Improving the enrichment setup

As the enrichment using glutamate as the carbon source did not keep the pH in the acidic treatments below the desired pH 6.1 during incubation, several tests were performed to find the cause and further improve the method. The threshold of pH 6.1 was based on previous research that demonstrated that N2OR lacks function below pH 6.1 and keeping pH stable below this threshold was therefore important (Bergaust et al., 2010; Liu et al., 2014).

To improve the enrichment setup, every step was examined in detail. It was hypothesized that the observed pH rise was caused by the glutamate additions. This is because reduction of glutamate produces ammonia (NH4+) and causes loss of the anionic glutamic acid.

4.2.1 Finding the reason for the rise in pH

First a test of how fumigated soil affected the pH was done. pH was measured frequently in fumigated and native K1 soil slurries over a period of 1.5 h. This was done aerobically at room temperature and with stirring at 600 rpm. More detailed pH measurements are shown in Table A6. pH did not rise drastically for the soils without any carbon additions, so inoculum of native soil into the fumigated soil were also tested. No rise in pH took place, but a reduction of 0.15 pH units was observed. To further examinate each step of the method, glutamate was given to the slurries. The results are shown in Figure 4.4.

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The results in Figure 4.4 showed no significant difference between the fumigated + natural and native K1 soil slurries, but a small rise in pH (~0.3 units) naturally for the soil without using a buffered medium. Addition of the glutamate stock caused pH to rise over 1 pH unit immediately after addition. The glutamate solution was not pH adjusted before use, most likely contributing to the rise. Measurements of pH after 48 h showed that the pH had increased to a value of over 8 for both fumigated and native soil. It was therefore concluded that the metabolism of glutamate and lack of pH adjustment was the reason for the observed pH rise.

4.2.2 Testing if phosphate buffer will keep pH stable

As an effort to buffer this pH rise, phosphate buffered (PB) medium was tested as a replacement for MilliQ water in the soil slurries. Three different PB concentrations were tested; 100 mM, 150 mM and 200 mM at a pH of 5.8 (acidic) and 7.4 (neutral). Native soil was used for these experiments and the slurries were incubated aerobically at room temperature with stirring (600

Figure 4. 4. pH measurements in aerobic soil slurries containing fumigated or live (native) K1 soil and MilliQ water before and after glutamate additions. Additions of 30mM 1 M Na-glutamate (pH 7) are indicated with a black vertical line. The box in the left corner shows pH immediately before and after glutamate was added. A indicates pH measured after glutamate was added and B indicates pH before addition of glutamate.

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rpm). The results are shown in Figure 4.5 and more detailed pH measurements are found in table A2, A3 and A4.

None of the buffer concentrations were high enough to keep pH below 6.1 for the acidic slurries (Figure 4.5). For the neutral slurries, pH continued to rise and only a concentration of 200 mM PB was enough to keep pH stable. The two highest buffer concentrations (150 mM, 200 mM) were also tested anaerobically since pH was expected to rise slower without oxygen present due to reduced metabolic activity. Only acidic slurries were tested under anaerobic conditions. The pH measurements are shown in Figure 4.6 and more detailed measurements are shown in table A5.

Figure 4. 5. pH measurements of acidic and neutral soil slurries using different phosphate buffer concentrations. Incubations were done aerobically and the slurries with 100 mM PB was incubated for 20 h while the 150 mM and 200 mM PB slurries was measured for 100 h. A threshold indicating the desired pH value is included. For acidic slurries this was at pH 6.1, while the neutral pH measurements had the threshold at 7.4.

Figure 4. 6. pH measurements for acidic soil slurries with buffer concentrations at 150 mM and 200 mM incubated anaerobically. A threshold was set at pH 6.1.

All vials stayed below the threshold for 100 h but was expected to increase further.

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While for this short testing period all slurries stayed below the threshold at 6.1, the experiment was expected to run for a longer period, and the pH was expected to continue to rise. It was therefore concluded that a buffered medium was likely not enough to reliably keep the pH stable when glutamate was the carbon source.

4.2.3 Exploring new carbon sources for the enrichment setup

As the pH did not remain acidic even with higher buffer concentrations, different carbon sources had to be explored. This was done using native K1 soil. Tryptic soy broth (TSB) and a hexose mixture (glucose, fructose and xylose) were selected as two possible options and tested on soil slurries that were incubated with 150 mM PB medium, at room temperature and with stirring at 600 rpm. The pH was measured for 40 h. The results are shown in Figure 4.7. and more details are found in table A6.

The findings in Figure 4.7 demonstrate that TSB caused the pH to rise above the threshold of the function of N2OR at pH 6.1, as well as the desired pH value for the neutral slurries at pH 7.4, while the hexose mixture managed to keep pH below the threshold levels for both neutral and acidic pH. Based on this, hexose mix was chosen as the carbon source for further enrichments.

Taken together, the experiments showed that pH increased when the cultures consumed glutamate and that the inclusion of phosphate buffer was not enough to keep pH below 6.1.

Figure 4. 7. pH measurements of native K1 soil slurries containing 150 mM PB and either TSB or hexose mixture as the carbon source. Thresholds at 6.1 for acidic slurries and 7.4 for neutral slurries are shown. Acidic slurries had an initial pH at 5.8, while neutral slurries started at pH 7.4.

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Thus, another carbon source had to be used. Testing different carbon sources lead to the decision that a mix of hexoses would be the best choice for keeping pH stable.