The effect of pH, sucrose and ammonium sulphate concentrations on the production of bacterial cellulose (Nata-de-coco) by Acetobacter xylinum

The effect of pH, sucrose and ammonium sulphate concentrations on the production of nata-de-coco, a form of bacterial cellulose, by Acetobacter xylinum was studied. Comparisons for physical properties like thickness, wet weight, water-holding capacity (WHC), moisture content and hardness, a textural parameter were done on nata-de-coco grown in tender coconut water medium supplemented with varying concentrations of sucrose and ammonium sulphate at different pH values. The results were analysed by fitting a second-order polynomial regression equation. Response surface methodology was used to study the effect of the three variables. The study showed that A. xylinum could effectively use sucrose as the sole carbon source in coconut water medium and that cellulose production was more dependent on pH than either sucrose or ammonium sulphate concentrations. Maximum thickness of nata was obtained at pH 4.0 with 10% sucrose and 0.5% ammonium sulphate concentrations. These conditions also produced good quality nata-de-coco with a smooth surface and soft chewy texture. The study will enable efficient utilization of coconut water, a hitherto wasted byproduct of coconut industry and will also provide a new product dimension to the aggrieved coconut farmers who are not getting the right price for their product.     

Effect of various carbon and nitrogen sources on cellulose synthesis by Acetobacter xylinum

The effect of various carbon and nitrogen sources on cellulose membrane production by Acetobacter xylinum was evaluated. Among the carbon sources, sucrose, glucose and mannitol were found to be suitable for optimum levels of cellulose production. The strain was able to utilize a wide range of protein and nitrogen sources such as peptone, soybean meal, glycine, casein hydrolysate, and glutamic acid for cellulose synthesis. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of pellicle proteins (PP) revealed electrophoretic bands of molecular masses in the range of 116–20 kDa. Furthermore, the strain can be useful for the removal of various nitrogenous and carbon substrates present in waste waters.     

Novel glycosyltransferase genes involved in the acetan biosynthesis of Acetobacter xylinum

Novel aceQ and aceR genes involved in the acetan biosynthesis of Acetobacter xylinum were newly isolated. The homology search with DNA Data Bank of Japan indicated that aceQ and aceR were glycosyltransferases. Their gene-disrupted mutants were obtained by homologous recombination using the tetracycline resistance gene and the electroporation method. By NMR and ESI-MS analyses, aceQ-disrupted mutant DQ was found to secrete a water-soluble polysaccharide harboring the -Man-GlcUA side chain and the aceR-disrupted mutant DR was found to secrete an acetan analog, lacking the terminal Rha residue. These results suggested that aceQ and aceR encode a glucosyltransferase and a rhamnosyltransferase, respectively. It was indicated that acetan analogs harboring various side chains can be generated easily by genetic engineering.     

Cloning and sequencing of the cellulose synthase catalytic subunit gene of Acetobacter xylinum

The gene for the catalytic subunit of cellulose synthase from Acetobacter xylinum has been cloned by using an oligonucleotide probe designed from the N-terminal amino acid sequence of the catalytic subunit (an 83 kDa polypeptide) of the cellulose synthase purified from trypsin-treated membranes of A. xylinum. The gene was located on a 9.5 kb HindIII fragment of A. xylinum DNA that was cloned in the plasmid pUC18. DNA sequencing of approximately 3 kb of the HindIII fragment led to the identification of an open reading frame of 2169 base pairs coding for a polypeptide of 80 kDa. Fifteen amino acids in the N-terminal region (positions 6 to 20) of the amino acid sequence, deduced from the DNA sequence, match with the N-terminal amino acid sequence obtained for the 83 kDa polypeptide, confirming that the DNA sequence cloned codes for the catalytic subunit of cellulose synthase which transfers glucose from UDP-glucose to the growing glucan chain. Trypsin treatment of membranes during purification of the 83 kDa polypeptide cleaved the first 5 amino acids at the N-terminal end of this polypeptide as observed from the deduced amino acid sequence, and also from sequencing of the 83 kDa polypeptide purified from membranes that were not treated with trypsin. Sequence analysis suggests that the cellulose synthase catalytic subunit is an integral membrane protein with 6 transmembrane segments. There is no signal sequence and it is postulated that the protein is anchored in the membrane at the N-terminal end by a single hydrophobic helix. Two potential N-glycosylation sites are predicted from the sequence analysis, and this is in agreement with the earlier observations that the 83 kDa polypeptide is a glycoprotein [13]. The cloned gene is conserved among a number of A. xylinum strains, as determined by Southern hybridization.     

