THE EFFECT OF TEMPERATURE AND NH 3 CONCENTRATION ON NH 3 OXIDISING ARCHAEA AND BACTERIA IN AGRICULTURAL SOIL

Marcus O. Bello. Microbiology Department, AdekunleAjasin University AkungbaAkoko, Ondo State, PMB 001, Nigeria. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History Received: 12 August 2018 Final Accepted: 14 September 2018 Published: October 2018

The aim of this study was to study the effect of temperature and NH 4 + concentration on NH 4 + oxidising activity and community structures of bacterial and archaeal NH 4 + oxidisers in Scottish soil. Microcosms were amended with low and high NH 4 + concentration and incubated at five different temperatures (15,20,25,30 and 35 o C) for 28 days. Nitrification activity, AOA and AOB growth increased with temperature and AOB growth were greatest at 30 o C in microcosms incubated with high NH 4 + concentration, while AOA growth was detected, irrespective of soil NH 4 + concentration and at all incubation temperature except 35 o C. AOB growth was detected at 35 o C even in microcosms incubated with low NH 4 + concentration. There was a greater change in the community structure of AOA and AOB with an increase in temperature only. This probably suggests that NH 4 + oxidation was dominated by AOA at low concentration between 15 -30 o C and at 35 o C AOB were able to grow when the competition with AOA for NH 4 + in soil was relieved by high temperature (35 o C) that inhibited AOA growth. The differences in AOA and AOB responses to temperature could suggest an additional niche differentiating factor between AOA and AOB. Temperature but not NH 4 + is one of the key environmental factors responsible for changes in the relative abundance and community structure of AOA and AOB in soil.
Further evidence suggests that NH 3 concentration differentially affects AOA and AOB in soil, as high concentrations of NH 4 + from cow urine and inorganic sources (200 μg NH 4 + N g -1 soil) stimulate the growth and NH 3 oxidation activities of AOB but inhibit AOA, leading to decreases in AOA:AOB, while low NH 3 concentration stimulates the growth of AOA but not AOB (Prosser, 2011;Nicolet al., 2011). Also, cultivated AOA generally have higher affinity for NH 3  Temperature is one of the major ecological factors that affect both the activity and abundance of AOA and AOB in soil (Prosser, 2011). NH 3 oxidation has been measured at high temperatures environments (up to 87 o C) and at winter temperatures as low as 2 o C (Prosser, 2011). Nitrification activity has been documented also in arctic soils with low temperature from 4 to , suggests that different genus of AOA and AOB could grow in soil at different temperature. However, even though studies have shown that AOA and AOB community compositions change with increase in temperature in soil at low NH 3 concentration, the interacting effect of temperature and highNH 3 concentration on NH 3 oxidation activity, growth and community structure of AOA and AOB in soil have not been studied. It was therefore hypothesised that: (a) NH 3 oxidation activity of AOA and AOB will increase in soil with high NH 3 concentration than at low NH 3 concentration, (b) NH 3 oxidation activity of AOA and AOB will increase with an increase in temperature, irrespective of soil NH 3 concentration and (c) changes in temperature and NH 3 concentration will cause a change in relative abundance and community structure of AOA and AOB in soil.

