Controlling the water content of never dried and reswollen bacterial cellulose by the addition of water‐soluble polymers to the culture medium

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  Controlling the water content of never dried and reswollen bacterial cellulose by the addition of water‐soluble polymers to the culture medium
  Controlling the Water Content of Never Dried andReswollen Bacterial Cellulose by the Addition of Water-Soluble Polymers to the Culture Medium MARIT SEIFERT, 1 STEPHANIE HESSE, 2 VASKEN KABRELIAN, 3 DIETER KLEMM 1 1 Institut fu¨r Organische Chemie und Makromolekulare Chemie, Friedrich-Schiller-Universita¨t,Lessingstraße 8, 07743 Jena, Germany 2 Institut fu¨r Optik und Quantenelektronik, Friedrich-Schiller-Universita¨t, Max-Wien-Platz 1, 07743 Jena, Germany 3 Section of Chemistry, Faculty of Science, Teshrien University, Latakia, Syria  Received 27 January 2003; accepted 13 June 2003 ABSTRACT:  For the modification of medically useful biomaterials from bacterially syn-thesized cellulose, fleeces of   Acetobacter xylinum  have been produced in the presence of 0.5, 1.0, and 2.0% (m/v) carboxymethylcellulose (CMC), methylcellulose (MC), andpoly(vinyl alcohol) (PVA), respectively, in the Hestrin–Schramm culture medium. Theincorporation of the water-soluble polymers into cellulose and their influence on thestructure, crystal modifications, and material properties are described. With IR andsolid-state  13 C NMR spectroscopy of the fleeces, the presence of the cellulose ethers andan increase in the amorphous parts of the cellulose modifications (NMR results) havebeen detected. The incorporation is represented by a higher product yield, too. Asdemonstrated by scanning electron microscopy, a porelike cellulose network structureforms in the presence of CMC and MC. This modified structure increases the waterretention ability (expressed as the water content), the ion absorption capacity, and theremaining nitrogen-containing residues from the culture medium or bacteria cells. Thewater content of bacterial cellulose (BC) in the never dried state and the freeze-dried,reswollen state can be controlled by the CMC concentration in the culture solution. Thefreeze-dried, reswollen BC–CMC (2.0%) contains 96% water after centrifugation,whereas standard BC has only 73%. About 98% water is included in a BC–MC com-posite in the wet state, and about 93% is included in the reswollen state synthesized inthe presence of 0.5, 1.0, or 2.0% MC. These biomaterial composites can be stored in thedried state and reswollen before use, reaching a higher water absorption than pure,never dried BC. The copper ion capacity of BC–CMC composites increases proportion-ally with the added amount of CMC. BC–CMC (0.5%) can absorb 3 times more copperions than srcinal BC. In the case of 0.5 and 1.0% PVA additions to the culture solution,this polymer cannot be detected in the cellulose fleeces after they are washed. Never-theless the presence of PVA in the culture medium effects a decreased product yield, aretention of nitrogen-containing residues in the material during purification, a reducedwater absorption ability, and a slightly higher copper ion capacity in comparison with Correspondence to:  D. Klemm (E-mail: dieter.klemm@uni- Journal of Polymer Science: Part A: Polymer Chemistry, Vol. 42, 463–470, (2004) © 2003 Wiley Periodicals, Inc. 463  srcinal BC. The water content of freeze-dried, reswollen BC–PVA (0.5%) is only 62%.  © 2003 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 42: 463–470, 2004 Keywords:  bacterial cellulose;  Acetobacter xylinum ; composites; water-soluble poly-mers; biomaterials; carboxymethylcellulose; methylcellulose; poly(vinyl alcohol); NMR;scanning electron microscopy (SEM) INTRODUCTION The bacterium  Acetobacter xylinum  (AX) pro-duces cellulose extracellularly under aerobic con-ditions. In contrast to plant cellulose, bacterialcellulose (BC) is distinguished by high crystallin-ity and purity (free of other biogenic compoundsand molecular inhomogeneities by isolation pro-cedures), high mechanical strength in the wetstate, and high water absorption ability. 1,2 To thisday, AX has been the most important microorgan-ism for investigations of BC biosynthesis andcrystallization 3–6 and the moldability of the cel-lulose material. 