Physico-Mechanical Properties of Chemically Treated Bacterial (Acetobacter xylinum) Cellulose Membrane

Bacterial cellulose obtained through fermentation by the Acetobacter xylinum is of superior functional quality in comparison to plant cellulose. Various alkali treatment methods were used to process bio-chemically complex pellicle into a clean cellulose membrane/sheet. The effect of potassium hydroxide, sodium carbonate and potassium carbonate was found to be milder on the final cellulose product in contrast to the widely used sodium hydroxide treatment. These novel treatment methods also caused improvement in the tensile strength of the membranes in comparison to sodium hydroxide. The overall quality of the 0.1 M sodium carbonate- and potassium carbonate-treated cellulose was superior, as the membranes displayed maximum tensile strength and elongation next to the native membrane. The low tensile strength of sodium hydroxide-treated membrane is attributed to its higher swelling characteristics in alkali. Further, the low swelling property of sodium carbonate- and potassium carbonate-treated membranes resulted in their high oxygen transmission rates (low oxygen barrier). Hunter lab colour parameters were determined to assess the effect of different alkali treatments on the colour characteristics of the membranes. Further, based on the high mechanical strength and comparatively low oxygen transmission rates, the processed cellulose membranes may find application as a bio- packaging material for controlled atmosphere packaging, where hydrophilic membranes with high oxygen barrier and water vapour permeation are desirable.     

Identification of a new gene in an operon for cellulose biosynthesis in Acetobacter xylinum

DNA sequencing of the region downstream of the cellulose synthase catalytic subunit gene of Acetobacter xylinum led to the identification of an open reading frame coding for a polypeptide of 86 kDa. The deduced amino acid sequence of this polypeptide matches from position 27 to 40 with the N-terminal amino acid sequence determined for a 93 kDa polypeptide that copurifies with the cellulose synthase catalytic subunit during purification of cellulose synthase. The cellulose synthase catalytic subunit gene and the gene encoding the 93 kDa polypeptide, along with other genes probably, are organized as an operon for cellulose biosynthesis in which the first gene is the catalytic subunit gene and the second gene codes for the 93 kDa polypeptide. The function of the 93 kDa polypeptide is not clear at present, however it appears to be tightly associated with the cellulose synthase catalytic subunit. Sequence analysis of the polypeptide shows that it is a membrane protein with a signal sequence at the N-terminal end and a transmembrane helix in the C-terminal region for anchoring it into the membrane.     

The mechanism of formation of Cellulose-like microfibrils in a cell-free system from Acetobacter xylinum

The mechanism of formation of cellulose-like microfibrils by a non-soluble, particulate enzyme and uridine diphosphoglucose (UDPG) in a cell-free system from Acetobacter xylinum was studied by transmission electron microscopy and X-ray diffraction. The suspension of particles to which the enzyme is adsorbed is composed of whole, dense ovoids, 50–250 nm long when wet, of fragments of the ovoids, and amorphous substance. There is a typical unit membrane around each ovoid but initially there is no trace of fibrillar material in the suspension. When the suspension of particles is incubated with UDPG, linear wisps of fibrils are produced which associate rapidly to form longer and wider threads, especially in 0.01 M NaCl. There is no visible attachment of the wisps to the particles. After 20 min incubation, threads with the typical morphology of cellulose microfibrils are formed that later tend to become entangled in clumps. The microfibrils are insoluble in hot, aqueous, alkaline solutions and resistant to the action of trypsin, but may be degraded by glusulase. After treatment with 1 M NaOH at 100° C or with cold 18% NaOH they show an X-ray diffraction pattern which resembles that of Cellulose II from mercerized, authentic bacterial cellulose. Incorporation of radioactive glucose into the insoluble residue is enhanced by drying of the cellulose microfibrils before alkaline digestion and especially by the addition of a gross excess of carrier cellulose after incubation. In this system there is no evidence for participation of linear, axial, synthesizing sites on the cell wall of the bacterium or for ordered, organized granules in the assembly of the microfibrils. That is, cellulose-like microfibrils may be formed in a cell-free system without the action of any of the previously suggested cell organelles. In addition, these observations are consistent with a previously described notion of a transient, hydrated, nascent, bacterial cellulose microfibril. The possibility that cellulose microfibrils of green plants may be formed in the same way is considered.N.R.C.C. 18314     