Construction and incubation of microcosms
Microcosms were constructed using sandy loam soil of pH 7.5 from an agricultural plot at the phase of a key arable crop rotation. The moisture content and soil pH were determined as described by Nicolet al. (2005) before setting up of the experiment. The air-dried soil was sieved (3.35-mm mesh) and stored at 4 o C for 2 weeks before use. The physicochemical characteristics of the soil used for the experiment are described by Kempet al. (1992) and Bartram et al. (2014). Triplicate soil microcosms were established in 250-ml, sterile glass bottles containing 50 g equivalent dry soil. The initial moisture content was adjusted to 30% (g water g -1 dry soil) with sterile distilled water. Microcosm experiments were performed with NH 4 + concentration and temperature as factors. Microcosms were divided into two sets, with one set amended to 100 µg N g -1 soil with (NH 4 ) 2 SO 4 (referred to as high NH 4 + 998 concentration), and the other set without NH 4 + amendment (referred to as low NH 4 + concentration). Each microcosm was covered with a butyl rubber stoppers and tightened with metal crimp tops to prevent the loss of moisture.
Microcosms were incubated at five different temperatures (15,20,25,30 and 35 o C) for 4 weeks. A non-destructive soil sample (2 g of soil) was taken twice weekly, concomitantly with microcosm aeration, and was analysed to determine the NH 4 + /NH 3 and NO 2 -+NO 3 concentrations. Microcosms with depleted NH 4 + concentration were directly spiked with NH 4 + that was lost due to NH 3 oxidation to maintain a stable NH 4 + concentration. The microcosms with low NH 4 + concentration received an equal volume of sterile distilled water only. The moisture content increased from 30% to approximately 34% at the end of the incubation. For each sampling point, microcosms were immediately re-capped after aeration. Soil samples (1 gram) were also taken weekly and immediately stored at -80 o C for nucleic acid extraction.
Chemical analysis NH 4 + /NH 3 and NO 2 -+NO 3 concentrations were determined in soil solution extracted from 1 g soil by mixing soil with 5 ml of 1 M KCl solution and centrifuging at 3,000 rpm for 15 minutes. Concentrations were measured colorimetrically using 96-well plates as described by Catãoet al. (2016). The NO 3 production was linear and nitrification rate (µg N g -1 day -1 ) was estimated as the gradient of linear regression of NO 3 concentration vs. time during incubation. Minimum nitrification rate (i.e. nitrification rate in soil without added NH 4 + ) and maximum nitrification rate (i.e. nitrification rate in soil supplied with high NH 4 + concentration when supplied NH 4 + was not exhausted) were determined in microcosms incubated with low and high NH 4 + concentration, respectively.

Abundance and growth of archaeal and bacterial NH 3 oxidisers
DNA was extracted from 0.5 g of soil stored at -80 o C by physicochemical lysis with bead-beating and 5% CTAB buffer, protein was removed using phenol:chloroform:isoamyl alcohol (25:24:1), while phenol was removed using chloroform:isoamyl alcohol (

Analysis of the change in community structure of archaeal and bacterial NH 3 oxidisers
Denaturing gradient gel electrophoresis (DGGE) analysis using amoA was done to determine the AOA and AOB community structure, as described in Gubry-Ranginet al. (2017). DGGE gels contained 20% (w/v) polyacrylamide and linear gradient between 15 to 55% denaturant for both archaea and bacteria amoA assays. A 10 µl combination of PCR product of either AOA or AOB and loading dye was loaded per sample. DGGE was performed at 60 o C and 75 Volts for a minimum of 960 minutes before fixing in the solution (10% ethanol and 0.5% acetate) for a minimum of 30 minutes. This was followed by silver-staining with silver nitrate (0.25 g per 250 ml of distilled water) for 20 minutes. The gels were developed in the developing solution (6.0 g of NaOH, 3 ml of 37% formaldehyde in 200 ml of distilled water) and scanned using an Epson GT9600 scanner with transparency unit (Epson Ltd, Hemel Hempstead, UK).

Statistical analyses
Statistical analyses were performed using the program R 3.4.0 (http://www.rproject.org/). Data for nitrification rate, pH and AOA and AOB growth were analysed independently using two-way ANOVA (Statistical Procedures for Agricultural Research package (agricolae)) with temperature and NH 4 + concentration as fixed factors. Tukey HSD multiple post-hoc tests were used to assess the significance of the differences among the means.

Nitrification rates
The concentration of NH 4 + was below the detection limit (0.1 µg N-NH 4 + g -1 of dry soil) in microcosms with low NH 4 + concentration at all incubation temperatures and times (Fig. 1a - concentration increased from 0 to 34 µg N g -1 within 10 days but then steadily decreased below the detection limit during the remaining incubation days (Fig. 1e). In microcosms supplemented with high NH 4 + concentration, the concentration of NH 4 + decreased with time at all temperatures ( Fig. 1a -e). The rate of decrease in NH 4 + concentration increased with temperature from 15 to 30 o C, was highest at 25 o C in microcosms incubated with high NH 4 + concentration.Unsurprisingly, NO 3 production under high NH 4 + treatment was higher in microcosms with high NH 4 + concentration than with low NH 4 + concentration. NO 3 concentration increased with time in all treatments ( Fig. 1f-j) Figure 2) and were not significantly different from the rate obtained at 35 o C(p< 0.001) (Fig. 2). According to the changes in NO 3 concentration, nitrification rate was lower in microcosms not supplemented with NH 4 + than those supplemented with high NH 4 + concentration (Fig. 2).The initial pH in all microcosms was not significantly different, independent of adjustment of soil moisture content and amendment with NH 4 + . However, soil pH significantly decreased co-ordinately with the final NO 3 concentration during incubation (Fig. 3).  1002

