International Scholarly Rearch Network
ISRN Microbiology
Volume2012,Article ID140951,4pages柳李商隐
doi:10.5402/2012/140951
Rearch Article
Pullulan Production by Aureobasidium pullulans ATCC201253 Cells Adsorbed onto Cellulo Anion and Cation Exchangers
Thomas P.West
Department of Biology and Microbiology,South Dakota State University,Brookings,SD57007,USA
Correspondence should be addresd to Thomas P.West,thomas.west@sdstate.edu
拜火教Received13July2012;Accepted30August2012
Academic Editors:I.Berber and G.Blaiotta
Copyright©2012Thomas P.West.This is an open access article distributed under the Creative Commons Attribution Licen, which permits unrestricted u,distribution,and reproduction in any medium,provided the original work is properly cited.
The anion exchanger phosphocellulo and the cation exchanger triethylaminoethyl cellulo were ud to immobilize cells of the fungus Aureobasidium pullulans ATCC201253and the adsorbed cells were subquently investigated for their ability to produce the polysaccharide pullulan using batch fermentation.The cells adsorbed on the triethylaminoethyl cellulo at pH7.5produced higher pullulan levels than tho cells immobilized on phosphocellulo at pH4.0for2cycles of168h at30◦C.Relative to the initial cycle of168h,pullulan production by the cells immobilized on the triethylaminoethyl cellulo decread slightly after 168h of the cond production cycle while pullulan production by the phosphocellulo-immobilized cells remained about the same after168h of the cond production cycle.
1.Introduction
Pullulan is an extracellular polysaccharide synthesized by the fungus Aureobasidium pullulans utilizing an excess of carbon with limiting nitrogen prent[1–4].The polysaccharide consists of prima
rily cross-linked maltotrio although a small proportion of maltotetrao residues has been detected in its structure[5–9].Due to its high water solubility and low viscosity,pullulan has numerous commercial applications including its u as a food additive,aflocculant,a blood plasma substitute,an adhesive,and afilm[8,10,11].
Immobilization of A.pullulans cells by adsorption onto solid supports has been examined in previous studies.The advantage of using immobilized cells is that they can be ud for more than a single cycle of polysaccharide production. When A.pullulans ATCC42023cells were immobilized by adsorption onto the solid support diatomaceous earth,the immobilized cells produced pullulan and were capable of being reutilized for a cond cycle of pullulan production[12,13].Similarly,A.pullulans ATCC42023 cells adsorbed onto sponge cubes were capable of veral cycles of polysaccharide production[14].Entrapped cells of A.pullulans can also be utilized to produce pullulan for at least two production cycles[15,16].A biofilm reactor using a plastic composite support found that pullulan production by immobilized A.pullulans ATCC201253cells was maximal at pH5.0after168h[17].Ion exchange resins, such as DEAE(diethylaminoethyl)cellulo and ECTEOLA (epichlorohydrin triethanolamine)cellulo,have been utilized to adsorb corn syrup-grown cells of A.pullulans ATCC42023,and the adsorbed cells produced pullulan for two production cycles[13,18,19].The cells
of the reduced pigmentation mutant strain A.pullulans ATCC201253have been adsorbed onto ion exchange resins and investigated relative to pullulan production[18,20].It was found that ATCC201253cells could be adsorbed at pH2.0onto ion exchangers to produce pullulan from corn syrup[18].
湛蓝天空In this study,pullulan production by corn syrup-grown cells of A.pullulans ATCC201253immobilized by adsorption onto the anion exchange resin phosphocellulo at pH4.0 or the cation exchange resin TEAE(triethylaminoethyl) cellulo at pH7.5using batch fermentation was compared. The effectiveness of utilizing the fungal cells adsorbed onto the ion exchangers for more than one cycle of batch pullulan production was also compared.
2.Materials and Methods
2.1.Strain and Media.Aureobasidium pullulans ATCC 201253was the strain ud in this work[20].The
composition of the culture medium was previously described [21].In the phosphate-buffered medium,corn syrup rved as the carbon source at afinal concentration of2.5%(w/v) while ammonium sulfate rved as the nitrogen source at a final concentration of0.06%(w/v).After inoculating batch cultures(50mL)with overnight cultures(0.5mL)grown in the same culture medium,ea
ch batch culture was shaken at 200rpm for48h at30◦C.
