Crystallinity of Partially N-Acetylated Chitosans

In order to search for a chitosan with low crystallinity, partially N-deacetylated chitins (PDC) and partially N-acetylated chitosans (PAC) with a low degree of N-acetylation (DAc) were examined by X-ray powder diffraction measurements. Three PDC samples, having less than 30% DAc and prepared by solid-state deacetylation, gave X-ray powder patterns showing the presence of α-chitin, a hydrated crystal of chitosan, or their mixture, respectively. When these PDC samples were treated with an acid-alkali, however, reduced crystallinity was observed. By annealing in water at 160 or 200°C, the latter PDC having DAc less than approx. 22% gave powder patterns indicating the presence of an anhydrous crystal which may spoil the chitosan’s functionality. In contrast, PAC prepared by N-acetylating pure chitosan (DAc=0%) in a swollen state, which can be expected to have random copolymers in the chain, was always less crystallized than PDC, this crystallinity depending on the molecular weight. In the case of high-molecular-weight PAC samples, whose DAc was in the range of 5–21%, the effect of high molecular weight on reducing crystallinity was larger than that of the degree of N-acetylation.     

Gel Permeation Chromatography of Polysaccharides: Universal Calibration Curve

Changes in the crystallinity and polymorph of chitosan, which may affect its functionality, by heating (up to 200°C) its water suspension were studied by X-ray diffraction measurements, using tendon chitosan prepared by N-deacetylation of a crab tendon chitin, and chitosan powders with various degrees of polymerization (DPv = 1,720–12,600) and N-acetylation (zero to 26%). It was found that the presence of hydrated polymorphs or anhydrous crystals in a chitosan sample could be examined easily by measuring the powder diffraction pattern of a sample. Chitosan with a low molecular weight or low degree of N-acetylation was highly crystallized, especially in the anhydrous form that is considered to spoil chitosan’s functionality, by heating.     

Synthesis, characterization, and antibacterial activity of N,O-quaternary ammonium chitosan

N,N,N-Trimethyl O-(2-hydroxy-3-trimethylammonium propyl) chitosans (TMHTMAPC) with different degrees of O-substitution were synthesized by reacting O-methyl-free N,N,N-trimethyl chitosan (TMC) with 3-chloro-2-hydroxy-propyl trimethyl ammonium chloride (CHPTMAC). The products were characterized by ¹H NMR, FTIR and TGA, and investigated for antibacterial activity against Staphylococcus aureus and Escherichia coli under weakly acidic (pH 5.5) and weakly basic (pH 7.2) conditions. TMHTMAPC exhibited enhanced antibacterial activity compared with TMC, and the activity of TMHTMAPC increased with an increase in the degree of substitution. Divalent cations (Ba²⁺ and Ca²⁺) strongly reduced the antibacterial activity of chitosan, O-carboxymethyl chitosan and N,N,N-trimethyl-O-carboxymethyl chitosan, but the repression on the antibacterial activity of TMC and TMHTMAPC was weaker. This indicates that the free amino group on chitosan backbone is the main functional group interacting with divalent cations. The existence of 100 mM Na⁺ slightly reduced the antibacterial activity of both chitosan and its derivatives.     

A chitinase with high activity toward partially<Emphasis Type="Italic"> N</Emphasis>-acetylated chitosan from a new,moderately thermophilic,chitin-degrading bacterium,<Emphasis Type="Italic"> Ralstonia</Emphasis> sp. A-471

