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The effects of growing bacterial cellulose (BC) in the presence of exopolysaccharides (EPS) extracted from Escherichia coli ATCC 35860 on the mechanical properties of BC have been studied. After harvesting and purifying the EPS, its composition was analyzed by gas chromatography-mass spectrometry. When adding the purified EPS into the culture media, another kind of EPS, composed of fructose, was produced by Gluconacetobacter xylinus ATCC 23769 and a minor portion of the added EPS was incorporated into cellulose fibrillar network. The characteristics of BC nanocomposites synthesized in the presence of purified EPS was systematically studied through tensile testing, x-ray diffraction, Fourier transform infrared spectroscopy, and field emission scanning electron microscopy. The results revealed that the EPS affected the cellulose-cellulose interactions during the physical aggregation of crystalline microfibrils, but did not impact the co-crystallization process during BC synthesis. The addition of 4 or 8 mg/L purified EPS into the culture media, was found to significantly improve the mechanical properties of BC nanocomposites while maintaining BC crystallinity and crystal size. [ABSTRACT FROM AUTHOR]

Bacterial cellulose (BC) is a promising biomaterial as well as a model system useful for investigating cellulose biosynthesis. BC produced under static cultivation condition is a hydrous pellicle consisting of an interconnected network of fibrils assembled in numerous dense layers. The mechanisms responsible for this layered BC assembly remain unknown. This study used calcofluor as a fluorescent marker to examine BC layer formation at the air/liquid interface. Layers are found to move downward into the media after formation while new layers continue to form at the air/liquid interface. Calcoflour is also known to reduce the crystallinity of cellulose, changing the mechanical properties of the formed BC microfibrils. Consecutive addition and accumulation of calcofluor in the culture medium is found to disrupt the layered assembly of BC. BC crystalllinity decreased by 22 % in the presence of 12 % calcofluor (v/v) in the medium as compared to BC produced without calcofluor. This result suggests that cellulose crystallinity and the mechanical properties which crystallinity provides to cellulose are major factors influencing the layered BC structure formed during biosynthesis. [ABSTRACT FROM AUTHOR]

Bacterial cellulose (BC) exhibits unique properties including high mechanical strength and high crystallinity. Improvement in the mechanical properties of BC is sought for many applications ranging from food to structural composites to biomedical materials. In this study, different additives including carboxymethyl cellulose (CMC), pectin, gelatin, cornstarch, and corn steep liquor were included in the fermentation media to alter the BC produced. Three different concentrations (1%, 3% and 5%) were chosen for each of the additives, with no additive (0%) as the control. The produced BC was then analyzed to determine tensile and compression modulus. Amongst the tested additives, BC produced in media containing 3% (w/v) pectin had the maximum compressive modulus (142 kPa), and BC produced in media containing 1% (w/v) gelatin exhibited the maximum tensile modulus (21 MPa). Structural characteristics of BC and BC-additive composites were compared using X-Ray diffraction (XRD). The crystal size and crystallinity of BC was reduced when grown in the presence of CMC and gelatin while pectin only decreased the crystallite size. This suggested that CMC and gelatin may be incorporated into the BC fibril structure. The field emission scanning electron microscopy (FESEM) images showed the increased micro-fibril aggregation in BC pellicles grown in the presence of additives to the culture media. [ABSTRACT FROM AUTHOR]

Gluconacetobacter hansenii, a Gram-negative bacterium, produces and secrets highly crystalline cellulose into growth medium, and has long been used as a model system for studying cellulose synthesis in higher plants. Cellulose synthesis involves the formation of β-1,4 glucan chains via the polymerization of glucose units by a multi-enzyme cellulose synthase complex (CSC). These glucan chains assemble into ordered structures including crystalline microfibrils. AcsA is the catalytic subunit of the cellulose synthase enzymes in the CSC, and AcsC is required for the secretion of cellulose. However, little is known about other proteins required for the assembly of crystalline cellulose. To address this question, we visually examined cellulose pellicles formed in growth media of 763 individual colonies of G. hansenii generated via Tn5 transposon insertion mutagenesis, and identified 85 that produced cellulose with altered morphologies. X-ray diffraction analysis of these 85 mutants identified two that produced cellulose with significantly lower crystallinity than wild type. The gene disrupted in one of these two mutants encoded a lysine decarboxylase and that in the other encoded an alanine racemase. Solid-state NMR analysis revealed that cellulose produced by these two mutants contained increased amounts of non-crystalline cellulose and monosaccharides associated with non-cellulosic polysaccharides as compared to the wild type. Monosaccharide analysis detected higher percentages of galactose and mannose in cellulose produced by both mutants. Field emission scanning electron microscopy showed that cellulose produced by the mutants was unevenly distributed, with some regions appearing to contain deposition of non-cellulosic polysaccharides; however, the width of the ribbon was comparable to that of normal cellulose. As both lysine decarboxylase and alanine racemase are required for the integrity of peptidoglycan, we propose a model for the role of peptidoglycan in the assembly of crystalline cellulose. [ABSTRACT FROM AUTHOR]

