-2004; doi: 10.1038/s41396-019-0408-x. Pankratova G, Hederstedt L, Gorton L. Extracellular electron transfer features of Gram-positive bacteria. Anal Chim Acta. 2019;1076:32-47; doi: 10.1016/j.aca.2019.05.007. Schmid MC, Hooper AB, Klotz MG, Woebken D, Lam P, Kuypers MM, et al. Environmental detection of octahaem cytochrome c hydroxylamine/hydrazine oxidoreductase genes of aerobic and anaerobic ammonium-oxidizing bacteria. Environ Microbiol. 2008;10(11):3140-9; doi: 10.1111/j.1462-2920.2008.01732.x. Arp DJ, Sayavedra-Soto LA, Hommes NG. Molecular biology and biochemistry of ammonia oxidation by Nitrosomonas europaea. Arch Microbiol. 2002;178(4):250-5; doi: 10.1007/s00203-002-0452-0. van Kessel MA, Speth DR, Albertsen M, Nielsen PH, Op den Camp HJ, Kartal B, et al. Complete nitrification by a single microorganism. Nature. 2015;528(7583):555-9; doi: 10.1038/nature16459. Stahl DA, de la Torre JR. Physiology and diversity of ammonia-oxidizing archaea. Annu Rev Microbiol. 2012;66:83-101; doi: 10.1146/annurev-micro-092611-150128. Bertini I, Cavallaro G, Rosato A. Cytochrome c : occurrence and functions. Chem Rev. 2006;106(1):90-115; doi: 10.1021/cr050241v. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-76082","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research","associatedPublications":[],"authors":[{"id":2287538,"identity":"f5c24646-3738-426a-a2a1-b735385bbf15","order_by":0,"name":"Shanghua Wu","email":"","orcid":"","institution":"Research Center for Eco-Environmental Sciences Chinese Academy of Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Shanghua","middleName":"","lastName":"Wu","suffix":""},{"id":2287539,"identity":"a41f3844-743d-4e1c-9d70-646bdc274377","order_by":1,"name":"Yuzhu Dong","email":"","orcid":"","institution":"Research Centre for Eco-Environmental Sciences Chinese Academy of Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yuzhu","middleName":"","lastName":"Dong","suffix":""},{"id":2287540,"identity":"f79640b5-426f-4ed3-9737-aadc86cfb6e4","order_by":2,"name":"Haonan Fan","email":"","orcid":"","institution":"Research Centre for Eco-Environmental Sciences Chinese Academy of Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Haonan","middleName":"","lastName":"Fan","suffix":""},{"id":2287541,"identity":"51764196-b2f0-4785-a69a-430ae5b1b54a","order_by":3,"name":"Ye Deng","email":"","orcid":"","institution":"Research Centre for Eco-Environmental Sciences Chinese Academy of Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ye","middleName":"","lastName":"Deng","suffix":""},{"id":2287542,"identity":"d50220e6-3fd1-46c3-8ccb-1a638b917e8d","order_by":4,"name":"Xuliang Zhuang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4UlEQVRIiWNgGAWjYDACdgY2MM1PvBZmqBbJBpK1GBwgVofBYfZnj3lq7thtvn344YMPfxjk+RuYnz3Ap0WymSHdmOfYs+Rt59KMDWe2MRjOOMBmboBPCz8zwzFpHrbDyWZneNikeRsYGDcw8LBJ4NPCxszYJs3z73CycQ8P++8/fxjsCWrhZ2YGGt522M6Ah4cNFBSJBLVINrOxSc7tO5wgcYbNWLK3TSJ5xmE2M7xaDI63P5N48+2wPX8P88MPP/7Y2Pa3Nz/DqwUGEhsgtAQopogD9kSqGwWjYBSMgpEIAA7KPK3SxKXnAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0001-5902-4267","institution":"Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Xuliang","middleName":"","lastName":"Zhuang","suffix":""}],"badges":[],"createdAt":"2020-09-11 10:21:47","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-76082/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-76082/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":2422776,"identity":"98e41c8d-0d85-4109-a015-89977aa39dec","added_by":"auto","created_at":"2020-09-15 21:02:27","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1082077,"visible":true,"origin":"","legend":"Concentrations of CO2 (a), NO (b), N2O (c), NH4+ (d) and NO3- (e) in surface agricultural soil incubated within Treatments 12C-B, 13C-B and 13C-BS after incubation for 28 days. The error bars of soil nitrogen contents indicate standard errors of triplicate microcosm incubations.","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-76082/v1/1.jpg"},{"id":2422778,"identity":"cc04c6db-5598-4c9c-aa13-79cc7155b8fe","added_by":"auto","created_at":"2020-09-15 21:02:27","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":471554,"visible":true,"origin":"","legend":"Abundance and community of AOA and AOB labelled in the heavy layers. (a and b) Quantitative distribution of the abundance of amoA genes in AOA (a) and AOB (b) across the buoyant density gradient of the fractionated DNA from soil incubated with either 12CO2 or 13CO2 after incubation for 28 days. The error bars indicate standard errors of triplicate microcosm incubations. (c and d) Communities of AOA (c), AOB and NOB (d) in the heavy layer DNA from the soil incubated with 13CO2 after incubation for 28 days. Treatments 13C-B 1 to 13C-B 3 were triplicates of microcosms.","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-76082/v1/2.jpg"},{"id":2422779,"identity":"019a3c27-386e-4359-812d-80f7fe21e16a","added_by":"auto","created_at":"2020-09-15 21:02:27","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":428125,"visible":true,"origin":"","legend":"Quantitative distribution of the amoA genes of complete nitrifying Nitrospira across the entire buoyant density gradient of the fractionated DNA from the soil incubated with either 12CO2 or 13CO2 after incubation for 28 days. The error bars indicate standard errors of triplicate microcosm incubations (Fig. 3).","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-76082/v1/3.jpg"},{"id":2422780,"identity":"291ba3c2-9349-42e3-b23f-f3a94e345ff5","added_by":"auto","created_at":"2020-09-15 21:02:27","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":284615,"visible":true,"origin":"","legend":"Network of bacteria and archaea in the heavy layer DNA from the soil incubated with 13CO2 and BA after incubation for 28 days. The red nodes belong to Bacillus, and the black nodes belong to