A moderately thermophilic bacterium, strain A-471, capable of degrading chitin was isolated from a composting system of chitin-containing waste. Analysis of the 16S rDNA sequence revealed that the bacterium belongs to the genus Ralstonia. A thermostable chitinase A (Ra-ChiA) was purified from culture fluid of the bacterium grown in colloidal chitin medium. Purification of the enzyme was achieved mainly by exploiting its binding to the colloidal chitin. The molecular mass of the enzyme was estimated to be 70 kDa and the isoelectric point approximately 4.7. N-terminal amino acid sequencing revealed a sequence of ADPYLKVAYYP, which had high homology (66% identity) with that of chitinase A1 from Bacillus circulans WL-12. The pH and temperature optima were determined to be 5.0 and 70°C, respectively. The enzyme was classified as a retaining glycosyl hydrolase and was most active against partially N-acetylated chitosans. Its activities towards the partially N-acetylated chitosans, i.e. chitosan 7B, chitosan 8B, and chitosan 9B, were about 11-fold, 9-fold, and 5-fold higher than towards colloidal chitin, respectively. Ra-ChiA cleaved (GlcNAc)₆ almost exclusively into (GlcNAc)_2. Activation of Ra-ChiA was observed by the addition of 1 mM Cu²⁺, Mn²⁺, Ca²⁺_, or Mg²⁺. Degradation of the partially N-acetylated chitosan produced oligosaccharides with a degree of polymerization ranging from 1–8; these are products that offer potential application for functional oligosaccharide production.     

Preparation and structure of chitosan soluble in wide pH range

Two kinds of chitosans, namely N-acetylated and N-deacetylated chitosan were prepared by the modified processes. They can dissolve in both acid and alkali solution. ¹³C NMR was used to study the basic solution of chitosan, and XRD, FT-IR and SEM were used to study the structure of N-acetylated and N-deacetylated chitosan. The result from X-ray diffraction showed that a transformation of crystal structure occurred during the N-acetylation or N-deacetylation process with the decrease of crystallinity and expansion of crystal lattices. FT-IR spectra revealed that the intermolecular and intramolecular hydrogen bonds were destroyed by both treatments and a looser structure was observed by the SEM. The lower crystallinity, the decreased intermolecular interactions, the more disordered and looser structure were easy for the permeation of LiOH/urea aqueous solution and coordinated with the breakage of intermolecular and intramolecular hydrogen bond by LiOH at low temperature, the prepared chitosans dissolved in LiOH/urea/H₂O mixture.     

Oscillatoria anguistissima: A Promising Cu2+ Biosorbent

Oscillatoria anguistissima rapidly adsorbs Cu²⁺ from aqueous solution. The adsorption of Cu²⁺ followed Freundlich Isotherm, and the amount of Cu²⁺ removed from solution increased with increasing Cu²⁺ concentration. The adsorption is pH dependent, and maximum Cu²⁺ removal occurs at pH 5. Of the various pretreatments, HCl treatment of the biomass increased the capacity for Cu²⁺ removal. Presence of Mg²⁺ and Ca²⁺ resulted in decline in the Cu²⁺ adsorption capacity of Oscillatoria cells. This species could also effectively remove Cu²⁺ from mine water containing 68.4 μg/ml of Cu²⁺ at pH 3.45. Received: 23 December 1996 / Accepted: 20 February 1997     

Fractionation and characterization of chitosan by analytical SEC and H NMR after semi-preparative SEC

Heterogeneity in molecular weight and degree of deacetylation (DDA) of chitosans from different sources and preparation methods were studied by fractionating chitosans, using semi-preparative SEC, and then determining molecular weight profiles of fractions by analytical SEC with multi-angle laser light scattering (SEC–MALLS), and degree of deacetylation (DDA) by ¹H NMR. Fractionation of two high molecular weight chitosans from different manufacturers, produced fractions that spanned a wide range of molecular weight (number-average M_n), from 65 to 400 kDa in one case, that was not evident when unfractionated material was directly analyzed by SEC providing M_n = 188 kDa and PDI = M_w/M_n = 1.73. In a second case, fractions ranged from 20 to 600 kDa with unfractionated M_n = 145 kDa and PDI = 1.83. Fractionation of low molecular weight chitosans also showed a broad range of molecular weight in the original material, however, the fractions obtained with the TSKgel G4000W column in the M_n range of 5–100 kDa were essentially monodisperse with PDIs between 1.0 and 1.4. The DDA of one low molecular weight chitosan (10 kDa) produced by nitrous acid degradation was dependent on the M_n of the fraction. This semi-preparative fractionation procedure revealed important compositional heterogeneities of chitosans not evident in unfractionated material, and permitted the production of monodisperse low molecular weight chitosans with homogeneous properties.     

