## 3 Experimental
### Bacterial Activity
#### 3.3.1 Sterilisation versus Pre-Oxidation  
To follow up on the hypothesis that microbial activity might be responsible for the mechanistic effects seen, i.e., NO\({}_{2}\)\(\rightarrow\) NO with no N retention in the filter transitioning to the desired NO\({}_{2}\) sorption, two sets of samples were prepared using either heat sterilisation at 110 \({}^{\circ}\)C in an oven or saturation with 0.1 w/w\({}^{\circ}\)H\({}_{2}\)O\({}_{2}\), which pre-oxidises any oxidisable chemical groups in the sample. The possibility that the NO\({}_{2}\)\(\rightarrow\)NO reaction may have been light (photon) catalysed was also considered. Each sample was then tested in a smaller filter box for convenience under both light and dark conditions: volume of 3 dm\({}^{3}\) filled with 33 pin-coated sheet samples (11 sticks with 3 sample sheets each) with a total filter mass (m) of approximately 60 g with an area exposure (A) of 0.54 m\({}^{2}\).  
Results showed that heat sterilisation only partly removed the RedOx effect, albeit noticeably. This was expected because heat sterilisation, though it killed bacteria and stopped regeneration of the effect, only partly oxidised the sample. In contrast, the H\({}_{2}\)O\({}_{2}\) treated sample completely lost the RedOx effect due to pre-oxidation. Removing light had no effect, such that light catalysis could be eliminated. The experimental trials and respective results are shown in Figure 24.  
Protein extraction showed that the thermally sterilised samples displayed protein content, which is a good indicator that bacterial cell wall breakdown fragments were present as a result of sterilisation. The absence of protein in the non-sterilised samples indicated that the presence of integral bacteria could not be ruled out.  
To confirm the bacterial and species content, samples of coated and uncoated paper were analysed with a special focus on denitrifying bacteria. Because high levels of bacteria were detected in the samples, cellulose breakdown bacteria were likely present (well-known contamination) and were analysed. An enrichment of denitrifying bacteria was then carried out separately.  
Figure 23: (**a**) Regeneration of the NO\({}_{2}\)\(\rightarrow\) NO effect between exposures while background NO level in the incoming air is constant, (**b**) long time repeated exposure (as shown Friday 5 th July–Tuesday 16 th July 2019) saturates the RedOx reaction.  
For the quantification of aerobic bacteria, 1 g of paper sample was mixed with 9 cm\({}^{3}\) of disruption buffer including tris and sodium lauroyl sarcosinate. Dilutions were spread on tryptic soy agar (TSA). After incubation for 2 d at 30 \({}^{\circ}\)C, the colony forming units per cm\({}^{3}\) sample (cfu-cm\({}^{-3}\)) were counted.  
For the enrichment of denitrifying bacteria, pieces of the papers (2 \(\times\) 2 cm\({}^{2}\)) were incubated in 12 cm\({}^{3}\) denitrification medium (3 g beef extract, 5 g peptone, and 1 g KNO\({}_{3}\)) for 3 days under anoxic conditions. For the samples of mineral GCC only and MNFC, 0.2 g and 0.4 g of the materials were used, respectively. Quantofix\({}^{\circledR}\) (Sigma Aldrich, St. Louis, MS, USA) peroxide test sticks were used to detect nitrate/nitrite. Dilutions of the enrichment cultures were spread on denitrification agar. After 3 days of incubation under anoxic conditions, colonies were selected for further analysis using a Bruker MS Biotyper (Bruker, Billerica, MA, USA). Conversion of nitrate to nitrite was detected in most of the enriched samples, which indicated the presence of denitrifying bacteria in the original samples. In some cases, species potentially capable of nitrate reduction were identified. The findings of the microbial analysis are summarised in Table 10.  
Figure 24: _Cont._