7–10  A growing field of recent research and applica-tions of biomaterials based on BC is the develop-ment of artificial materials for implantation. 10 For this purpose, bacterially synthesized cellulose(BASYC) is designed tubularly directly during cultivation. These shaped products are applied ascovers in experimental micronerve surgery and,most importantly, as blood vessel interpositionswith an inner diameter of about 1 mm. A highwater content, a low degree of roughness of theinner surface, and a complete vitalization of BASYC in rat experiments have demonstratedthe enormous potential of the biomaterial formedical applications. 10 The formation and structure of BC can be con-trolled by the variation of the components of thenutrient medium and the cultivation conditions.Tajima et al. 11 cocultivated a cellulose producing an AX strain with another AX type producing awater-soluble polymer (WSP) extracellularly. A composite of BC with up to 30 wt % WSP wasbuilt. In most cases, WSPs such as carboxymeth-ylcellulose (CMC) or methylcellulose (MC) havebeen added to the culture medium. They are in-corporated into the cellulose material at concen-trations of up to 30 wt %. 12 The produced BC–CMC composites are 2 to 3 times more transparentfor visible light (500 nm) than pure BC, 12 and sim-ilar composites possess a higher specific absorptioncapacity for lead ions than BC and CMC. 13,14 Brown 15 reported an increased water absorptionability of BC–CMC and BC–MC. As described byTakaietal., 16 BC–CMCiscompletelydegradablebycellulases, whereas only 20–40% of BC–MC is de-graded. Elsewhere, the influence of WSPs added toa culture medium on the cellulose modifications I  and I   has been discussed. 17,18 For the modification of medically useful bioma-terials from BASYC, fleeces of AX have been pro-duced in the presence of 0, 0.5, 1.0, and 2.0% (m/v)CMC, MC, and poly(vinyl alcohol) (PVA), respec-tively, in the Hestrin–Schramm culture medium.The incorporation of WSPs into cellulose andtheir influence on the structure, crystal modifica-tions, and material properties are described. Theamount and purity of the fleeces have been mea-sured, and the composites have been character-ized by solid-state  13 C NMR and IR spectroscopy,scanning electron microscopy, the water content,and the copper ion capacity. EXPERIMENTAL Materials and Methods The nutrient medium, according to Hestrin andSchramm, 19 contained 0.60 g of   D -glucose, 0.10 g of disodium hydrogen phosphate, 0.03 g of citricacid, 0.15 g of yeast extract, and 0.15 g of bac-topeptone in 30 mL of distilled water. The AX ( Gluconoacetobacter xylinus ) strain was obtainedfrom the strain collection of the Institute of Bio-technology at Leipzig (Professor H.-P. Schmauder).This strain could produce high-molecular-weightcellulose with a degree of polymerization of 7000–8000from D -glucosewithayieldofabout40–50%. 10 The WSPs added to the medium were CMC sodiumsalt (Fluka) with a degree of substitution of 0.60–0.95atpH5.5–8.5(1%inwater),MC(AcrosOrgan-ics) with a viscosity of 12–18 cP (2% in water), andPVA (Merck-Schuchardt). AX was precultivated in 30 mL of the Schramm–Hestrin medium for 7 days at 28 °C, and 1 mL of this preculture solution was inoculated into 30-mLsamples of the Hestrin–Schramm medium plus 0,0.5, 1.0, or 2.0% (m/v) WSP. After 14 days of incubation at 28 °C understatic conditions, the samples were heated insteam at 121 °C, and the BC fleeces were har- 464  SEIFERT ET AL.   vested and prepared for further investigations.The general procedure consisted of washing in 50mL of distilled water three times, boiling in 20 mLof a sodium dodecyl sulfate solution for 20 min at80–90°C,washingin50mLofdistilledwaterthreetimes, boiling in 20 mL of a sodium dodecyl sulfatesolution for 20 min at 80–90 °C, washing in 50 mLof distilled water three times, and boiling in 50 mLof distilled water for 10 min at 80–90 °C two times.Never dried, highly swollen fleeces were usedfor the measurement of the water content. For theother analytical methods, the fleeces were freeze-dried. Analytical Investigations Elemental Analysis  The CHN elemental analyses were taken on aLeco CHNS-932. NMR Spectroscopy  For cross-polarity/magic-angle-spinning   13 C