AOA. ","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-76082/v1/4.jpg"},{"id":2422781,"identity":"90087106-d8f6-43f7-b3e2-c83d236ba5b7","added_by":"auto","created_at":"2020-09-15 21:02:27","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":245166,"visible":true,"origin":"","legend":"Abundances of AOA in the MFC systems. The error bars indicate standard errors of triplicate Q-PCRs. ","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-76082/v1/5.jpg"},{"id":2422782,"identity":"7af7d191-32ba-41db-9340-5d25d3079634","added_by":"auto","created_at":"2020-09-15 21:02:27","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":640143,"visible":true,"origin":"","legend":"Potential relations between BA and AOM and regulations in the nitrification process. (a) Changes in the nitrification process after BA was inoculated. The solid stars indicate the dominant species in the nitrification process with BA or without BA. (b) The potential relations between BA and the three ammonia oxidizers[48].","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-76082/v1/6.jpg"},{"id":15669393,"identity":"5404394c-b410-4d3e-bead-63d00bb68c40","added_by":"auto","created_at":"2021-11-18 13:53:12","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":999870,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-76082/v1/27c61e0e-24e6-4a6b-8cb0-f1fbf3b7f8a3.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eResuscitation of inactive ammonia-oxidizing archaea and complete nitrifiers by extracellular electrons from a heterotrophic bacterium, \u003cem\u003eBacillus amyloliquefaciens\u003c/em\u003e\u003c/p\u003e","fulltext":[{"header":"Background","content":" \u003cp\u003eNitrous oxide (N\u003csub\u003e2\u003c/sub\u003eO) is the third most abundant and long-lived greenhouse gas in the atmosphere. Anthropogenic emissions are the most important sources of global N\u003csub\u003e2\u003c/sub\u003eO production, comprising nearly 30\u0026ndash;45% of total emissions, with approximately two-thirds of this derived from agriculture and soil[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMicrobial transformations of nitrogenous compounds, especially heterotrophic denitrification and chemolithoautotrophic aerobic nitrification, are the two dominant contributors to N\u003csub\u003e2\u003c/sub\u003eO emissions[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Classically, heterotrophic denitrification has been considered to be the major biological source and sink of N\u003csub\u003e2\u003c/sub\u003eO in the terrestrial nitrogen cycle, but N\u003csub\u003e2\u003c/sub\u003eO emissions from nitrification are thought to be of equal importance to those from denitrification in recent decades. N\u003csub\u003e2\u003c/sub\u003eO results directly from the ammonia-oxidizing activity of traditional nitrifiers and indirectly from the activities of ammonia-oxidizing bacteria, ammonia-oxidizing archaea, and the newly discovered complete nitrifier (complete ammonia oxidation; comammox) \u003cem\u003eNitrospira inopinat\u003c/em\u003ea[\u003cspan additionalcitationids=\"CR2 CR3 CR4 CR5 CR6 CR7 CR8\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Nitrifier denitrification may account for up to 100% of N\u003csub\u003e2\u003c/sub\u003eO emissions from ammonia in soils and is more significant than classical denitrification at low oxygen concentrations, especially fluctuating aerobic-anaerobic conditions[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Given predicted increases in farmland and fertilizer application (EPA reference), agricultural soils are likely to contribute up to 59% of total N\u003csub\u003e2\u003c/sub\u003eO emissions by 2030[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], increasing the need to explore approaches to manage and reduce N\u003csub\u003e2\u003c/sub\u003eO emissions from agricultural systems[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn our previous work, a strain of \u003cem\u003eBacillus amyloliquefaciens\u003c/em\u003e (BA) that significantly reduced N\u003csub\u003e2\u003c/sub\u003eO emissions by 35%-50% after inoculation was isolated from rice rhizosphere, and the results suggested that the mitigation was mostly due to the inhibition of nitrification[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. However, the detailed process of inhibition and relations of organisms behind the regulation of BA have not been studied, especially those between heterotrophic BA and three groups of autotrophic AOM.\u003c/p\u003e \u003cp\u003eAOA \u003cem\u003eamoA\u003c/em\u003e genes are abundant and widespread in soils, frequently outnumbering AOB \u003cem\u003eamoA\u003c/em\u003e genes[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], and comammox bacteria are also abundant in some soils[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The relative abundance and dominance of these three types of ammonia-oxidizing microorganisms (AOM) are affected by abiotic factors, including pH, temperature and moisture content[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], and by several nitrification inhibitors. AOM may also be influenced by interactions with other microorganisms, e.g., through cross-feeding and quorum sensing[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], and by direct or indirect electron transfer between species. The latter is rarely studied and can be investigated using microbial fuel cells (MFCs). \u003cem\u003eGeobacter sulfurreducens\u003c/em\u003e and \u003cem\u003eShewanella oneidensis\u003c/em\u003e are the two most commonly used model strains in MFCs because of their good efficiency in transferring electrons; in MFCs, protons are the only thing allowed to pass through the membrane between the anode and cathode chambers and complete the circuit[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, we constructed microcosms to investigate the influence of the heterotrophic bacterium BA on nitrification and three different types of nitrifiers. We also constructed MFCs to study the possible extracellular electron transfer between BA and AOA. Together, these results suggest a complex regulation strategy in which heterotrophic bacteria affect autotrophic organisms, and extracellular electron transfer may be a possible link between the two types of microbes.