49
Large-Scale Production of BNC
the. aeration. of. cultures. of. most. G. xylinus. strains. by. stirring. or. shaking.
gives. rise. to. spontaneous. non-cellulose-producing. mutants,. a. phenotype.
gains.an.advantage.when.oxygen.is.available.in.abundance.(Krystynowicz.
et.al..2002;.Ross.et.al..1991)..The.transformation.from.cellulose-producing.to.
non-cellulose-producing.bacteria.cells.was.also.reported.by.Park.et.al..(2004).
for.Gluconacetobacter hansenii PJK..They.reasoned.this.was.because.of.shear.
forces,.resulting.in.the.inhibition.of.BNC.formation.
Hestrin. and. Schramm. (1954). reported. and. described. in. detail. a. spontaneous. occurrence. of. Cel − . mutants. in. agitated. cultures.. They. isolated. morphologically.different.types.of.Gluconacetobacter.colonies,.showing.different.
abilities.to.synthesize.cellulose..They.were.mentioned.by.Yamada.(2000).as.
Cel + ,. which. are. fully. able. to. synthesize. cellulose. (round,. gelatinous,. and.
markedly.convex.colonies),.whereas.Cel − .describes.non-cellulose-producing.
mutants.(generally.flat.and.dull.colonies).with.or.without.the.ability.to.revert.
to.Cel + .forms.
The.optimal.culture.conditions.for.effective.BNC.production.were.investigated. in. detail. by. Krystynowicz. et. al.. (2002).. They. argued. that. Cel + . colonies.dominate.under.stationary.culture.conditions,.not.allowing.for.uniform.
medium.aeration,.and.explained.the.phenomenon.by.Gluconacetobacter.cell.
behavior.upon.culturing.under.different.conditions..In.static.conditions,.the.
cellulose-synthesizing.cells.move.toward.the.oxygen-rich.medium–air.interface,. where. they. form. a. BNC. membrane. that. limits. access. of. oxygen. into.
the.lower.parts.of.the.culture..In.agitated.cultures,.cell.growth.is.preferred.
instead. of. polymer. synthesis. due. to. the. uniform. aeration,. which. results.
in. Cel − . domination. in. the. culture.. In. order. to. prove. this. hypothesis,. they.
compared.BNC.production.under.stationary.culture.conditions.and.under.
intensive. agitation. and. aeration.. They. found. out. that. “the. major. obstacle.
encountered.in.agitated.and.highly.aerated.cultures.of.G. xylinus is.the.tendency. of. cellulose. producing. strains. to. revert. to. non-cellulose. producing.
mutants.(Cel − .mutants),.which.contributes.to.a.decline.in.BNC.production.”
Moreover,.Krystynowicz.et.al..(2002).examined.the.influence.of.different.
medium.compositions.on.the.occurrence.of.G. xylinus E25.mutants.both.in.
stationary.and.agitated.culture.conditions..In.experiments.with.stationary.
cultures,.the.type.of.carbon.source.and.presence.or.absence.of.ethanol.had.
no.significant.impact.on.Cel − .mutant.occurrence..In.contrast,.in.experiments.
with. agitated. cultures,. Cel − . mutants. were. observed. immediately. after. the.
first.culture.transfer,.and.their.numbers.increased.continuously.during.subsequent.transfers,.reaching.a.maximum.value.of.80%.after.the.third.transfer..
Because.of.this,.only.a.few.G. xylinus.strains.have.been.found.to.be.suitable.
for.agitated.cultivation.of.BNC.(e.g.,.ATCC.53582;.Czaja.et.al..2004b;.Toyosaki.
et.al..1995)..In.this.context,.it.was.also.discussed.by.Jung.et.al..(2005).as.well.
as.Park.et.al..(2003).that.the.addition.of.ethanol.to.the.culture.medium.may.
prevent.the.transformation.process.
In. contrast. to. this,. Ben-Bassat. et. al.. (1992). determined. that. agitated. cultures. exhibit. substantially. reduced. byproducts. such. as. gluconic. and.