NMRwith two-pulse phase modulation (TPPM) decou-pling(4-mmrotor),thespectrawereobtainedwithaBruker400-MHzwide-borespectrometer(AMX400WB) under a static magnetic field of 9.4 T. Thesamples were rotated with a frequency of 6.5 kHz,the repetition time was 2 s, and the cross-polarity(CP) time was 1 ms. IR Spectroscopy  The spectra were obtained with a Nicolet/Impact400. The freeze-dried sample (1 mg) was mixedwith 200 mg of KBr. Scanning Electron Microscopy  The freeze-dried samples were cooled by nitrogenand broken, and the surface of the fracture wascoated with gold. The sectional area was in-spected by the registration of secondary electronswith a DSM 940A scanning electron microscope(Zeiss, Germany) with the DISS photorecording system (Point Electronic, Germany) and a picturesize of 1000 pixels    1000 pixels. Water Content  The water content of the never dried and freeze-dried samples was determined according to thewell-known procedure of Jayme and Rothamel. 20 Samples were stirred in 100 mL of distilled waterfor 2 h at 30 °C, centrifuged with G3 sinters at4000 rpm for 15 min ( m wet ), and dried at 105 °Cuntil a constant weight ( m dry ) was obtained. Foran easy demonstration of the water content of theBC and composites, the values were calculatedaccording to ( m wet    m dry )/  m wet    100. Copper Ion Capacity  The absorption capacity of the freeze-dried BCcomposites for copper ions was measured by EISUGmbH (Wolfen, Germany). The samples werestirred in a CuSO 4  solution of a defined coppercontent for 3 h. The copper part left in the solu-tion was determined by titration with ethyl-enediaminetetraacetic acid. The copper content(m/m) was calculated in relation to the mass of the freeze-dried samples. RESULTS AND DISCUSSION To develop biomaterials of the BASYC type for fur-ther medical applications, we designed compositesofthisBCwithCMC,MC,andPVA.Forsystematicinvestigations, BC fleeces were produced in culturemedia containing 0, 0.5, 1.0, or 2.0% (m/v) of theseWSPs. After cultivation, the samples were heatedin steam at 121 °C, washed carefully with waterand a sodium dodecyl sulfate solution, and usedneverdried(watercontentdetermination)orfreeze-dried (other analytical methods). The presence of the WSPs in the purified composites could be de-tectedbysolid-stateNMRandIRtechniques.More-over, the NMR analysis allowed the specification of cellulose crystal modifications (I   /I  ) in relation tothe amorphous parts. To obtain information on thenetwork structure of BASYC-type composites, weinspected the cross sections of freeze-dried fleeceswith a scanning electron microscope. In the pres-ence of WSPs, the structure of the biomaterial isquitedifferentfromthatoftheoriginalBC.Becauseof the distinct porelike structure, it was importanttomeasurethewaterretentionability(expressedaswater content) and the interaction with ions. Withrespect to the absorption of heavy metals, copperions were used. The specific structure of the com-posites influenced the removability of nitrogen-con-tainingmoleculesfromthenutrientmediumorbac-teria cells. The nitrogen content of the purified,freeze-dried fleeces was determined by elementalanalysis. Moreover, the WSPs added to the culturesolutions affected the product yield. WATER CONTENT OF BACTERIAL CELLULOSE  465  Amount and Purity of the Composites With the described culture conditions (0.6 g of  D -glucose, 30 mL of the culture medium, and 14days of cultivation at 28 °C), purified and freeze-dried standard BC fleeces of about 300 mg couldbe obtained, corresponding to a  D -glucose utiliza-tion of around 50%.For samples of BC fleeces formed in the pres-ence of 1.0% WSP, yields of 127% (CMC as anadditive) and even 157% (MC) in relation to stan-dard BC (100%) were obtained. In accordancewith the IR and  13 C NMR results, these WSPswere included in the cellulose materials. In con-trast, the BC–PVA (1.0% additive) fleeces werecharacterized by a remarkably lower yield of 73%after the same kind of cultivation and workupprocedure. We assumed an influence of the PVA additive on BC formation. In the case of 2.0% PVA addition, a fleece with a 97% yield in relation tostandard BC was produced. The  13 C NMR spectrashowed a PVA content in this composite.For standard BC without the addition of