Physicochemical characterization of chitin and chitosan from crab shells

Crab chitosan was prepared by alkaline N-deacetylation of crab chitin for 60, 90 and 120 min and the yields were 30.0-32.2% with that of chitosan C120 being the highest. The degree of N-deacetylation of chitosans (83.3–93.3%) increased but the average molecular weight (483–526 kDa) decreased with the prolonged reaction time. Crab chitosans showed lower lightness and WI values than purified chitin, chitosans CC and CS but higher than crude chitin. With the prolonged reaction time, the nitrogen (8.9–9.5%), carbon (42.2–45.2%) and hydrogen contents (7.9–8.6%) in chitosans prepared consistently increased whereas N/C ratios remained the same (0.21). Crab chitosans prepared showed a melting endothermic peak at 152.3–159.2 °C. Three chitosans showed similar microfibrillar crystalline structure and two crystalline reflections at 2θ = 8.8–9.0° and 18.9–19.1°. Overall, the characteristics of three crab chitosans were unique and differed from those of chitosan CC and CS as evidenced by the element analysis, differential scanning calorimetry, scanning electron microscopy and X-ray diffraction patterns.     

Biosorption Performance of Multimetal Resistant Fungus Penicillium chrysogenum XJ-1 for Removal of Cu2+ and Cr6+ from Aqueous Solutions

Adsorption for heavy metals via biomaterials such as fungal biomass presents a practical remediation technique for polluted water. Among all known filamentous fungi, Penicillium chrysogenum is widespread in nature and can serve as a biosorbent for heavy metals. In the current study, the ability of P. chrysogenum XJ-1 to remove copper (Cu²⁺) and chromium (Cr⁶⁺) from water was evaluated. The maximum biosorption capacity of XJ-1 for Cu²⁺ reached 42.83 ± 0.57 mg g^?1 dry biomass at pH 5.0 after the equilibrium time of 1.5 h. The maximum biosorption capacity for Cr⁶⁺ at pH 3.0 reached 52.69 ± 1.68 mg g^?1 dry biomass after the equilibrium time of 1.5 h. The biosorption data of XJ-1 biomass were well fitted to the Freundlich isotherm model and the pseudo-second-order Lagergren kinetic model. Laundry powder-treated and HCl-treated XJ-1 biomass significantly enhanced its adsorption capacity to Cu²⁺ and Cr⁶⁺, respectively. HCl and NaOH were suitable desorbents for Cu²⁺/Cr⁶⁺ loading biomass, respectively. Fourier transform infrared spectroscopy analyses revealed that hydroxyl, amine, and sulfonyl groups on the biosorbent contributed to binding Cu²⁺ and Cr⁶⁺ and that carbonyl and carboxyl groups were also vital binding sites of Cu²⁺. Scanning electron microscopy and energy-dispersive x-ray (SEM-EDX) analyses confirmed that considerable amounts of metals were precipitated on the cell surface of XJ-1. Our results suggested that XJ-1 might be used to purify multimetal-contaminated water. This low-cost and eco-friendly biomass of XJ-1 seems to have a broad use in the restoration of metal-contaminated water.     