Behavior of Freezable Bound Water in the Bacterial Cellulose Produced by Acetobacter xylinum: An Approach Using Thermoporosimetry

The aim of the study is to examine thermal behavior of water within reticulated structure of bacterial cellulose (BC) films by sub-ambient differential scanning calorimetry (DSC). BC films with different carbon source, either manitol (BC (a)) or glycerol (BC (b)), were produced by Acetobacter xylinum using Hestrin and Shramm culture medium under static condition at 30 ± 0.2°C for 3 days. BC samples were characterized by electron scanning microscopy and X-ray diffraction spectroscopy. The pore analysis was done by B.H.J. nitrogen adsorption. The pre-treated with 100% relative humidity, at 30.0 ± 0.2°C for 7 days samples were subjected to a between 25 and −150°C-cooling–heating cycle of DSC at 5.00°C/min rate. The pre-treated samples were also hydrated by adding 1 μl of water and thermally run with identical conditions. It is observed that cellulose fibrils of BC (a) were thinner and reticulated to form slightly smaller porosity than those of BC (b). They exhibited slightly but non-significantly different crystalline features. The freezable bound water behaved as a water confinement within pores rather than a solvent of polymer which is possible to use thermoporosimetry based on Gibb–Thomson equation to approach pore structure of BC. In comparison with nitrogen adsorption, it was found that thermoporosimetry underestimated the BC porosity, i.e., the mean diameters of 23.0 nm vs. 27.8 nm and 27.9 nm vs. 33.9 nm for BC (a) and BC (b), respectively, by thermoporosimetry vs. B.H.J. nitrogen adsorption. It may be due to large non-freezable water fraction interacting with cellulose, and the validity of pore range based on thermodynamic assumptions of Gibb–Thomson theory.     

Effects of mixing conditions on the production of microbial cellulose byAcetobacter xylinum

Microbial cellulose has many potential applications due to its excellent physical properties. The production of cellulose fromAcetobacter xylinum in submerged culture is, however, beset with numerous problems. The most difficult one has been the appearance of negative mutants under shaking culture conditions, which is deficient of cellulose producing ability. Thus genetic instability ofAcetobacter xylinum under shaking culture condition made developing a stable mutant major research interest in recent years. To find a proper type of bioreactor for the production of microbial cellulose, several production systems were developed. Using a reactor system with planar type impeller with bottom sparging system, it was possible to produce 5 g/L microbial cellulose without generating cellulose minus mutants, which is comparable to that of static culture system.     

Cloning of a gene involved in cellulose biosynthesis in Acetobacter xylinum: Complementation of cellulose-negative mutants by the UDPG pyrophosphorylase structural gene

Summary Three cellulose-negative (Cel^-) mutants of Acetobacter xylinum strain ATCC 23768 were complemented by a cloned 2.8 kb DNA fragment from the wild type. Biochemical analysis of the mutants showed that they were deficient in the enzyme uridine 5-diphosphoglucose (UDPG) pyrophosphorylase. The analysis also showed that the mutants could synthesize (1-4)-glucan in vitro from UDPG, but not in vivo from glucose. This result was expected, since UDPG is known to be the precursor for cellulose synthesis in A. xylinum. In order to analyze the function of the cloned gene in more detail, its biological activity in Escherichia coli was studied. These experiments showed that the cloned fragment could be used to complement an E. coli mutant deficient in the structural gene for UDPG pyrophosphorylase. It is therefore clear that the cloned fragment must contain this gene from A. xylinum. This is to our knowledge the first example of the cloning of a gene with a known function in cellulose biosynthesis from any organism, and we suggest the gene be designated celA.     

