29
Effect of Cultivation on BNC Biomaterials
nitrogen. concentration. (Cardinal. and. Hedrick. 1947). and. can. be. used. as.
a. nitrogen. source. in. culture. media. for. microorganisms.. Different. CSL.
batches.have.been.shown.to.differ.in.total.nitrogen.and.lactic.acid.. content.
(Noro.et.al..2004),.which.can.have.a.great.impact.on.the.production.of.BNC..
According.to.Noro.et.al.,.40.mL.CSL/L.growth.media.is.the.optimal.concentration.for.optimal.buffering.capacity..With.40.mL/L.CSL,.the.pH.was.
shown. to. stay. constant. at. 4.5. during. the. whole. cultivation. time,. which.
also.seemed.to.be.optimal.for.BNC.production.(Noro.et.al..2004)..This.is.in.
accordance.with.Verschuren.et.al..(2000).showing.that.the.optimal.pH.for.
BNC.production.is.between.pH.4.and.5..Moreover,.Verschuren.et.al..used.
three.different.batches.of.CSL.in.their.study.and.concluded.that.pH.was.
the.factor.that.affected.cellulose.production,.since.the.amount.of.nitrogen,.
lactic.acid,.and.fructose,.respectively,.were.set.to.the.same.concentrations.
in.all.three.CSL.batches.
Genetic Modifications of G. xylinus for
Enhanced Production of Bacterial Cellulose
Genetic.modification.of.G. xylinus.may.be.a.way.to.increase.BNC.production..
Environmental.factors.such.as.nutrient.limitations.and.other.factors.such.as.
stirring.during.cultivation.and.oxygenation.can.induce.spontaneous.mutations.of.the.bacteria,.which.thereby.may.lose.the.ability.to.produce.cellulose.
Transfection of G. xylinus with the pBla-VHb-122 Plasmid
negative.and.obligate.aerobe.bacterium.that.lives.in.a.microaerobic.environment..During.evolution.these.bacteria.developed.a.way.to.utilize.oxygen.more.
efficiently..This.occurs.via.the.expression.of.bacterial.hemoglobin,.whose.primary.function.is.to.enhance.respiration.in.the.bacteria..Vitreoscilla.hemoglobin.
(VHb).enhances.respiration.by.capturing.oxygen.and.facilitates.the.transfer.
of.oxygen.to.the.terminal.oxidase.(Tsai.et.al..1996)..The.gene,.vgb,.encoding.
the.hemoglobin.in.Vitreoscilla.was.isolated,.sequenced,.and.characterized.in.
1988.by.Dikshit.and.Webster,.and.has.successfully.been.expressed.in.different.
. heterologous.hosts,.such.as.Escherichia coli.(Chien.et.al..2006)..The.expression.
of.the.gene.in E. coli.enhances.growth.and.product.formation..The.plasmid.
has.successfully.been.amplified.in.E. coli.and.introduced.into.G. xylinus.by.
electroporation.(Setyawati.et.al..2007)..Chien.et.al..(2006).. demonstrated.that.
the. expression. of. VHb. enhances. BNC. production. in. G. xylinus.. G. xylinus.
expressing.VHb.(VHb+).have.a.higher.specific.growth.rate.under.both.. aerobic.
and.anaerobic.conditions..The.VHb+.bacteria.exhibited.no.obvious.lag.phase.