Antibacterial activity of quaternary ammonium chitosan containing mono or disaccharide moieties: Preparation and characterization

The 9 quaternary ammonium chitosans containing monosaccharides or disaccharides moieties were successfully synthesized by reductive N-alkylation then quaternized by N-(3-chloro-2-hydroxypropyl) trimethylammonium chloride (Quat-188). The chemical structures of quaternary ammonium chitosan derivatives were characterized by ATR-FTIR and ¹H NMR spectroscopy. The degree of N-substitution (DS) and the degree of quaternization (DQ) were determined by ¹H NMR spectroscopic method. It was found that the DS was in the range of 12–40% while the DQ was in the range of 90–97%. The results indicated that the O-alkylation was occured in this condition. Moreover, all quaternary ammonium chitosan derivatives were highly water-soluble at acidic, basic, and neutral pH. Minimum inhibitory concentration (MIC) antibacterial studies of these materials were carried out on Escherichia coli (Gram-negative) and Staphylococcus aureus (Gram-positive) bacteria compared to quaternary ammonium N-octyl and N-benzyl chitosan derivatives. The quaternary ammonium mono and disaccharide chitosan derivatives showed very high MIC values which were in the range of 32 to >256 μg/mL against both bacteria. Also it was found that the antibacterial activity decreased with increasing the DS. This was due to the increased hydrophilicity of mono and disaccharide moieties. On the other hand, the low MIC values (8–32 μg/mL) were obviously observed when the DS of quaternary ammonium N-octyl and N-benzyl chitosan derivatives was lower than 18%. The results showed that the presence of hydrophobic moiety such as the N-benzyl group enhanced the antibacterial activity compared to the hydrophilic moiety against both bacteria.     

N-(carboxymethylidene)chitosans and N-(carboxymethyl)chitosans: Novel chelating polyampholytes obtained from chitosan glyoxylate

Glyoxylic acid, added to aqueous suspensions of chitosan, causes immediate dissolution of chitosan and gel formation within 3–4 h if the pH is 4.5–5.5. Solutions at lower pH values gel after 2 min of warming at 60–80°. Chitosan glyoxylate solutions brought to alkaline pH with sodium hydroxide do not precipitate chitosan. Evidence is given that a Schiff base, namely N-(carboxymethylidene)chitosan, is formed. N-(Carboxymethylidene)chitosans are reduced by sodium cyanoborohydride at room temperature to give N-(carboxymethyl)chitosans, obtained as white, free-flowing powders, soluble in water at all pH values. A series of N-(carboxymethyl)chitosans having various degrees of acetylation and N-carboxymethylation was obtained, and characterized by viscometry, elemental analysis, and i.r. spectrometry. For the fully substituted N-(carboxymethyl)chitosans, the pK′ is 2.3, the pK″ is 6.6, and the isoelectric point is 4.1. The addition of N-(carboxymethyl)chitosan to solutions (0.2–0.5mm) of transition-metal ions produces immediate insolubilization of N-(carboxymethyl)chitosan-metal ion chelates.     

Fixed-bed biosorption of Cu2+ by polyacrylonitrile-immobilized dead cells of Saccharomyces cerevisiae

Dead cells of Saccharomyces cerevisiae 54 were immobilized by entrappment in polyacrylonitrile. The beads obtained were used to adsorb copper in an up-flow fixed-bed column. The effect of polymer content and cell loading were studied to optimize the porosity and the efficiency in copper removal of the biosorbent beads in a batch system. The optimal concentration of the polyacrylonitrile was assumed to be 12%(w/v) and a concentration of 0.5 g cell dry weight in 1 g polymer was most effective in adsorption of Cu²⁺. The adsorption capacity of this biosorbent was 27 mg Cu²⁺/g dry biomass at 200 mg/l initial concentration of copper ions. Adsorption of Cu²⁺ in a batch system was studied using different initial concentrations of the solute. The optimal conditions in the up-flow column of the following parameters were determined: flow rate, bed height, and initial concentration of Cu²⁺ of the solutions. Results of fixed-bed biosorption showed that breakthrough and saturation time appeared to increase with the bed height, but decrease with the flow rate and the initial concentration. The linearized form of the Thomas equation was used to describe dynamic adsorption of metal ions. As a result, the adsorption capacity of the batch system and the column system was compared. Desorption of copper ions was achieved by washing the column biomass with 0.1 M HCl at an eluent flow rate of 1 ml/min. The reusability of the immobilized biomass was tested in five consecutive adsorption-desorption cycles. The regenerated beads retained over 45% of their original adsorption capacity after five A/D cycles. This revised version was published online in August 2006 with corrections to the Cover Date.     