aWSP, nitrogen could not be detected by elementalanalysis. This meant that the described purifica-tion removed protein remainders from the nutri-ent medium and from cells of AX All samples of the CMC, MC, and PVA series contained nitrogenin the range of 0.11–0.35%. This indicated thatthe WSP additives affected the retention of theprotein residues under the standard washing pro-cedure. Solid-State  13 C NMR Spectroscopy In Figure 1, the  13 C NMR spectra of freeze-driedBC composites in the solid state are shown. Thesamples were measured with CP, and the CPexperiments were executed continuously withoutthe conditions being changed. Nevertheless, theoptimized CP setup could be changed a little bythe warming of the probe head. The differencespectra are the results of actual spectra minus thestandard BC spectrum for the detection of a pos-sible portion of WSPs in the composites.The samples of the BC–CMC series showed acommon NMR spectrum of BC. The differencespectrum indicated that fewer parts of CMC Figure 1.  Solid-state  13 C NMR spectra of freeze-dried BC fleeces formed with the addition of differentamounts of WSP and their difference spectra to BC.From top to bottom per series are shown the spectra of pure WSP, BC–WSP (0.5%), BC–WSP (1.0%), and BC–WSP (2.0%) followed by the corresponding differencespectrum. 466  SEIFERT ET AL.  might be adhered to the BC material, althoughthe CMC peak at 176 ppm could not be resolved inall BC–CMC spectra. Anyway, it is also possiblethat the signals in the difference spectra werecaused by changes in the CP conditions. In thiscase, the intensities are hardly comparable.However, in the spectrum of BC–MC (0.5%), acontent of MC was observable (peaks at 85 and 61ppm). These signals became more intensive athigher WSP additions. Besides, the C1 signalchanged. Our point of interest was determining if it was due to a change in the    and    modifica-tions as Yamamoto and Horii reported 17 or rathera signal of incorporated MC.For BC–PVA composites, only the differencespectrum of BC–PVA (2.0%) minus BC disclosedless PVA incorporated into the fleece. In BC–PVA (0.5 and 1.0%), no PVA was detectable.For the determination of the I   /I   fraction andtheamorphouspart,theC1andC4positionsweredecomposed into several Gaussian lines for C  (C   ), C   (C   ), and amorphous parts (see Fig. 2). As the CP experiments were executed continu-ously without changes in the conditions, the CPstimulations of C   and C   could be consideredidentical. The reason for this is their uniform  1 Hsurroundings, which allowed us to determine therelative parts of C   and C   with a high probabil-ity. The fitted integral values for each part werenormalized, and the results for C1 are illustratedin Figure 3.For the BC–CMC series, there was a noticeabledecreaseintheC1  part,whereastherewasalmostno change in the C1   fraction. There are signs of increases in C1   negligible in the scope of the mea-surement precision. For C4 (not shown), there werenosignificantdifferencesintheI   /I  fraction.First,there was no decrease in the C4  signal observable,and second, there were signs of increasing C4   negligible in the scope of the measurement preci-sion. A clear decrease in C4 was determined forC4    and C4  , but in this case, no exact statementsare possible because these resonances overlap.Therefore, it is obvious that the decrease in C   wascoupled to an increase in the amorphous parts.For BC–MC composites, a clear decrease in theC1   (Fig. 3) and C4   (not shown) parts could beobserved. Besides, the integrals of C1   and C1   ,as well as the communal resonance of C4    andC4  , decreased noticeably, whereas almost nochanges occurred for C4   . Therefore, the de-crease in C   and C   was most likely caused by anincrease in the amorphous parts.In the BC–PVA set, no significant changes inthe C   and C   parts and no differences in theI   /I   fraction were observable. IR Spectroscopy To detect the WSPs in the purified and freeze-dried composites, we compared the IR spectrawith the spectra of pure BC and WSP. Figure 2.  Demonstration of C1 resonance decomposition into Gaussian lines for theC1  , C1  , C1   , and amorphous parts. WATER CONTENT OF BACTERIAL CELLULOSE  467
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