AOA and AOB growth
The growth of AOB and AOA were determined by plotting the changes in amoA abundances over time (i.e., by subtracting the amoA abundance at day 0 from amoA abundance at day 28).AOA growth was detected irrespective of NH 4 + concentration from 15 to 30 o C (Fig. 4A) and there was no significant effect of NH 4 + concentration on AOA growth (p = 0.77). At 35 o C, there was no detectable growth of AOA irrespective of NH 4 + concentration. Only temperature had a significant effect on the growth of AOA after 28 days of incubation (p< 0.001). Growth of AOB was detected only in the treatment with high NH 4 + concentration (Fig. 4B).AOB growth increased significantly with temperature from 15 to 30 o C and was greatest at 30 o Cbut decreased at 35 o C after 28 days of incubation (Fig. 4B) (p < 0.001). AOB growth were better in microcosms incubated with high NH 4 + concentration compare to AOA, while AOB growth were not detectable in microcosms incubated with low NH 4 + concentration. Even though with low growth of AOB in microcosms incubated with high NH 4 + concentration, the growth of AOA were not detectable irrespective of NH 4 + concentration at 35 o C. The change in community structure of archaeal and bacterial NH 3 oxidisers DGGE was performed to determine the effect of temperature andNH 4 + concentration on community structure within AOA and AOB showed that intensity and number of detected AOA amoA bands increased from 15 to 30 o C. However, the intensity and the number of detectable bands was low at 35 o C (Fig. 5A). There was a greater change in the community structure of AOA at 20, 25  The temperature ranges for NH 3 oxidation activity by NH 3 oxidisers under low and high NH 4 + concentration differed, i.e., the optimum temperature for NH 3 oxidation activity by NH 3 oxidisers in microcosms incubated with low and high NH 4 + concentrationwas35 and 25 o C, respectively. This confirms that both NH 4 + concentration and temperature influence nitrification rate in soil. Also, there is an evidence of interaction between NH 4 + concentration and temperature on nitrification activity of NH 3 oxidisers in soil. The observed decrease in soil pH is a function of increase in the nitrification activity and increased soil acidity during incubation, as found previously by (Verhammeet al., 2011).  , 2011). Hence, the differences in AOA and AOB responses to temperature could suggest an additional niche differentiating factor between AOA and AOB. It is not the conclusion of the present study that AOA cannot grow in soil at temperatures higher than 30 o C. For instance, Ca. N. franklandus with an optimum growth temperature of 40 o C, have been isolated from soil with similar pH as those used in the present study (Lehtovirta-Morley еtаl., 2016). The tolerance of Ca. N. franklandus to high incubation temperature could have been selected for, by the different chemical compositions and lack of possible toxic metabolites from other organisms that are active against Ca. Nitrosocosmicus at temperature above 30 o C in culture medium compared, to the soil environment. Temperature but not NH 4 + concentration caused changes in community structures of AOA and AOB. These observations are supported by previous studies, which demonstrated that the relative abundance and community structure of AOA and AOB change with increase in temperature (Gubry-Ranginet al., 2017; Tourna et al., 2008).

Conclusion:-
It was demonstrated in the present studies that the NO 3 production is higher in soil that received high NH 4 + concentration (i.e., highly fertilised soil) compared to the soil without additional NH 4 + supply. High NO 3 production due to a high NH 4 + -based fertiliser used in commercial agricultural practice will result in increased leaching of NO 3 from soil to groundwater and surface water thereby causing water pollution. This also contribute to the decrease in anticipated crop production despite the yearly increase in NH 4 + -based fertiliser that is applied in commercial agriculture and account for nearly 70% loss of NH 4 + -based fertiliser applied in commercial agricultural soil. High nitrification activity in commercial agriculture practice leads to a decrease in soil pH, especially in soil with low buffering capacity due to NO 3 production will increase soil acidification. This will result to an increased requirement for liming by farmers to restore soil pH. The cost of soil liming will indirectly increase the cost of crop production.