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Biomimetic Mineralization of Apatite on Bacterial Cellulose
having. been. flash. frozen. in. liquid. nitrogen. (namely. native. BC). and. some.
were.used.for.TEMPO-mediated.oxidation.
Surface Modification by TeMPO-Mediated Oxidation (BC-TeMPO)
TEMPO-mediated. oxidation. was. performed. to. introduce. surface. carboxyl.
groups. at. the. C6. primary. hydroxyl. groups. of. the. cellulose. chains.. Briefly,.
the.never-dried.BC.pellicles.(dry.weight.of.0.15.±.0.003.g).were.suspended.
in.distilled.water.(80.ml).containing.TEMPO.(0.0075.g).and.sodium.bromide.
(0.075 g).under.continuous.stirring..An.appropriate.amount.of.sodium.hypochlorite.solution.corresponding.to.2.42.mmole/g.cellulose.was.added.slowly.
and.the.pH.of.the.reaction.was.maintained.at.10.5.by.addition.of.0.5.M.NaOH..
The.oxidation.was.carried.out.for.approximately.2.h,.when.no.more.NaOH.
consumption. was. observed.. Then,. 2. ml. of. ethanol. was. added. to. quench.
the. oxidation,. followed. by. the. addition. of. 0.1N. HCl. to. adjust. the. reaction.
mixture.to.reach.neutral.pH..The.oxidized.BC.pellicles.were.washed.with.
a.large.amount.of.distilled.water.to.remove.residual.reagents.and.subjected.
to.freeze-drying,.as.mentioned.above.(namely.BC-TEMPO)..The.carboxylate.
content.measured.by.conductimetric.titration.was.0.25.mmol/g.cellulose.
Some.of.the.TEMPO-oxidized.BC.pellicles.recovered.after.washing.with.
distilled. water. were. immersed. in. 0.1. M. calcium. chloride. (CaCl 2 ). solution.
with.stirring.at.room.temperature.for.6.h.to.exchange.the.sodium.counterion.
of.the.carboxylate.groups.to.calcium..The.ion-exchanged.TEMPO-oxidized.
BC.pellicles.were.subjected.to.freeze-drying.(BC-TEMPO-Ca).after.washing.
with. distilled. water.. The. amount. of. calcium. ions. in. BC-TEMPO-Ca. measured. by. X-ray. fluorescence. analysis. (MESA-500,. Horiba. Co.,. Japan). was.
0.22.mmol/g.cellulose..With.a.carboxylate.content.of.0.25.mmol/g.cellulose,.
the. ion. exchange. ratio. was. approximately. 1:1.. Therefore. the. formation. of.
BC-COOCa + .was.predominant.over.(BC-COO) 2 Ca.(Saito.and.Isogai.2005).
Microbial Synthesis of BC from Modified Culture Medium (BC-glcNAc)
The.standard.culture.medium.was.modified.by.the.addition.of.amino.sugar.
N-acetylglucosamine.(GlcNAc).as.a.source.of.sugar.together.with.glucose.in.
different.compositions.(Table 11.1).(Nge.et.al..2010a)..As.shown.in.Figure 11.1,.
adaptation. of. Acetobacter. cells. to. the. presence. of. the. amino. sugar. was. performed. by. five. repeated. transfers. of. 3-day-old. inoculum. into. the. medium.
containing. different. GlcNAc. concentrations.. The. 3-day-old. inoculum. was.
selected.based.on.cell.density.estimated.by.measuring.the.optical.density.at.
600.nm..The.volume.ratio.of.inoculum.to.medium.was.1:10.for.all.compositions.
and.the.static.culture.fermentation.was.carried.out.as.described.previously.
The.effect.of.sugar.composition.on.the.rate.of.cellulose.production.is.shown.
in.Figure 11.2a..The.cellulose.dried.mass.produced.in.the.presence.of.glucose.
(AJ2000).increased.over.14.days.and.reached.6.41.mg/ml,.with.an.exponential. mass. production. from. days. 3–7. followed. by. a. slower. production. rate.