Cellulose synthesized by Acetobacter xylinum in the presence of multi-walled carbon nanotubes

The structure of bacterial cellulose is affected by the bacterial strain used, culture media and cultivation conditions. In this study, acid-treated multi-walled carbon nanotubes (MWNTs) were added into a static culture medium and their effect on bacterial cellulose structure was studied by scanning electron microscopy (SEM), atomic force microscopy (AFM), Fourier transform infrared spectroscopy (FT-IR), CP/MAS (13)C NMR and X-ray diffractometry. The bacterial cellulose ribbons and the MWNTs interwound and formed a three-dimensional network architecture. Band-like assemblies with sharp bends and rigidity were also produced in the presence of MWNTs. The intermolecular hydrogen bonds in bacterial cellulose produced in the presence of MWNTs were weakened. The crystal structure, cellulose I(alpha) content, crystallinity index (CrI) and crystallite size all changed. The results may suggest that the acid-treated MWNTs containing hydroxyl groups interact with the sub-elementary bacterial cellulose fibrils, subsequently interfering with the aggregation and crystallization.     

A non-extractable triterpenoid of the hopane series in Acetobacter xylinum

Abstract Cells of the bacterium Acetobacter xylinum were analysed for their residual triterpenoid content after exhaustive lipid extraction using chloroform/methanol. Whereas the well known bacteriohopanetetrol mixture was present in the extract, the cells still contained a single bacteriohopanepentol cyclitol ether which could be detected either as the primary alcohol derivative after H₅IO₆ oxidation followed by NaBH₄ reduction of the already extracted cells or as the octa-acetate by direct acetylation followed by solvent extraction. This is the first evidence of a probable selective complexation of a hopanoid via non-covalent linkages to other cell constituents.     

Phosphate accumulation of <Emphasis Type="Italic">Acetobacter xylinum</Emphasis>

The cells of Acetobacter xylinum decreased phosphate concentration in the medium from 5 to 2.5 or 0.3 mM during incubation in the presence of Mg²⁺ and glucose, or Mg²⁺ and casamino acids, respectively. The prevalence of orthophosphate or polyphosphate in the biomass of A. xylinum depends on the medium composition. Under phosphate uptake in the presence of glucose, the content of orthophosphate in the biomass changed little, while that of polyphosphate increased fourfold. At incubation with casamino acids, the content of orthophosphate increased 15 times, while that of polyphosphate increased only 2.5 times. Some part of orthophosphate in this case seems to be bound with the cell surface. The polyphosphate chain length in the cells of A. xylinim increases under phosphate uptake. This increase is more noticeable in the presence of glucose. Casamino acids can be replaced by α-ketoglutaric acid in combination with (NH₄)₂SO₄, or arginine, or glutamine, the catabolism of which results in formation of NH₄ ⁺ and α-ketoglutarate.     

Influential factors to enhance the moving rate of Acetobacter xylinum due to its nanofiber secretion on oriented templates

Acetobacter xylinum, a bacterium which secretes a cellulose nanofiber, moves due to the inverse force of extrusion of the fiber, which accordingly correlates with the fiber production rate. To improve the production, the moving rate of the bacterium was focused to examine the influential factors on the substrates for culture and additives in the culture medium. From the real-time video analysis, the oriented template having a strong interaction with the secreted cellulose nanofibers proved to be suitable for the bacteria to move faster. Furthermore, addition of carboxymethylcellulose sodium salt (CMC) to the culture medium cause the bacteria to move faster in the culture medium. In this case, secreted cellulose nanofiber formed different from a normal cellulose nanofiber. The above result could provide an understanding how the formation of cellulose nanofibers contributes to the production rate as well as the bacterial moving rate.     

