Development of zinc tolerance by the ammonia oxidising community is restricted to ammonia oxidising Bacteria, rather than Archaea

摘要

Both ammonia oxidizing Bacteria (AOB) and ammonia oxidizing Archaea (AOA) are abundant in soil, but, their relative contributions to soil nitrification are still debated and different studies have reported contrasting results. It is therefore still unclear under which soil conditions AOB or AOA dominate nitrification. Zinc contamination of soils has a pronounced effect on nitrification in soil, however previous studies have shown that this effect is transient due to the development of a tolerant nitrifying community. This study investigates the influence of long-term zinc (Zn) stress on the nitrifying community and determines the role of the Archaea and Bacteria in nitrification of soils exposed to Zn. To assess this, a grassland soil was artificially spiked with 1300 and 2400 mg Zn kg-1, as ZnCl2, and incubated outside, receiving each 4 months 150 mg NH4+-N kg-1. Nitrification was inhibited by Zn after incubation for 1 week and 2 months. However, after 12 months Zn exposure, nitrification in both Zn-contaminated treatments was restored to the initial nitrification rate of the uncontaminated soil. The soil ammonia oxidiser community in both Zn treatments developed larger Zn tolerance than the uncontaminated soil. Soil was sampled after 12 months incubation and the samples were subsequently subjected to Stable Isotope Probing (SIP) of DNA by incubating 20 g of pre-incubated soil under 13CO2 atmosphere at 30 °C for 24 days in gas-tight sealed bottles. Ammonia was supplied at days 0, 8 and 16 (100 mg N kg-1 soil) following injection of 6 ml of 13CO2 or 12CO2, in triplicate. Bottles were opened twice weekly to ensure aerobic conditions, resealed and CO2 concentrations were reestablished. Soil was sampled at day 24, total DNA was extracted from soil and DNA extracts were subjected to isopycnic centrifugation in a CsCl gradient (Beckman Coulter ultracentrifuge, rotor VTI 65.2, 24h, 186000 g) separating 13C-labelled DNA from 12C labelled DNA into different fractions. Only active populations, i.e. populations that autotrophically consumed CO2, incorporate 13C into their DNA. The abundance of ammonia oxidizing Bacteria and Archaea was measured in each fraction by qPCR of amoA genes, using primers amoA-1F and amoA-2R to target AOB and primers Crenamo23F and Crenamo616R to target AOA. 13C was assimilated by AOB in the uncontaminated soil, as indicated by a higher relative abundance of AOB in heavier fractions than in 12C-incubated samples. This clear shift was not observed for the AOA. After Zn exposure for 12 months a similar pattern was observed in both Zn treatments. These results show that, although ammonia oxidising Archaea dominate the number of nitrifying microorganisms in the uncontaminated soil, ammonia oxidizing Bacteria are responsible for soil nitrification. Stress conditions, arising from chronic zinc exposure, increased the relative abundance of AOB to AOA and did not change the predominance of AOB in soil nitrification activity. These results indicate that the recovery of nitrification after long-term Zn exposure and development of Zn tolerance in this soil is due to the activity of AOB, rather than AOA.