\u003c/p\u003e "},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n\u003cp\u003e\u003cstrong\u003eSoil microcosms and stable isotope probing (SIP)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe influence of BA on nitrification was investigated in soil microcosms consisting of 10\u0026nbsp;g of sieved acidic soil (mesh size of 3.35\u0026nbsp;mm) from Southwest China used in our previous work[\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e]. Microcosms were adjusted to 60% maximum water-holding capacity in 120-ml serum bottles, sealed with rubber stoppers and aluminium caps and incubated at 30\u0026nbsp;\u0026deg;C in the dark for 4 weeks. The headspace was sampled and adjusted to 5% (vol/vol) CO\u003csub\u003e2\u003c/sub\u003e by injection through the rubber septum, and each bottle was flushed for 5\u0026nbsp;min weekly with synthetic air (20% O\u003csub\u003e2\u003c/sub\u003e, 80% N\u003csub\u003e2\u003c/sub\u003e) between sampling and injection. For every week, 100\u0026nbsp;\u0026micro;g urea-N g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e dry weight soil was added by liquid to each bottle.\u003c/p\u003e\n\u003cp\u003eThree treatments were established, each in triplicate microcosms. In Treatment 12C-B, the headspace contained \u003csup\u003e12\u003c/sup\u003eC-CO\u003csub\u003e2,\u003c/sub\u003e and active BA was inoculated. The inoculum was grown in liquid batch culture to stationary phase in M9 Minimal Medium (\u003csup\u003e13\u003c/sup\u003eC-glucose was used in treatments of \u003csup\u003e13\u003c/sup\u003eC-CO\u003csub\u003e2\u003c/sub\u003e to avoid additional \u003csup\u003e12\u003c/sup\u003eC into the microcosms) at 30\u0026nbsp;\u0026deg;C and 180\u0026nbsp;rpm for 28\u0026nbsp;h. Cells were separated by centrifugation at 8000\u0026nbsp;rpm and washed three times with sterilized water to remove the remaining culture medium. The final cell suspension was adjusted to 3\u0026thinsp;\u0026times;\u0026thinsp;10\u003csup\u003e8\u003c/sup\u003e CFU ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and 1\u0026nbsp;ml of this suspension was added weekly by injection through the rubber septum into each microcosm for 3 consecutive weeks. Treatment 13C-B was identical to Treatment 12C-B, except that the headspace contained \u003csup\u003e13\u003c/sup\u003eC-CO\u003csub\u003e2\u003c/sub\u003e. Treatment 13C-BS was identical to Treatment 13C-B but was inoculated with \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e sterilized by autoclaving as a control.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSoil chemical analysis\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/div\u003e\n\u003cp\u003eSoil pH was determined in water at a ratio of 1:2.5 (w/v) using a pH meter (Accumet Excel XL 60, Fisher Scientific, Singapore). The NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e and NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e concentrations were determined after extraction in 2\u0026nbsp;M KCl (soil:solution ratio of 1:10 (w/v)) and measured using a continuous flow analyser (SAN++, Skalar, Breda, Holland).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInstantaneous CO\u003csub\u003e2\u003c/sub\u003e, NO and N\u003csub\u003e2\u003c/sub\u003eO measurements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eN\u003csub\u003e2\u003c/sub\u003eO and CO\u003csub\u003e2\u003c/sub\u003e concentrations in the headspace were measured using an Agilent gas chromatograph (Agilent Technologies Inc., Wilmington, DE) equipped with a micro-TCD and a flame ionization detector, respectively. NO concentration was measured using a NO-NO\u003csub\u003e2\u003c/sub\u003e-NO\u003csub\u003ex\u003c/sub\u003e chemiluminescent analyser (42i Thermo Environmental Instruments Inc., USA)[\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDNA extraction and SIP gradient fractionation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDNA was extracted from 0.5\u0026nbsp;g of dry soil using the FastDNA SPIN kit for soil (MP Biomedicals, Santa Ana, CA, USA) according to the manufacturer\u0026rsquo;s instructions. The extracted DNA was dissolved in 80 \u0026micro;L of sterile, deionized, nuclease-free water and divided into two parts: one part was stored at 4\u0026nbsp;\u0026deg;C prior to quantitative PCR and the other was stored at -20\u0026nbsp;\u0026deg;C for further use.\u003c/p\u003e\n\u003cp\u003eThe quantity and purity of soil DNA were determined by a Nanodrop ND-1000 UV-Vis Spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). SIP fractionation was performed as described previously[\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e]. For each sample, 3.5\u0026nbsp;\u0026micro;g of soil DNA was mixed with CsCl (buoyant density of 1.725\u0026nbsp;g ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) before ultracentrifugation at 44,100 r.p.m. (~\u0026thinsp;177,000 \u003cem\u003egav\u003c/em\u003e) in a Vti 65.2 rotor (Beckman Coulter, cat. no. 362754) at 20\u0026nbsp;\u0026deg;C for 44\u0026nbsp;h with vacuum and maximum acceleration and without braking (requiring an additional 2\u0026nbsp;h to stop). DNA fractionation was carried out by displacing the gradient medium with sterile water from the top of the ultracentrifuge tube using an NE-1000 single syringe pump (New Era Pump Systems Inc., Farmingdale, NY, USA) with a precisely controlled flow rate of 0.375\u0026nbsp;ml min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Up to 15 DNA gradient fractions were generated with equal volumes of approximately 380\u0026nbsp;ml, and a 65-ml aliquot of each fraction was used for refractive index measurement