Microbial studies on nitrification from permafrost environments…

Tina Sanders, Claudia Fiencke and Eva-Maria Pfeiffer Introduction

Nitrogen as well as carbon cycling in arctic ecosystems is dominated by physi-cal and biogeochemiphysi-cal controls which are unique to the generally cold-dominated environment. Drastic seasonal fluctuations in temperature, a short growing season, cold soil temperature and the occurrence of permafrost are some of the obvious physical controls on nitrogen cycling and biological activity.

Most of the nitrogen accumulates in the organic substance in response to low soil temperatures, excessive soil moisture and low soil oxygen concentration (Gersper et al., 1980, Marion and Black, 1987; Nadelhoffer et al., 1991, Schimel et al., 1996). Standing crop in tundra vegetation store about 2 times more nitro-gen than temperate grasslands (Van Cleve and Alexander, 1981) but through the low N-mineralisation rates and lack of N-input by N-fixation and N-pollution the soils are nitrogen deficient and rely to a large extent on internal recycling (McCown, 1978).

N-cycling in the soil is crucial for growth of plants and microorganisms. Imbal-ances in N-cycling due to nitrate leaching, nitrogen oxide release and increase the methane emission (Adamsen and King, 1993; Carini et al., 2003). Most of the N-transformations were catalyzed by microorganisms.

Nitrification, the microbiological oxidation of ammonia to nitrate via nitrite, occu-pies a central position within the terrestrial nitrogen cycle. Aerobic chemolitho-autotrophic ammonia and nitrite oxidizing bacteria (AOB and NOB) represent the most important group of nitrifying bacteria (Fiencke et al., 2005). As a result of nitrate and acid formation, the nitrification process has various direct and indi-rect implications for soil systems. It increases the loss of soil nitrogen due to leaching of nitrate and volatilization of nitrogen gases directly or by denitrifica-tion. As a result the nitrogen supply to plants is influenced.

Recently, it has been detected that beside bacteria (AOB) also archaea (AOA) participate in the process of ammonia oxidation as they have been found in dif-ferent soils and habitats (Nicol and Schleper, 2006; Leininger et al, 2006). One representative specimen of the AOA was cultivated in enrichment culture (Kön-neke et al., 2005). In some habitats more archaea than bacteria genes were detected (Leininger et al., 2006). At this moment it is not definitely clear which group of microorganisms take the decisive role in the N-cycle.

Generally, nitrifying bacteria are found in the upper layer of soils, especially the rhizosphere where organic matter is mineralized, and ammonia and oxygen are present. But the slow growth rates and difficulties in recovering pure cultures have hampered cultivation-dependent approaches to investigate the number, community composition and dynamics of nitrifiers in soil. The number and

turn-over rate is therefore determined by traditionally methods like most-probable-number (MPN) technique and activity tests.

Material and Methods

Field investigations on nitrification were carried out on Samoylov in July and August 2007. Soil samples were taken from two polygons, at the polygon rim and polygon center, at 3 depths (0-5, 5-15, 15-25 cm) (Figure 3.3-1 A and B).

From the fresh samples potential ammonia oxidizing activities were measured at about 4 to 6°C for 32 days by ISO DIN 15685. This ISO standard is normally used for activated sludge and soils in moderate climates. So a modified method was developed which takes the conditions in permafrost soils under considera-tion. For the ISO-method 25 g of fresh soil sample in 100 ml medium with 0, 75 mM ammonia sulfate as substrate is used. In the adapted method less soil sample (5 g) is used and both ammonia and nitrite as substrate are applied.

Therefore in the modified activity tests beside the potential ammonia oxidation, the second step of nitrification, the nitrite oxidation was measured.

Figure 3.3-1: A: Soil sample polygon rim; B: Soil sample polygon center

The microbial DNA of the fresh polygon samples was isolated by PowerSoil DNA Isolation kit and was transported to University of Hamburg. The DNA iso-lated in Siberia will be compared to the DNA isoiso-lated after transportation. In this DNA mixture screens for bacteria, archaea and ammonia oxidizing organisms will be carried out. The screening will be performed by fingerprint analysis dena-turing gradient gel electrophoresis (DGGE) with probes against their key en-zymes and 16S rRNA. Furthermore it is planned to adapt the method of real time PCR to quantify the gene copies of ammonia oxidizing bacteria and ar-chaea.

After transportation of frozen and unfrozen soil samples, measurement of cell numbers of nitrifiers by the most probably number test (MPN) and further activ-ity tests at different temperatures are planned. Enrichment and isolation of the ammonia oxidizing bacteria and archaea will be carried out under low tempera-tures and by use of small concentrations of substrate. To enrich archaea, growth of bacteria will be inhibited by addition of the antibiotic streptomycin.

27 cm

27 cm

A B

The main aim is to understand how nitrification takes place in Permafrost soils of the arctic tundra and to clarify if ammonia oxidizers of the domain Bacteria or Archaea dominate.

Preliminary results

In fresh soil samples of the polygonal tundra, potential ammonia oxidation was measured by ISO DIN 15685-test. During the test period (32 days) ammonia oxidation could only detected in soil samples from the polygon rim (5-15 cm) (Figure 3.3-2). The other soil samples still offered no or very little activities.

Polygon rim; 5 - 15 cm

0,00 50,00 100,00 150,00 200,00

0 5 10 15 20 25 30 35 Time [d]

Nitrite [µM]

ISO DIN 15865

Figure 3.3-2: Potential ammonia oxidation activity in soil samples of the polygon rim at 5 – 15 cm measured by test ISO DIN test. This means an activity of 50.1 ng N-nitrite/g dw*h.

Polygon rim, 5-15 cm

0 200 400 600 800

0 10 20 30 time [d]

Nitrite [µM]

NOB

Figure 3.3-3: Potential nitrite oxidation activity in soil samples of the polygon rim at 5 – 15 cm measured by the modified method. This means an activity of 147.9 ng N-nitrite/g dw*h.

In the modified approaches no ammonia oxidation but nitrite oxidation activities were detected. In the samples of the polygon rim (0–5 cm and 5–15 cm) and polygon center (0–5 cm) a decrease of nitrite was measured. The complete

consumption of nitrite can be shown in the sample polygon rim (5 – 15 cm) (Figure 3.3-3).

The activity tests will be repeated with transported soil samples, the method modified once more and the test adapted as far as possible to situ conditions.

In previous tests on samples from the Lena Expedition 2005 potential ammonia oxidizing activity was shown in all depths of polygon rim and center (Sanders, 2006).

3.4 Morphology and properties of recent gelisols and