2.2.Cell Immobilization on Ion Exchangers.Phosphocellu-lo or TEAE cellulo(1g)was pretreated by quentially washing with200mL of0.25N HCl and0.25N NaOH in a sterile250mL Erlenmeyerflask.The resins were then resus-pended in0.5N HCl(200mL)overnight to sterilize them [22,23].Following washing the resins at least three times with sterile water(200mL),the water was removed from the resins and the resins could be utilized for cell adsorp-tion.Phosphocellulo was suspended in50mL of culture medium(pH4.0)containing2.5%(w/v)corn syrup while the TEAE cellulo was suspended in50mL of culture medium(pH7.5).After each suspension was inoculated with medium containing about105fungal cells/mL(from48h batch cultures),eachflask was shaken(100rpm)for48h at30◦C.Each ion exchange resin with the adsorbed fungal cells was collected by low-speed centrifugation and washed twice with0.85%NaCl.After each resin was suspended in 50mL of culture medium(pH6.0)containing2.5%(w/v) corn syrup,the immobilized cells in theflasks were shaken (125rpm)for168h at30◦C.Pullulan production by fungal cells immobilized at pH4.0on phosphocellulo or at pH7.5 on TEAE cellulo was monitored at24h intervals.Following thefirst cycle of168h,each cellulosic ion exchanger was col-lected by low-speed centrifugation,washed,and again sus-pended in culture medium(pH6.0)containing2.5%(w/v) corn syrup.The cultures were shak
en(125rpm)for a cond cycle of168h at30◦C during which the pullulan levels were measured at an interval of24h.
2.3.Assays and Statistical Analysis.To monitor polysaccha-ride production,culture medium(2mL)was removed and the sample was centrifuged(14,600g,30min,4◦C)with the supernatant being ud for the pullulan determinations. To one volume of the pullulan-containing supernatant,two volumes of95%ethanol were added to precipitate the poly-saccharide.The precipitated pullulan was collected on 0.45µm HVLPfilters(25mm diameter).Allfilters were dried to constant weight at105◦C and reweighed to deter-mine pullulan concentrations[21].The viable fungal cell concentration of the adsorbed cells on each ion exchange resin was determined as stated previously[18].Pullulan con-centrations were expresd as g/l and reprent the mean of three independent determinations.The Student’s t-test was ud during statistical analysis.
3.Results and Discussion
Culture medium pH has been shown to affect fungal cell adsorption onto the ion exchange resins DEAE cellulo, phosphocellulo,TEAE cellulo,and ECTEOLA学习美甲
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Fermentation time (h)
Figure1:Pullulan levels(g/l)produced over a period of168h during cycle1by Aureobasidium pullulans ATCC201253cells adsorbed on triethylaminoethyl cellulo( )or phosphocellulo ( ).Error bars indicate the standard deviations of mean data values.
[13,18,19].The culture medium pH ud to adsorb the A.pullulans ATCC201253cells onto the resins
was2.0 which was bad upon obrved pullulan production by the immobilized cells incubated in medium(pH6.0)contain-ing3%corn syrup[13,19].In this study,it was determined that a higher concentration of cells could be adsorbed when the culture medium pH was elevated.The concentration of ATCC201253cells adsorbed at pH4.0onto the phos-phocellulo was determined to be1.4×105±0.9×105 (mean±standard deviation)colony-forming units/mg dry weight of resin which is higher than the concentration of 3.2×104±0.1×104(mean±standard deviation)colony-forming units/mg dry weight of resin noted previously for adsorption onto phosphocellulo at pH2.0[18].Similarly, the concentration of ATCC201253cells adsorbed at pH7.5 onto the TEAE cellulo was found to be5.4×106±6.5×106 (mean±standard deviation)colony-forming units/mg dry weight of resin which is higher than the concentration of 1.2×105±0.1×105(mean±standard deviation)colony-forming units/mg dry weight of resin obrved previously for adsorption onto TEAE cellulo at pH2.0[18].With the concentration of adsorbed cells being incread by elevating the culture medium pH ud for cell adsorption,it was of interest to compare pullulan production by ATCC201253 cells adsorbed onto phosphocellulo at pH4.0and onto TEAE cellulo at pH7.5.
With respect to both ion exchange resins,the ability of the immobilized cells to produce pullulan when incubated in medium(pH6.0)containing3%corn syrup during the initial cycle of polysaccharide
production for168h using batch fermentation was investigated.As can be en in Figure1,the cells adsorbed on phosphocellulo produced pullulan after24h and continued to produce it for120h during the initial cycle of production.Pullulan production by the cells adsorbed on TEAE cellulo was noted after 24h and continued to be produced for144h during the initial cycle of production(Figure1).Pullulan production by the immobilized cells appeared to increa little after 144h for cells adsorbed on either ion exchanger(Figure1).