using an AR200 digital hand-held refractometer (Reichert, Inc., Buffalo, NY, USA). The fractionated DNA was purified and dissolved in 30 \u0026micro;L of sterile, deionized, nuclease-free water[\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantitative PCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFunctional marker genes (bacterial and archaeal \u003cem\u003eamoA\u003c/em\u003e) and \u003cem\u003eamoA\u003c/em\u003e of \u003cem\u003eNitrospira\u003c/em\u003e clade A were quantified using Premix Ex Taq (TaKaRa, Japan) and gene-specific primers. The primers used for \u003cem\u003eNitrospira\u003c/em\u003e clade A were coma-244f/coma-659r. Details of plasmid standards, gene-specific qPCR primers, reaction mixtures and thermal programmes are described in previous studies[\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e]. Each sample was quantified in duplicate using the CFX Connect\u0026trade; Real-Time PCR Detection System (Bio-Rad laboratories). The algorithm that was used to calculate the efficiency (E) and threshold cycle (CT) was based on the kinetics of each individual reaction.\u003c/p\u003e\n\u003cp\u003eBA abundance was determined using Q-PCR based on the method reported by Yong \u003cem\u003eet al.\u003c/em\u003e [\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e]. Primers targeting the \u003cem\u003epgsB\u003c/em\u003e gene were used for Q-PCR: pgsB726-f (5\u0026rsquo;-TGGCGCCATGAGA-ATCCT-3\u0026rsquo;), pgsB791-r (5\u0026rsquo;-GCAAAGCCGTTTACGAAATGA-3\u0026rsquo;) and pgsB-probe (5\u0026rsquo;-FAM-CCGCTGCTCAGCACGAAGGAGC-TAMRA-3\u0026rsquo;). Thermal cycling consisted of an initial denaturation at 95\u0026nbsp;\u0026deg;C for 30\u0026nbsp;s, followed by 40 cycles of denaturation at 95\u0026nbsp;\u0026deg;C for 5\u0026nbsp;s and annealing and extension at 60\u0026nbsp;\u0026deg;C for 34\u0026nbsp;s.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHigh-throughput sequencing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe heavy fractions (considered as the active species including ammonia oxidizers and their mostly closely linked species incorporating \u003csup\u003e13\u003c/sup\u003eC into their genomes during the DNA-SIP incubation) of Treatment 13C-B were sent for 16S rRNA sequencing of both bacteria and archaea. To construct a network to investigate the niches of AOA and AOB, we used universal primers for bacteria and archaea. The V3-V4 region of the bacterial 16S rRNA gene was amplified using the primers 341F (5'-CCTAYGGGRBGCASCAG \u0026minus;\u0026thinsp;3') and 806R (5'-GGACTACNNGGGTATCTAAT \u0026minus;\u0026thinsp;3')[\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e]. The V4 region of the archaeal 16S rRNA gene was amplified using the primers 524F10extF (5'-TGYCAGCCGCCGCGGTAA-3') and Arch958RmodR (5'-YCCGGCGTTGAVTCCAATT-3')[\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e]. Fragments from each soil sample were sequenced using MiSEq.\u0026nbsp;Following gene amplification, 3\u0026nbsp;\u0026micro;l of the PCR product was used for agarose gel electrophoresis (1%) to confirm the results of amplification. PCRs of 4 replicates for each sample preparation were combined and quantified with PicoGreen. From each sample, 200\u0026nbsp;ng of the PCR product was collected and pooled with other samples for one sequencing run. The pooled mixture was purified with a QIAquick Gel Extraction Kit (QIAGEN Sciences, Germantown, MD, USA) and re-quantified with PicoGreen. The purified mixture was then diluted and loaded as described in the MiSeq Reagent Kit Preparation Guide (Illumina, San Diego, CA, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConstruction of a microbial fuel cell (MFC) system\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA MFC system was constructed to assess possible extracellular electron transfer between heterotrophic BA and autotrophic AOM. Two-bottle MFC reactors (total volume of each bottle was 100\u0026nbsp;mL) were constructed as described by McAnulty in 2017[\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e]. The anode side was the soil used in this experiment, and the cathode side was subjected to 3 different treatments: BA, sterile BA as the negative control and \u003cem\u003eShewanella oneidensis\u003c/em\u003e MR-1 as the positive control. The MFC systems were incubated at 30\u0026nbsp;\u0026deg;C in the dark for 14 days.\u003c/p\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n\u003cp\u003e\u003cstrong\u003eData analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter assigning each sequence to the appropriate sample according to its barcode and allowing up to two mismatches, a total of 30,000 reads from both ends were obtained as a partitioned run for each sample. All the OTUs belonging to ammonia-oxidizers were picked up from the bacterial and archaeal OTU table after classification. Network analysis was performed and visualized using Cytoscape (Version 3.72). All statistical analyses were carried out in the R platform (3.6.2, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.r-project.org\u003c/span\u003e\u003c/span\u003e). The Shapiro\u0026ndash;Wilk test and Bartlett test were used to check whether the data conformed to normality and homoscedasticity, respectively. The post hoc Tukey HSD test was used to check if the relationship between two sets of data was statistically significant.