## 3 Results and Discussion
### Optimization of CoPc Immobilization Conditions  
For real applications, the optimum CoPc immobilization content on the BC substrate is desired to enhance its catalytic reaction efficiency without increasing the cost. The immobilized CoPc content directly depends on the amount of attached spacer; hence, the reaction condition for spacer attachment should be carefully monitored prior to CoPc immobilization. The attachment levels of different spacers on the oxidized BC substrate as a function of reaction time is illustrated in Figure 4. The attached amount of all spacers increased almost directly proportionally with time, from 0 to 4 h, suggesting that the spacer attachment rate was initially constant. It is interesting that there appears to be a direct correlation between the attachment level and the spacer \begin{table}
\begin{tabular}{c c c c c} \hline  & **C (mol \%)** & **O (mol \%)** & **N (mol \%)** & **Co (mol \%)** \\ \hline BC & 63.13 & 36.87 & - & - \\ Oxidized BC & 65.51 & 34.49 & - & - \\ TP-attached BC & 63.55 & 29.61 & 6.84 & - \\ Glutaraldehyde-activated BC & 64.53 & 28.56 & 6.91 & - \\ CoPc@TP-BC & 72.44 & 7.54 & 19 & 1.01 \\ \hline \end{tabular}
\end{table} 
Table 1: Chemical compositions of BC during the functionalization processes. Figure 3: XPS spectra of (a): bacterial cellulose (BC); (b): oxidized BC; (c): tetraethylenepentamine (TP)-attached BC; (d): glutaraldehyde-activated BC and (e): CoPc@TP-BC. The window included shows, in detail, the Co region of CoPc@TP-BC. length, with longer spacers having lower amounts of spacer attached. The difference in attachment level increased with time. After reacting for 10 h, the attached amounts of TT, TP, and PH spacers were ~2.2 mmol/g, ~2 mmol/g and ~1.7 mmol/g, which were ca. 10%, 15% and 30% lower than that of the DT spacer (~2.4 mmol/g), respectively. Further prolongation of the reaction time did not consistently increase the attached spacer content on oxidized BC. Considering both the attachment level and the attachment efficiency, the optimum attachment time was set to 10 h.  
Our previous work illustrated that under optimum oxidation conditions, oxidized BC contains 14.13% (wt./wt.) of aldehyde groups (ca. 5 mmol/g, slightly lower than the theoretical maximum content of 18.1% (wt./wt.)) [25], while the amount of attached spacer was lower than 2.5 mmol/g; that is, less than half of the aldehyde groups were effectively utilized for the attachment of spacers. The relatively low-efficiency usage of aldehyde groups can be ascribed to two reasons. The first reason is the difficulty of producing heterogeneous reactions and the incompleteness characteristic of macromolecular reactions. The second reason may be attributed, at least partly, to the steric hindrance effect; that is, when the amount of attached spacer increased to a certain level, the already fixed spacers prevented further progress of the attachment reaction. It is reasonable to assume that the most obvious steric hindrance effect arises from PH, i.e., the longest spacer. Given the above factors, the attachment levels for all of the spacers were much smaller than the theoretical value, and the PH spacer had the lowest attached content. The spacer-attached BC substrates were further activated with glutaraldehyde, a classic bifunctional chemical crosslinker, for the immobilization of the CoPc catalyst. The immobilized CoPc contents were affected by many factors, such as the contents of the attached spacers and the activation levels. Figure 5a demonstrates the influence of initial glutaraldehyde concentration on the immobilized CoPc content for DT-attached BC. The immobilized CoPc content increased in association with the increase of glutaraldehyde concentration. It is reasonable to assume that with sufficient activation reagents, more binding sites were provided for CoPc immobilization. Meanwhile, the amount of DT spacer decreased drastically, which statistically represents the successfully activation of glutaraldehyde for CoPc immobilization.