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

For the modification of medically useful biomaterials from bacterially synthesized 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), and poly(vinyl alcohol) (PVA), respectively, in the Hestrin-Schramm culture medium. The incorporation of the water-soluble polymers into cellulose and their influence on the structure, crystal modifications, and material properties are described. With IR and solid-state ¹³C NMR spectroscopy of the fleeces, the presence of the cellulose ethers and an increase in the amorphous parts of the cellulose modifications (NMR results) have been detected. The incorporation is represented by a higher product yield, too. As demonstrated by scanning electron microscopy, a porelike cellulose network structure forms in the presence of CMC and MC. This modified structure increases the water retention ability (expressed as the water content), the ion absorption capacity, and the remaining nitrogen-containing residues from the culture medium or bacteria cells. The water 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. The freeze-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 composite in the wet state, and about 93% is included in the reswollen state synthesized in the presence of 0.5, 1.0, or 2.0% MC. These biomaterial composites can be stored in the dried state and reswollen before use, reaching a higher water absorption than pure, never dried BC. The copper ion capacity of BC-CMC composites increases proportionally with the added amount of CMC. BC-CMC (0.5%) can absorb 3 times more copper ions than original 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. Nevertheless the presence of PVA in the culture medium effects a decreased product yield, a retention of nitrogen-containing residues in the material during purification, a reduced water absorption ability, and a slightly higher copper ion capacity in comparison with original 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     

Cp/mas 13c nmr and Electron Diffraction Study of Bacterial Cellulose Structure Affected by Cell Wall Polysaccharides

Acetobacter xylinum was cultured in Schramm–Hestrin medium containing pectin (pectin medium), xylan (xylan medium), or glucomannan (mannan medium). X-ray diffractometry revealed that xylan and glucomannan affected the size of the cellulose crystals and their d-spacing values. Solid-state cross polarization magic angle spinning carbon-13 nuclear magnetic resonance spectroscopy indicated that the ratio of cellulose I was reduced by the addition of polysaccharides. These effects were more remarkable on the cellulose in the mannan medium than that in the xylan medium, and were scarcely observed in the pectin medium. Electron diffraction analysis revealed that these effects on hemicelluloses along cellulose microfibrils are continuous in the mannan medium and discontinuous in the xylan medium. These findings suggest that the uronic acid in the polysaccharides prevents interactions with cellulose leading to alterations of the structure of the cellulose crystal.     

COMMUNICATION: Culture conditions producing structure entities composed of Cellulose I and II in bacterial cellulose

Culture conditions for the production of Cellulose I and/or II structures have been investigated by transmission electron microscopy using smooth colonies of Acetobacter xylinum ATCC23769. Cells prepared from smooth colonies produce the band material composed of Cellulose II in phosphate buffer (pH 7) at 4 °C. In contrast, the same cells produce the normal twisting ribbons of Cellulose I when the incubation temperature is raised to 28 °C. The band material is also produced at 4 °C in 2% buffered glucose solution and in the standard Hestrin-Schramm medium. This revised version was published online in August 2006 with corrections to the Cover Date.     

Affinity of Hemicellulose for Cellulose Produced by Acetobacter Xylinum

Cellulose composites were produced by culturing Acetobacter aceti subsp. xylinum (ATCC 53524, agitation tolerant strain) under shaking and agitating conditions in the presence of 2% pine or beech Björkman lignin-carbohydrate complexes (LCCs) or six different types of hemicellulosic polysaccharides including glucuronoxylan, glucomannan, O-acetyl-glucuronoxylan, arabinoglucuronoxylan, arabinogalactan and xyloglucan. Hemicellulosic polysaccharide contents in cellulose composites were similar in spite of the differences in culture, shaking and agitating conditions. On the basis of hemicellulosic polysaccharide contents and X-ray diffraction patterns after extraction by dilute NaOH solution, glucomannan family polysaccharides were found to have the highest affinity to bacterial cellulose. Composites with neutral and acidic LCCs were resistant against alkali while high lability of their delignified carbohydrates against alkali indicates the importance of lignin for formation of cellulose-hemicellulose-lignin framework of plant secondary cell-walls.     

Spectral assignments and anisotropy data of cellulose I(alpha): 13C-NMR chemical shift data of cellulose I(alpha) determined by INADEQUATE and RAI techniques applied to uniformly 13C-labeled bacterial celluloses of different Gluconacetobacter xylinus strains

Solid-state (13)C-NMR spectroscopy was used to characterize native cellulose pellicles from two strains of Gluconacetobacter xylinus (ATCC 53582, ATCC 23769), which had been statically cultivated in Hestrin-Schramm (HS) medium containing fully (13)C-labeled beta-D-glucose-U-(13)C(6) as the sole source of carbon. For both samples, the (13)C-NMR chemical shifts were completely assigned for each (13)C-labeled site of cellulose I(alpha) with the aid of 2D refocused INADEQUATE NMR. To determine the principal chemical shift tensor components, a pulse sequence based on the recoupling of anisotropy information (RAI) was applied at 10 kHz MAS. The detailed (13)C tensors of cellulose I(alpha) from different bacterial celluloses are thus available now for the first time, and these results have been compared with previously published data of nonenriched material and with theoretical predictions.     