Effect of addition of sodium alginate on bacterial cellulose production by <Emphasis Type="Italic">Acetobacter xylinum</Emphasis>

Bacterial cellulose (BC) production by Acetobacter xylinum NUST4.1 was carried out in the shake flask and in a stirred-tank reactor by means of adding sodium alginate (NaAlg) into the medium. When 0.04% (w/v) NaAlg was added in the shake flask, BC production reached 6.0 g/l and the terminal yield of the cellulose was 27% of the total sugar initially added, compared with 3.7 g/l and 24% in the control, respectively. The variation between replicates in all determinations was less than 5%. During the cultivation in the stirred-tank reactor, the addition of NaAlg changed the morphology of cellulose from the irregular clumps and fibrous masses entangled in the internals to discrete masses dispersing into the broth, which indicates that NaAlg hinders formation of large clumps of BC, and enhances cellulose yield. Because the structure of cellulose is changed depending on the culture condition such as additives, structural characteristics of BC produced in the NaAlg-free and NaAlg medium are compared using scanning electron microscopy (SEM), fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD). SEM photographs show some differences in reticulated structures and ribbon width and FT-IR spectra indicate that there is the hydrogen bonding interaction between BC and NaAlg, then X-ray diffraction (XRD) analysis reveals that BC produced with NaAlg-added has a lower crystallinity and a smaller crystalline size. The results show that enhanced yields and modification of cellulose structure occur in the presence of NaAlg.     

Cloning and sequencing of the levansucrase gene from Acetobacter xylinum NCI 1005.

The levansucrase gene (lsxA) was cloned from the genomic DNA of Acetobacter xylinum NCI 1005, and the nucleotide sequence of the lsxA gene (1,293 bp) was determined. The deduced amino acid sequence of the lsxA gene showed 57.4% and 46.2% identity with the levansucrases from Zymomonas mobilis and Erwinia amylovora, respectively, while only 35.2% identity with that from Acetobacter diazotrophicus. The gene product of lsxA (LsxA) that was overproduced in E. coli coded for a polypeptide of molecular mass 47 kDa. The LsxA released glucose and produced polysaccharide from sucrose, the structure of which was analyzed by nuclear magnetic resonance spectroscopy and determined to be a beta-(2,6)-linked polyfructan.     

Cloning and sequencing of the beta-glucosidase gene from Acetobacter xylinum ATCC 23769.

The beta-glucosidase gene (bglxA) was cloned from the genomic DNA of Acetobacter xylinum ATCC 23769 and its nucleotide sequence (2200 bp) was determined. This bglxA gene was present downstream of the cellulose synthase operon and coded for a polypeptide of molecular mass 79 kDa. The overexpression of the beta-glucosidase in A. xylinum caused a tenfold increase in activity compared to the wild-type strain. In addition, the action pattern of the enzyme was identified as G3ase activity. The deduced amino acid sequence of the bglxA gene showed 72.3%, 49.6%, and 45.1% identity with the beta-glucosidases from A. xylinum subsp. sucrofermentans, Cellvibrio gilvus, and Mycobacterium tuberculosis, respectively. Based on amino acid sequence similarities, the beta-glucosidase (BglxA) was assigned to family 3 of the glycosyl hydrolases.     

Isolation of α-glucan and lipopolysaccharide fractions from Acetobacter xylinum

A cellular phenol-water extract of Acetobacter xylinum NRC 17007 was fractionated on Sepharose 4 B. The fraction eluting with the void volume consisted to about 95% of glycogen-like material. The lipopolysaccharide fraction was of lower molecular weight and had the following composition (%, w/w): Mannose, 42; glucose, 7; galactose, 3.8; heptose, 2; 2-keto-3-deoxy-octonate, 1.2; glucosamine, 3.3; phosphate, 4.5; total fatty acids, 3.9. Among the fatty acids, 3-hydroxy-tetradecanoic acid was present, and 2-hydroxy-hexadecanoic acid predominated.