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n\u003cp\u003e\u003cstrong\u003eInhibition of nitrification by BA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe influence of BA on nitrification was assessed by measuring differences in NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e, NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e, CO\u003csub\u003e2\u003c/sub\u003e, NO and N\u003csub\u003e2\u003c/sub\u003eO concentrations during incubation of microcosms (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). The increases in NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e, NO and N\u003csub\u003e2\u003c/sub\u003eO concentrations in Treatments 12C-B and 13C-B were significantly less than those in Treatment 13C-BS. The nitrate concentration in Treatments 13C-B and 12C-B was significantly (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) lower than that in 13C-BS after incubation for 28 days. The nitric oxide concentration in 13C-B and 12C-B was significantly (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) higher than that in 13C-BS. The N\u003csub\u003e2\u003c/sub\u003eO concentration in 13C-B and 12C-B was significantly (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) higher than that in 13C-BS. In contrast, the CO\u003csub\u003e2\u003c/sub\u003e concentration decreased after inoculation of microcosms with active BA. The CO\u003csub\u003e2\u003c/sub\u003e concentration in 13C-B and 12C-B was significantly (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) higher than that in 13C-BS. These factors all lead to the potential inhibition of the nitrification process by BA. However, NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e in Treatments 13C-B and 12C-B was also less than that in Treatment 13C-BS. The ammonium concentration in Treatments 13C-B and 12C-B was significantly (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) lower than that in Treatment 13C-BS.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDNA-SIP\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv id=\"Sec13\" class=\"Section3\"\u003e\n\u003cp\u003eTo identify which ammonia oxidizers incorporated \u003csup\u003e13\u003c/sup\u003eCO\u003csub\u003e2\u003c/sub\u003e during incubation, both archaeal and bacterial \u003cem\u003eamoA\u003c/em\u003e genes were quantified in DNA from different fractions (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ea and b). The quantitative abundance of AOB was greatest around a buoyant density of ~\u0026thinsp;1.71\u0026ndash;1.72\u0026nbsp;g ml \u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in Treatment 12C-B and ~\u0026thinsp;1.73\u0026ndash;1.75\u0026nbsp;g ml \u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in Treatments 13C-B and 13C-BS. In contrast, the archaeal abundance was greatest at ~\u0026thinsp;1.71\u0026ndash;1.72\u0026nbsp;g ml \u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in 12C-B and 13C-BS and ~\u0026thinsp;1.73\u0026ndash;1.75\u0026nbsp;g ml \u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in 13C-B. This indicates that bacterial \u003cem\u003eamoA\u003c/em\u003e was labelled in Treatment 13C-BS, while in Treatment 13C-B and 12C-B, both ammonia-oxidizing archaea and bacteria were labelled after incubation for 28 days.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSequencing of 16S rRNA genes in the heavy fractions indicated that neither the AOA nor AOB were the most abundant species in the \u003csup\u003e13\u003c/sup\u003eC-labelled 16S rRNA-based communities. Within Thaumarchaeota, 93.6% of the \u003csup\u003e13\u003c/sup\u003eC-labelled AOA community fell within the Soil Crenarchaeotic Group and 1.5% within South African Gold Mine Gp 1. Within the bacteria, 80% of the AOB and NOB sequences fell within the \u003cem\u003eNitrospira\u003c/em\u003e group, while few traditional AOB, e.g., \u003cem\u003eNitrosococcus\u003c/em\u003e, or NOB, e.g., \u003cem\u003eNitrobacter\u003c/em\u003e, were observed in labelled DNA (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ec and d).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eActivity of comammox after stimulation by BA\u003c/strong\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n\u003cp\u003eGiven the relatively high proportion of \u003cem\u003eNitrospira\u003c/em\u003e 16S rRNA sequences in \u003csup\u003e13\u003c/sup\u003eC-labelled DNA, specific \u003cem\u003eamo\u003c/em\u003eA primers for comammox were quantified in DNA from heavy fractions. This analysis indicated that comammox, especially \u003cem\u003eNitrospira\u003c/em\u003e clade A, was active in our BA-induced systems (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). The relative abundance of \u003cem\u003eNitrospira\u003c/em\u003e clade A was greatest around a buoyant density of ~\u0026thinsp;1.71\u0026ndash;1.72\u0026nbsp;g ml \u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in Treatments 12C-B and 13C-BS and at ~\u0026thinsp;1.73\u0026ndash;1.75\u0026nbsp;g ml \u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in Treatment 13C-B. Moreover, we amplified clade B at first but did not obtain any positive results.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNetwork of the microcosms\u003c/strong\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n\u003cp\u003eIn order to elucidate the niche of BA after inoculation into the microcosms, we constructed a network of microorganisms, including archaea and bacteria (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). The network showed that both nitrifiers and BA were not heavily connected to other organisms because of their small size in the whole community. However, BA and Thaumarchaeota were linked indirectly by \u003cem\u003eMicrobacteriaceae\u003c/em\u003e, and the edge indicated positive relations between both BA-\u003cem\u003eMicrobacteriaceae\u003c/em\u003e and \u003cem\u003eMicrobacteriaceae\u003c/em\u003e-Thaumarchaeota.