Analytical Investigations of Bacterial Cellulose

The cultivation of the bacterium Acetobacter xylinus AX 5 was carried out in the common Hestrin-Schramm medium containing D -glucose as C-source and citric acid as buffer component. HPLC studies proved to be convenient methods to investigate the stability and interactions of these constituents in the starting culture liquid. Within the initial sterilization step and limited by the citric acid, up to 6% of the D -glucose was partially isomerized to D-fructose and degraded to dark-yellow products. In static culture, A. xylinus AX 5 produces cellulose extracellularly on the surface of this medium. Solid-state NMR spectroscopy represents a suitable analytical method to characterize the supramolecular structure of the bacterial cellulose in never-dried, air-dried, and freeze-dried states. It could be demonstrated that the drying process reduces the degree of crystallinity in the range of about 12% without changes in the Iα/β ratio of these cellulose modifications.     

Production of bacterial cellulose from alternate feedstocks

Production of bacterial cellulose by Acetobacter xylinum ATCC 10821 and 23770 in static cultures was tested from unamended food process effluents. Effluents in cluded low-solids (LS) and high-solids (HS) potato effluents, cheese whey permeate (CW), or sugar beet raffinate (CSB). Strain 23770 produced 10% less cellulose from glucose than did strain 10821 and diverted more glucose to gluconate. Unamended HS, CW, and CSB were unsuitable for cellulose production by either strain, and LS was unsuitable for production by strain 10821. However, strain 23770 produced 17% more cellulose from LS than from glucose, indicating that unamended LS could serve as a feedstock for bacterial cellulose.     

2-Ketogluconic acid production by acetobacter pasteurianus

Production of 2-ketogluconic acid in batch fermentation was investigated.Acetobacter pasteurianus ATCC 6438, which produces selectively 2-ketogluconic acid only, was used. The optimal pH for glucose dehydrogenation to gluconate by resting cells was 5.0 and for gluconate dehydrogenation to 2-ketogluconate 4.25. When glucose medium was used, the 89% yield was achieved after 90 h. For the optimal productivity, medium containing glucose and gluconate with the molar glucose:gluconate ratio 7.4 was proposed, and the yield of 92% after 56 h was achieved. This composition of medium led to the elevation of dissolved oxygen concentration during fermentation. It consequently resulted in elevated gluconate dehydrogenase activity being discussed as the rate-limiting activity of the batch production.     

TEM study of band-like cellulose assemblies produced by Acetobacter xylinum at 4 °C

Two kinds of band-like cellulose assemblies, coarse and densestructures, are produced in Hestrin–Schramm medium at 4 °Cusing smooth colonies isolated from Acetobacter xylinumATCC23769, whereas ribbon cellulose assemblies are produced by the samecoloniesat 28 °C. The dense and the coarse band-like assembliesconsist of many strand-like cellulose entities (strands) and areextruded perpendicularly to the long axis of the bacterial cell. In an earlystage of incubation at 4 °C, the dense band-like assembly isproduced and the number of strands decreases gradually with increasingincubation time at 4 °C, probably because the number of activeTC subunits decreases as a result of the low-temperature shock for thebacteria.In contrast, the coarse band-like assembly is clearly observed after about 1h of incubation at 4 °C. The number of strands inthe coarse band-like assembly is about one third of that of the dense band-likeassembly and does not change during the incubation time of about 6h. In the selected-area electron diffraction (ED) experiment, thedense band-like assembly gives crystalline reflections corresponding to thecellulose II type crystal, while the coarse band-like assembly does not giveanycrystalline reflections under the same ED conditions.     

In Situ crystallization of bacterial cellulose I. Influences of polymeric additives,stirring and temperature on the formation celluloses I α and I β as revealed by cross polarization/magic angle spinning (CP/MAS)13C NMR spectroscopy

To obtain further information about the formation of cellulose I and I, cross polarization/magic angle spinning (CP/MAS)¹³C NMR spectroscopy was used to study the effects of polymeric additives, stirring and culture temperature on the I When xyloglucan (XG) or carboxymethyl cellulose sodium salt (CMC) was added to the incubation medium, the amount of cellulose I decreased markedly, from a normal level of 64% to as low as 30%, with the most additive giving the lowest levels of I. Moreover, stirring causes mixtures containing even small amounts of XG to have a large effect. These results suggest that CMC or XG interferes with the aggregation of fibrillar units into the normal ribbon assemblies. It may be that there is a strain associated with this aggregation that results in the higher-energy I form. Thus, cellulose I may grow preferentially when the strain caused by aggregation is not present. Lower temperatures (36–10 °C) gave an increase in I (from 56 to 72%).