Large-Scale Production of BNC
the. aeration. of. cultures. of. most. G. xylinus. strains. by. stirring. or. shaking.
gives. rise. to. spontaneous. non-cellulose-producing. mutants,. a. phenotype.
gains.an.advantage.when.oxygen.is.available.in.abundance.(Krystynowicz.
et.al..2002;.Ross.et.al..1991)..The.transformation.from.cellulose-producing.to.
non-cellulose-producing.bacteria.cells.was.also.reported.by.Park.et.al..(2004).
for.Gluconacetobacter hansenii PJK..They.reasoned.this.was.because.of.shear.
forces,.resulting.in.the.inhibition.of.BNC.formation.
Hestrin. and. Schramm. (1954). reported. and. described. in. detail. a. spontaneous. occurrence. of. Cel − . mutants. in. agitated. cultures.. They. isolated. morphologically.different.types.of.Gluconacetobacter.colonies,.showing.different.
abilities.to.synthesize.cellulose..They.were.mentioned.by.Yamada.(2000).as.
Cel + ,. which. are. fully. able. to. synthesize. cellulose. (round,. gelatinous,. and.
markedly.convex.colonies),.whereas.Cel − .describes.non-cellulose-producing.
mutants.(generally.flat.and.dull.colonies).with.or.without.the.ability.to.revert.
to.Cel + .forms.
The.optimal.culture.conditions.for.effective.BNC.production.were.investigated. in. detail. by. Krystynowicz. et. al.. (2002).. They. argued. that. Cel + . colonies.dominate.under.stationary.culture.conditions,.not.allowing.for.uniform.
medium.aeration,.and.explained.the.phenomenon.by.Gluconacetobacter.cell.
behavior.upon.culturing.under.different.conditions..In.static.conditions,.the.
cellulose-synthesizing.cells.move.toward.the.oxygen-rich.medium–air.interface,. where. they. form. a. BNC. membrane. that. limits. access. of. oxygen. into.
the.lower.parts.of.the.culture..In.agitated.cultures,.cell.growth.is.preferred.
instead. of. polymer. synthesis. due. to. the. uniform. aeration,. which. results.
in. Cel − . domination. in. the. culture.. In. order. to. prove. this. hypothesis,. they.
compared.BNC.production.under.stationary.culture.conditions.and.under.
intensive. agitation. and. aeration.. They. found. out. that. “the. major. obstacle.
encountered.in.agitated.and.highly.aerated.cultures.of.G. xylinus is.the.tendency. of. cellulose. producing. strains. to. revert. to. non-cellulose. producing.
mutants.(Cel − .mutants),.which.contributes.to.a.decline.in.BNC.production.”
Moreover,.Krystynowicz.et.al..(2002).examined.the.influence.of.different.
medium.compositions.on.the.occurrence.of.G. xylinus E25.mutants.both.in.
stationary.and.agitated.culture.conditions..In.experiments.with.stationary.
cultures,.the.type.of.carbon.source.and.presence.or.absence.of.ethanol.had.
no.significant.impact.on.Cel − .mutant.occurrence..In.contrast,.in.experiments.
with. agitated. cultures,. Cel − . mutants. were. observed. immediately. after. the.
first.culture.transfer,.and.their.numbers.increased.continuously.during.subsequent.transfers,.reaching.a.maximum.value.of.80%.after.the.third.transfer..
Because.of.this,.only.a.few.G. xylinus.strains.have.been.found.to.be.suitable.
for.agitated.cultivation.of.BNC.(e.g.,.ATCC.53582;.Czaja.et.al..2004b;.Toyosaki.
et.al..1995)..In.this.context,.it.was.also.discussed.by.Jung.et.al..(2005).as.well.
as.Park.et.al..(2003).that.the.addition.of.ethanol.to.the.culture.medium.may.
prevent.the.transformation.process.
In. contrast. to. this,. Ben-Bassat. et. al.. (1992). determined. that. agitated. cultures. exhibit. substantially. reduced. byproducts. such. as. gluconic. and.