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAOA in the MFC system\u003c/strong\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n\u003cp\u003eTo further investigate whether AOA provides positive feedback to electron transfer from the outside, we constructed a MFC system, and the abundance of AOA was quantified by Q-PCR. The abundance of AOA in the presence of BA was significantly higher than that in Treatment SBA (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e), but it was not significantly higher between \u003cem\u003eShewanella\u003c/em\u003e and SBA. The abundance of \u003cem\u003eamoA\u003c/em\u003e in Treatment C was 2.62\u0026thinsp;\u0026times;\u0026thinsp;10\u003csup\u003e5\u003c/sup\u003e\u0026nbsp;g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e dry weight soil, which was much lower than that in the other three treatments with inoculation of both active and sterile microbes. The abundance of AOA in Treatment BA was 6.65\u0026thinsp;\u0026times;\u0026thinsp;10\u003csup\u003e5\u003c/sup\u003e\u0026nbsp;g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e dry weight soil, which was significantly higher than that in the presence of \u003cem\u003eShewanella\u003c/em\u003e and Sterile BA (6.08\u0026thinsp;\u0026times;\u0026thinsp;10\u003csup\u003e5\u003c/sup\u003e (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and 5.71\u0026thinsp;\u0026times;\u0026thinsp;10\u003csup\u003e5\u003c/sup\u003e (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), respectively).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study suggests that the three autotrophic AOM, AOA, AOB and comammox bacteria \u003cem\u003eNitrospira\u003c/em\u003e clade A, were active in ammonia oxidation. The system involved a heterotrophic bacterium (HB), BA, which suggested a special relation between the HB and autotrophic AOM in the soil to be confirmed in the future. This provides compelling evidence that the regulation of the extraneous HB on the nitrification process involved the enhancement of oxidation of NH\u003csub\u003e2\u003c/sub\u003eOH to NO through AOA and \u003cem\u003eNitrospira\u003c/em\u003e clade A and the inhibition of the production of N\u003csub\u003e2\u003c/sub\u003eO through AOB, leading to a significant increase in NO and mitigation of N\u003csub\u003e2\u003c/sub\u003eO in the nitrification process. It also provides evidence that the potential regulating strategy is extracellular electron transfer between HB and AOM, especially AOA.\u003c/p\u003e\n\u003cp\u003ePrevious studies have suggested a complex relation between heterotrophic bacteria and autotrophic AOM, including competition for NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e and cooperation where organic carbon is low[\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e]. There are different ways microbes interact, e.g., cross-feeding, co-metabolism, cell-to-cell communication[\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e] and interspecies extracellular electron transfer (IEET)[\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e]. In our constructed microcosms, we found the co-activity of AOA, AOB and the newly discovered comammox in the ammonia-oxidizing process. After BA was inoculated into the microcosms, it successfully resuscitated the inactive ammonia-oxidizing archaea and complete nitrifier \u003cem\u003eNitrospira\u003c/em\u003e in the soil, which was previously dominated by ammonia-oxidizing bacteria[\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eNitrification is a two-step process:[\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e] ammonia (NH\u003csub\u003e3\u003c/sub\u003e) is oxidized via hydroxylamine (NH\u003csub\u003e2\u003c/sub\u003eOH) to nitrite (NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e) and, subsequently, nitrite is oxidized to nitrate (NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e). Within these two steps, two processes contribute to N\u003csub\u003e2\u003c/sub\u003eO emissions. The first is aerobic N\u003csub\u003e2\u003c/sub\u003eO formation from the abiotic reaction of the intermediates NH\u003csub\u003e2\u003c/sub\u003eOH, NO and NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e, termed chemodenitrification. NH\u003csub\u003e3\u003c/sub\u003e is first oxidized to NH\u003csub\u003e2\u003c/sub\u003eOH by the enzyme ammonia monooxygenase (AMO), which belongs to the superfamily of copper-dependent membrane-bound monooxygenases[\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e]. Subsequently, NH\u003csub\u003e2\u003c/sub\u003eOH is oxidized to NO by hydroxylamine dehydrogenase (HAO) under oxic and anoxic conditions[\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e]. NH\u003csub\u003e2\u003c/sub\u003eOH released in this process may be oxidized to N\u003csub\u003e2\u003c/sub\u003eO by oxidants, including Fe\u003csup\u003e3+\u003c/sup\u003e and MnO\u003csub\u003e2\u003c/sub\u003e, while NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e and NO may be reduced by reductants such as Fe\u003csup\u003e2+\u003c/sup\u003e, Cu\u003csup\u003e2+\u003c/sup\u003e or humic substances[\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e]. The second is nitrifier-denitrification, an enzymatic process in which NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e is reduced to N\u003csub\u003e2\u003c/sub\u003eO via NO; reduction of NO to N\u003csub\u003e2\u003c/sub\u003eO is catalysed by two classes of cytochrome \u003cem\u003ec\u003c/em\u003e nitric oxide reductases (NORs). Chemodenitrification is associated with ammonia oxidation by ammonia-oxidizing bacteria (AOB), ammonia-oxidizing archaea (AOA) and comammox, while nitrifier denitrification has only been reported in AOB[\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e], and all genome-sequenced AOA and comammox lack NOR[\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e]. The inoculation of BA and the change in AOM status \u003cem\u003ein situ\u003c/em\u003e obviously changed the rate of nitrification. The consumption of CO\u003csub\u003e2\u003c/sub\u003e and production of N\u003csub\u003e2\u003c/sub\u003eO and NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e were lower in Treatment 13C-B compared to Treatment 13C-BS, indicating that BA inhibited the nitrification process[\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e43\u003c/span\u003e]. However, NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e remained