Effect of dietary chitosans on trace iron, copper and zinc in mice

Dietary chitosans with different molecular weight M_w and the degree of deacetylation DDA (high molecular weight chitosan HCS with M_w 7.60 × 10⁵ and DDA 85.5%, middle molecular weight chitosan MCS with M_w 3.27 × 10⁴ and DDA 85.2%, chito-oligomer COS with M_w 0.99 × 10³ and DDA 85.7% and water-soluble chitosan WSC with M_w 3.91 × 10⁴ and DDA 52.6%) were used at the 1.05% level to feed mice for 90 days. Afterwards no pathological symptoms, clinical signs or deaths were observed. The body weight of mice in chitosan group and control group showed no significant difference. Although HCS, COS and WSC had no significant effect on the level of Fe, Zn and Cu in the tested mice’s liver, spleen, heart and kidney, MCS significantly increased the level of Fe, Zn and Cu in liver. Therefore dietary ingestion of chitosan did not depress the level of Fe, Zn and Cu in mice.     

Chitosan/cellulose-based beads for the affinity purification of histidine-tagged proteins

Chitosan/cellulose-based beads (CCBs) for the affinity purification of histidine-tagged proteins were prepared from chitosan/cellulose dissolved in ionic liquid as a solvent, and their structures were characterized by Fourier transform infrared spectroscopy, transmission electron microscopy, and thermogravimetric analysis. The affinity purification was used to separate hexahistidine-tagged (his-tagged) enhanced green fluorescent protein (EGFP) from Escherichia coli. The results showed that Zn²⁺–CCB exhibited more specific adsorption capacity toward the target protein compared with Ni²⁺–CCB and Cu²⁺–CCB. The maximum adsorption of EGFP was 1.84?mg/g of Zn²⁺–CCB, with 90% purity under the optimized conditions (ionic strength (1.0?M NaCl), pH (7.2) and imidazole concentration (500?mM)). In addition, a regeneration method for the sorbent was further developed by washing with ethylenediaminetetraacetic acid disodium and then reimmobilizing with metal ions. This technique is an alternative method for the purification of his-tagged proteins, making the process more economical, fast, stable, and large batch.     

Colorimetric assay of chitosan in presence of proteins and polyelectrolytes by using o-phthalaldehyde

A novel approach of colorimetric quantification of chitosan based on the derivatization reaction of its primary amino groups with o-phthalaldehyde and a thiol – N-acetyl-l-cysteine has been developed. The reaction of equal volumes of sample solution and the reagent solution was allowed to proceed for 1 h, and then the absorbance values were measured at 340 nm against the reference solution. The procedure conditions have been optimized for chitosan assay in the presence of polyanionic electrolyte dextran sulphate (pH 8.9, the reagent solution: 4.0 mM o-phthalaldehyde, 2.6 mM N-acetyl-l-cysteine, 0.25 M NaCl). The method has proven to be convenient and reliable for quantitative determination of either the concentrations of chitosans of various molecular weights or their degree of deacetylation. The different reactivity of chitosans and proteins can be used in order to determine chitosan in presence of the protein. This approach ensured accurate assay within the chitosan concentrations ranging from 0.01 to 0.15 mg/ml and could be applied for quantitative analysis of chitosan in protein-loaded microparticles.     