Fermentation time (h)
P u l l u l a n (g /L )
Figure 2:Pullulan levels (g/l)produced over a period of 168h during cycle 2by Aureobasidium pullulan
s ATCC 201253cells adsorbed on triethylaminoethyl cellulo ( )or phosphocellulo ( ).Error bars indicate the standard deviations of mean data values.
The pullulan levels produced during the initial cycle of 168h by the cells adsorbed onto TEAE cellulo were higher than the polysaccharide levels produced by the cells adsorbed onto phosphocellulo (Figure 1).The di fference in pullulan production after 96h,120h,144h,and 168h between the cells adsorbed onto phosphocellulo at pH 4.0and the cells adsorbed onto TEAE cellulo at pH 7.5was statistically significant (P <0.01).As can be en in Figure 2,pullulan levels produced during the cond cycle of 168h by the cells adsorbed onto TEAE cellulo were higher than the polysac-charide levels produced by the cells adsorbed onto phospho-cellulo which was also noted during the initial cycle of pro-duction.The di fference in pullulan production after 120h between the cells adsorbed onto phosphocellulo at pH 4.0and the cells adsorbed onto TEAE cellulo at pH 7.5was statistically significant (P <0.01).For the cells adsorbed on the phosphocellulo or TEAE cellulo,there was no statisti-cal di fference in pullulan production after 168h between the first and cond production cycle.It appeared that pullulan production by the immobilized cells on either ion exchanger was equally e ffective for two cycles of production for 168h.The pH 4.0-adsorbed ATCC 201253cells onto phosphocel-lulo ud in this study produced le
ss polysaccharide than the pH 2.0-adsorbed cells on phosphocellulo during the initial production cycle of 168h [18].In contrast,the pH 4.0-adsorbed ATCC 201253cells ud in this work produced higher polysaccharide levels on phosphocellulo than the pH 2.0-adsorbed cells during the cond production cycle of 168h [18].The pH 7.5-adsorbed ATCC 201253cells on TEAE cellulo ud in this study produced higher polysac-charide levels than the pH 2.0-adsorbed cells on phosphocel-lulo during both production cycles of 168h [18].
Prior studies have investigated the immobilization of A .pullulans ATCC 42023cells using the ion exchange resins DEAE cellulo or ECTEOLA cellulo and the ability of the immobilized cells to produce pullulan using batch fermentation [13,19].ATCC 42023cells were adsorbed onto DEAE cellulo or ECTEOLA cellulo at pH 2.0[13,19].Pullulan production by the ATCC 201253cells adsorbed
on TEAE cellulo ud in this study was more similar to production by the DEAE cellulo-adsorbed ATCC 42023cells than the ECTEOLA cellulo-adsorbed ATCC 42023cells since the cond cycle of production was lower than the initial production cycle [13,19].Although the findings from this study showed that the phosphocellulo-adsorbed cells produced less polysaccharide than ECTEOLA cellulo-adsorbed cells,pullulan production by the ECTEOLA cellu-lo-adsorbed cells w
as similar to pullulan production by the phosphocellulo adsorbed cells becau pullulan levels decread relatively little during the cond production cycle compared to the initial production cycle [19].
4.Conclusions
The cation exchanger TEAE cellulo appeared to be a more e ffective adsorption support to immobilize A .pullulans ATCC 201253cells when the cells were adsorbed at pH 7.5instead of pH 2.0.The anion exchanger phosphocellulo was less e ffective than TEAE cellulo for pullulan production by ATCC 201253cells independent of whether adsorption occurred at pH 2.0or 4.0.The ATCC 201253cells adsorbed at pH 4.0onto phosphocellulo were able to produce pullulan at comparable levels during both production cycles unlike the pH 2.0-adsorbed ATCC 201253cells where pullulan production diminished during the cond production cycle.Further study of A .pullulans cell immobilization using anion and cation exchangers for pullulan production will be necessary to identify the most e ffective ion exchanger that adsorbs high fungal cell concentrations and produces high pullulan levels for veral cycles.
Acknowledgments
This work was supported by funds from the South Dakota AES and from US Department of Agriculture Grant no.94-37501-0884and NIFA Grant no.2011-67010-20051.The technical assistance of Beth Nemmers was greatly appreci-ated.
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