Results and Discussion(CNC)
In this page, we will show all of our experimental results about CNCs and evaluate the experimental situation. By doing this, we attempt to find a direction to improve our research in the future.
Preparation of CNCs
According to the quenching effect from 10-fold cold DI water, we got the raw product solutions (Figure 1). With abundant raw cellulose fibers existing, the solutions are a little turbid.
Figure 1 raw CNC solutions
Then, after centrifugation, we prepared the CNC Suspension. We used red laser pointer to irradiate DI water and the CNC Suspension, respectively, and only the CNCs forms the Tyndall effect, which proved the existence of CNCs conveniently. (Figure 2)
Figure 2 CNC suspension
Freeze-drying to get final product
After overnight freeze-drying process, we got the final product CNCs in the end. (Figure 3)
Figure 3 Solid CNCs
Thermal Gravimetric Analyzer (TGA)
TGA was carried out to observe the thermal characteristics of the CNCs (Figure 4). Evaporation of water led to the first stage of gradual weight loss. The onset temperature which CNCs began to degrade was around 223 ℃. The most obvious weight loss occurred at 393 ℃ while the literature value is 313 ℃[1], indicating the high thermal stability of CNCs we made.
Figure 4 TGA analysis of CNCs
TEM and SEM observation
The pure CNC will crystallize in aqueous solutions and thus forming a square shape (Figure 5), which can be a standard to recognize whether bacteria are embedded in the CNC.
Figure 5 TEM images of CNCs
In the Figure 6a, 6b taken under TEM, it’s obvious that the fibers of CNC are attached to the surface of E.coli, which reveals that the CNCs have successfully wrapped E.coli. Meanwhile the profile of CNCs has been displayed in Figure 6c, its sphere is extremely smooth while that of CNCs with E.coli is relatively rough. The red arrow of Figure 6d clearly indicates the location of E.coli.
Figure 6 TEM and SEM observation with E.coli
In same process, we observed the embedding situation of Streptomycete as well. In the Figure 7b, different from the pure Streptomycete which has smooth fibers (Figure 7a), the embedding in CNCs results in Streptomycete’s surface having abundant granular substance (CNCs). On the other hand, the size of Streptomycete colony were extremely expanded after the embedding in CNCs (Figure 7c, 7d), which further revealed the success of embedding.
Figure 7 TEM and SEM observation with Streptomycete
The images of Figure 8 show the growth of the CNC fibers. During the freeze-drying process, water infiltrated into the CNCs microspheres and formed multiple hydrogen bonds with CNCs, which caused the formation of mischcrystal under low temperature and had a structure of three-dimensional network. Water sublimated during freeze-drying so the porous CNCs skeleton was left. Therefore, we observed that the fibers formed by CNCs became more and more coiled while more and more slimy matters were adhered to the surface of the fibers with the increasing amount of bacteria. These proved the bacteria were embedded into the CNC fibers and had an indirect influence on the features of CNC fibers.
Figure 8 CNC fibers with E.coli
Dynamic Light Scattering (DLS)
The Figure 9 reveals the embedding situation of E.coli with CNCs. E represents the pure E.coli. 4 h-CNC-E represent the microsphere of E.coli with CNCs. CNC is the microsphere with E.coli which has been stored in 4 ℃ for 20 days, and we guess the E.coli are dead and the CNC shell collapsed inward.
Figure 9 Dynamic Light Scattering of E, CNC and 4 h-CNC-E
The Figure 9 indicates that 4 h-CNC-E occurs obviously self-assembly in general. The average particle sizes of each kind of compound are shown on the Table 1.
Through simple subtraction, we can get the thickness of CNC on the surface of E.coli:
Thickness = (1513.8 – 1317.1)/2 = 0.9835 nm
Reference
1 Zhou, J. et al. Synthesis of multifunctional cellulose nanocrystals for lectin recognition and bacterial imaging. Biomacromolecules 16, 1426-1432, doi:10.1021/acs.biomac.5b00227 (2015).