This study characterized the cellulosic and non-cellulosic exopolysaccharides (EPS) produced by four Gluconacetobacter strains. The yields of bacterial cellulose and water-soluble polysaccharides were dependent on both carbon source and Gluconacetobacter strain. The carbon substrate also affected the composition of the free EPS. When galactose served as an exclusive carbon source, Gluconacetobacter xylinus ( G. xylinus ) ATCC 53524 and ATCC 700178 produced a distinct alkaline stable crystalline product, which influenced the crystallization of cellulose. Gluconacetobacter hansenii ( G. hansenii ) ATCC 23769 and ATCC 53582, however, did not exhibit any significant change in cellulose crystal properties when galactose was used as the carbon source. Microscopic observation further confirmed significant incorporation of EPS into the cellulose composites. The cellulosic network produced from galactose medium showed distinctive morphological and structural features compared to that from glucose medium. [ABSTRACT FROM AUTHOR]

In this work, Family 1 cellulosebinding modules CBMCel7Aand CBMCel6Awere heterologouslyexpressed and purifiedfrom Escherichia coli, and the bindingproperties between these CBMs and cellulose substrates were studied.Cellulose nanowhiskers (CNWs, the crystalline portion of cellulose),microcrystalline cellulose Avicel PH101 (partially crystalline cellulose),and phosphoric acid swollen cellulose (PASC, amorphous cellulose)were used as representative models for cellulose to better understandthe binding interactions between the CBMs and different regions ofnative cellulose. Isothermal titration calorimetry (ITC) was combinedwith adsorption-isotherm experiment to analyze the thermodynamicsof CBM binding to various cellulose substrates. N2adsorptionand static light scattering (SLS) data were used to estimate the accessiblesurface area of cellulose which was then used for ITC data analysis.A new method of determining the cellulose molarity based on the availablesurface area for CBM binding was developed, which allows differentcellulose substrates to be compared for binding experiments. The ITCresults showed that the binding constant (Ka) to crystalline CNWs was ∼105M–1for CBMCel7A, while ∼106M–1for CBMCel6A, suggesting a higher binding affinity ofCBMCel6Ato CNWs. For Avicel, lower binding constants forboth CBMs were observed, and weak bindings to PASC were characterized,suggesting that the binding between CBMCel7A,Cel6Aandcellulose to some extent relates to the crystallinity of cellulose.Additionally, the binding reactions were driven by a favorable enthalpychange, offset partially by an unfavorable entropy change. It is suggestedthat CBMCel6Apreferentially binds to the reducing endof cellulose chain, while CBMCel7Adoes not show such endbinding specificities. Cello-oligosaccharides less than two glucoseunits did not bind with CBMs, and improved binding affinities wereobserved for cello-oligosaccharides with longer glucose units. [ABSTRACT FROM AUTHOR]

The pellicle formation, crystallinity, and bundling of cellulose microfibrils produced by bacterium Gluconacetobacter xylinus were studied. Cellulose pellicles were produced by two strains (ATCC 53524 and ATCC 23769) for 1 and 7 days; pellicles were analyzed with scanning electron microscopy (SEM), X-ray diffraction (XRD), vibrational sum-frequency-generation (SFG) spectroscopy, and attenuated total reflectance infrared (ATR-IR) spectroscopy. The bacterial cell population was higher at the surface exposed to air, indicating that the newly synthesized cellulose is deposited at the top of the pellicle. XRD, ATR-IR, and SFG analyses found no significant changes in the cellulose crystallinity, crystal size or polymorphic distribution with the culture time. However, SEM and SFG analyses revealed cellulose macrofibrils produced for 7 days had a higher packing density at the top of the pellicle, compared to the bottom. These findings suggest that the physical properties of cellulose microfibrils are different locally within the bacterial pellicles. [ABSTRACT FROM AUTHOR]