in the soil, and the higher emissions of NO in Treatment 13C-B after inoculation did not support the inhibition of nitrification. The concentrations of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e in Treatment 13C-B were less than those in Treatment 13C-BS, and the concentration of NO was slightly higher in 13C-B, which led to an enhancement of the ammonia-oxidizing process. The analysis of ammonia-oxidizing microorganisms in this system helped us to resolve this problem. Our soil used in this work was agricultural soils collected from a typical acidic soil area in China, in which AOB play a dominant role, as previous works have suggested[\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e44\u003c/span\u003e]. The results of SIP in Treatment 13C-BS confirmed the dominance of AOB in our experimental soils. However, the results also suggested that both AOA and \u003cem\u003eNitrospira\u003c/em\u003e clade A become other active organisms that oxidize NH\u003csub\u003e3\u003c/sub\u003e to NH\u003csub\u003e2\u003c/sub\u003eOH in Treatment 13C-B. This indicated that the initially inactive and feeble AOA in agricultural soils was resuscitated by the inoculation of BA and began to be active in function with AOB \u003cem\u003ein situ\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eThe labelled AOA in Treatment 13C-B mainly belong to Soil Crenarchaeotic Group, which indicates their dominant function in the ammonia-oxidizing process. After that, the AOB in Treatment 13C-B were sequenced, and we found that only a few AOB were classified, including \u003cem\u003eNitrosospira\u003c/em\u003e and \u003cem\u003eNitrosococcus\u003c/em\u003e. We wondered if any other species participated in this process, especially the newly isolated comammox. Interestingly, the results of Q-PCR of comammox showed that \u003cem\u003eNitrospira\u003c/em\u003e clade A was labelled in the heavy fractions. Taken together, after the inoculation of BA into the acidic soils, the whole ammonia-oxidizing process was redefined; instead of the sole activity of AOB as usual, both AOA and comammox were more active and made use of NH\u003csub\u003e3\u003c/sub\u003e.\u003c/p\u003e\n\u003cp\u003eThe results further revealed the abnormal increase in the production of NO in Treatment 13C-B. As we have mentioned before, NO is an important intermediate in the nitrification process both in biotic and abiotic pathways[\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e45\u003c/span\u003e]. However, in almost all the AOA and comammox genomes sequenced to date, no canonical nitric oxide reductases (NORs) have been detected, despite the wide presence of a nitrite reductase gene (\u003cem\u003enirK\u003c/em\u003e) in AOA. Although cytochrome P450 and other enzymes possibly involved in the production of N\u003csub\u003e2\u003c/sub\u003eO and acting as NOR may be detected in the future, it can only take action in some AOA in the presence of excess nitrite[\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e46\u003c/span\u003e]. After the inoculation of BA and the recovery of AOA and comammox in the soil, more NO was produced in the microcosms (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ea).\u003c/p\u003e\n\u003cp\u003eThe results and relations between the inoculated BA and ammonia-oxidizing microorganisms indicate a complex competence and cooperation among these species. BA is a widely isolated heterotrophic bacterium that holds a good NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e affinity in oligotrophic environments, and the inoculation of BA consumes NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e in soil, which makes it more competitive for nitrification by AOM[\u003cspan class=\"CitationRef\"\u003e47\u003c/span\u003e]. This in turn provides opportunities for AOA and comammox, whose affinity for ammonium is better than AOB[\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e48\u003c/span\u003e], and makes the co-function of AOM in soils possible[\u003cspan class=\"CitationRef\"\u003e49\u003c/span\u003e]. In addition, the inoculation of BA into the soils changed the original niche \u003cem\u003ein situ\u003c/em\u003e, and new relations between BA and the ammonia-oxidizers were constructed. The network showed that BA and the AOA Thaumarchaeota are at the edge of the network, and they are indirectly and positively connected by a \u003cem\u003eMicrobacteriaceae\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eBoth BA and \u003cem\u003eMicrobacteriaceae\u003c/em\u003e contain genes encoding multiheme cytochrome \u003cem\u003ec\u003c/em\u003e, which is good for the transport of electrons and facilitates ammonia oxidation, especially AOA[\u003cspan class=\"CitationRef\"\u003e50\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e52\u003c/span\u003e]. To date, no \u003cem\u003ecytochrome c\u003c/em\u003e has been found in AOA compared to its wide expression in AOB and comammox[\u003cspan class=\"CitationRef\"\u003e53\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e55\u003c/span\u003e]. Previous studies have confirmed the influence of IEET on anaerobic methane oxidizing microbes[\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e], which is similar to ammonia-oxidizing microbes. Therefore, we constructed a MFC system to investigate the potential IEET between BA and AOA. Although the MFC systems were carried out for only 14 days, the abundance of AOA in Treatment BA was significantly higher than that in the control. This indicated that the electrons transferred from BA were as high as the typical \u003cem\u003eShewanella\u003c/em\u003e at the cathode side, and the electrons may play a positive role in the abundance of AOA in the soil. Of course, this is only preliminary evidence for the IEET between these two species, but the difference between the 3 treatments did provide evidence that AOA reply positively to the electron transferred from other organisms.