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 NH2OH to NO through AOA and Nitrospira clade A and the inhibition of the production of N2O through AOB, leading to a significant increase in NO and mitigation of N2O 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 NH4+ 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 (NH3) is oxidized via hydroxylamine (NH2OH) to nitrite (NO2−) and, subsequently, nitrite is oxidized to nitrate (NO3−). Within these two steps, two processes contribute to N2O emissions. The first is aerobic N2O formation from the abiotic reaction of the intermediates NH2OH, NO and NO2−, termed chemodenitrification. NH3 is first oxidized to NH2OH by the enzyme ammonia monooxygenase (AMO), which belongs to the superfamily of copper-dependent membrane-bound monooxygenases[35]. Subsequently, NH2OH is oxidized to NO by hydroxylamine dehydrogenase (HAO) under oxic and anoxic conditions[1, 4, 7]. NH2OH released in this process may be oxidized to N2O by oxidants, including Fe3+ and MnO2, while NO2− and NO may be reduced by reductants such as Fe2+, Cu2+ or humic substances[36]. The second is nitrifier-denitrification, an enzymatic process in which NO2− is reduced to N2O via NO; reduction of NO to N2O 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 CO2 and production of N2O and NO3− were lower in Treatment 13C-B compared to Treatment 13C-BS, indicating that BA inhibited the nitrification process[40–43]. However, NH4+ 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 NH4+ 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 NH3 to NH2OH 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 NH3.
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 N2O 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. 6a).
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 NH4+ affinity in oligotrophic environments, and the inoculation of BA consumes NH4+ 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 NH4+ (competence resource) and the facilitation of electron transport (energy supplying), a hypothesis that warrants further experimental elucidation (Fig. 6b).