\u003c/p\u003e\n\u003cp\u003eAltogether, this suggests that BA is regulated by all autotrophic AOM in acidic soils via the competence of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e (competence resource) and the facilitation of electron transport (energy supplying), a hypothesis that warrants further experimental elucidation (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eb).\u003c/p\u003e"},{"header":"Conclusions","content":" \u003cp\u003eThis is a novel discovery of the resuscitation of AOA and newly found complete nitrifiers under the originally dominant AOB in the ammonia-oxidizing process. The results were detected based on the complex interactions between microbes. In our work, after the inoculation of BA, the whole nitrification in soil was changed, the ammonia-oxidizing process was inhibited; in addition, the emission of N\u003csub\u003e2\u003c/sub\u003eO was much lower, and the production of NO increased owing to the lack of NOR in AOA and comammox. We provide a possible explanation for the special relationship between BA and AOM; both the competence of ammonium with AOB and the facilitation of electron transport with AOA may have led to these results. However, far more research needs to be performed, not only to testify and confirm the relations and electron transfer between these microorganisms but also to determine if some other microbes are involved in this process. Additionally, the mechanisms by which BA regulates the ammonia-oxidizing process at metabolic levels should be elucidated.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSequences data of 16S rRNA high-throughput sequencing are available in NCBI Sequence Read Archive repository database (www.ncbi.nlm.nih.gov/sra) under accession number SRR12587975 to SRR12588174.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by the National Natural Science Foundation of China (Nos. 41907273, 31670507 and 91951108).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eS.W. conducted the experiments with the help of Y.D. and F.H. S.W. and Y.D. analyzed the data with the help of Y.D.. S.W. wrote the manuscript. S.W. and X.Z. initiated the project and directed the research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank Professor James I. Prosser for his general help in revising the manuscript and provide strains for further study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAffiliations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eKey Laboratory of Environmental Biotechnology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eShanghua Wu, Yuzhu Dong, Haonan Fan, Ye Deng \u0026amp; Xuliang Zhuang\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCollege of Resources and Environment, University of Chinese Academy of Sciences, Beijing 100049, China\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eShanghua Wu, Yuzhu Dong, Haonan Fan, Ye Deng \u0026amp; Xuliang Zhuang\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eKits KD, Jung MY, Vierheilig J, Pjevac P, Sedlacek CJ, Liu S, et al. 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Chem Rev. 2006;106(1):90-115; doi: 10.1021/cr050241v.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"ammonia-oxidizing bacteria and archaea, complete nitrifiers, Bacillus amyloliquefaciens, extracellular electron transfer, nitrous oxide, nitric oxide","lastPublishedDoi":"10.21203/rs.3.rs-76082/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-76082/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e Nitrification in the soil is dominated by ammonia-oxidizing archaea (AOA), ammonia-oxidizing bacteria (AOB) and the newly discovered complete nitrifiers, which are members of complex communities. Complicated relations have been found in microbial communities, but the relations between heterotrophic bacteria and autotrophic ammonia oxidizers (AOM) are unclear, and experimental models are lacking. Here, we constructed a microcosm based on DNA stable isotope probing that was established between a heterotrophic bacterium, \u003cem\u003eBacillus amyloliquefaciens\u003c/em\u003e (BA), and the soil autotrophic AOM. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eThe inoculation of BA changed the activities of three indigenous nitrifiers. The originally marginal AOA and complete nitrifiers were active in the nitrification process as well as the AOB, which led to mitigation of N\u003csub\u003e2\u003c/sub\u003eO and an increase in NO. The network analysis showed that the inoculated BA was indirectly and positively linked to AOA through a \u003cem\u003eMicrobacteriaceae\u003c/em\u003e that contains genes encoding proteins responsible for transporting electrons. Further, the microbial fuel cell system indirectly confirmed the potential regulation of extracellular electron transfer (EET) between these two species. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003eWith our findings, a useful model to investigate the relations between heterotrophic bacteria and AOM was constructed, and evidence of EET in complex regulations was provided.\u003c/p\u003e","manuscriptTitle":"Resuscitation of inactive ammonia-oxidizing archaea and complete nitrifiers by extracellular electrons from a heterotrophic bacterium, Bacillus amyloliquefaciens","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2020-09-15 21:02:25","doi":"10.21203/rs.3.rs-76082/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2471a366-ca8b-4e6a-a44d-bd48c54c1e8f","owner":[],"postedDate":"September 15th, 2020","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":506699,"name":"General Microbiology"}],"tags":[],"updatedAt":"2020-11-27T20:08:32+00:00","versionOfRecord":[],"versionCreatedAt":"2020-09-15 21:02:25","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-76082","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-76082","identity":"rs-76082","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}