Effect of ultrasonic treatment on the biochemphysical properties of chitosan

Four chitosans with different molecular weights and degrees of deacetylation degree and 28 chitosans derived from these initial chitosans by ultrasonic degradation have been characterized by gel permeation chromatography (GPC), FT-IR spectroscopy, X-ray diffraction and titrimetric analyses. Antimicrobial activities were investigated against E. coli and S. aureus using an inhibitory rate technique. The results showed that ultrasonic treatment decreased the molecular weight of chitosan, and that chitosan with higher molecular weight and higher DD was more easily degraded. The polydispersity decreased with ultrasonic treatment time, which was in linear relationship with the decrease of molecular weight. Ultrasonic degradation changed the DD of initial chitosan with a lower DD (<90%), but not the DD of the initials chitosan with a higher DD (>90%). The increased crystallinity of ultrasonically treated chitosan indicated that ultrasonic treatment changed the physical structure of chitosan, mainly due to the decrease of molecular weight. Ultrasonic treatment enhanced the antimicrobial activity of chitosan, mainly due to the decrease of molecular weight.     

Antioxidant,Cytotoxic and Antimicrobial Activity of Chitosan Preparations Extracted from Ganoderma Lucidum Mushroom

Two chitosan extracts were prepared by chemical and enzymatic treatment of Ganoderma lucidum mushroom, as an alternative source to crustacean shells. The molecular weight of the enzymatic extract was lower than that of the chemical one and of shrimp chitosan, as determined by viscosity measurements. Characteristic signals were identified in the ¹H‐NMR spectra and high deacetylation degree indicated good physico‐chemical properties for both mushroom chitosan extracts. The scavenging capacity of mushroom chitosan extracts was moderate against the synthetic radicals of 2,2′‐azinobis(3‐ethylbenzothiazoline‐6‐sulfonic acid) (ABTS) and 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH), but higher values were observed for the enzymatic extract, compared to the chemical extract and shrimp chitosan. In vitro cytotoxicity was evaluated in L929 mouse fibroblast cell lines and the results of MTT assay showed good cytocompatibility in the tested range of concentrations. The growth of Gram‐positive bacteria was inhibited more than Gram‐negative bacteria in the presence of mushroom chitosan extracts, in particular by the chemical one, indicating their efficiency as antimicrobial agents. All these results strengthen the evidence of mushroom polysaccharide preparations availability for biomedical applications.     

Isolation of highly copper‐tolerant fungi from the smelter of the Naganobori copper mine,an historic mine in Japan

Aims: Copper is a critical metal of modern industry, and is the most widespread heavy metal contaminant in wastewater. Therefore, isolation of copper‐tolerant microbes having the potential as biosorbent is fascinating not only from an environmental microbiology, but also from a biotechnology view point. In this study, we attempted to isolate highly copper‐tolerant microbes from soil samples of the Nabanobori copper mine, the oldest mine in Japan. Methods and Results: As a result of an enrichment culture, two fungal strains were isolated from soil of the smelter remains. The isolates could grow in a maximum of 200 mmol l^?l Cu²⁺, and grew under a wide pH range. The Cu²⁺‐binding capacity of nontreated biomass of the isolates was around 35 mg Cu²⁺ g^?1‐biomass. Analysis of 18S rDNA suggested that the isolates belong to the Aspergillus/Penicillium clade, but they represented a distinct lineage against known neighbours. Conclusion: The isolates were highly copper‐tolerant, and their Cu²⁺‐binding capacity was comparable to well‐studied fungal sorbents. The isolates were implied as novel species. Soil of the historic old mine under weather‐beaten conditions might be a suitable source for metal‐tolerant microbes. Significance and Impact of the Study: The present results advance our understanding of metal‐tolerant microbes, and offer a new tool for both environmental control and metal recovery operations.     

Comparison of the Adsorption Capabilities of Myriophyllum spicatum and Ceratophyllum demersum for Zinc,Copper and Lead

Industrial wastewaters contain various heavy metal components and therefore threaten aquatic bodies. Heavy metals can be adsorbed by living or non‐living biomass. Submerged aquatic plants can be used for the removal of heavy metals. This paper exhibits the comparison of the adsorption properties of two aquatic plants Myriophyllum spicatum and Ceratophyllum demersum for lead, zinc, and copper. The data obtained from batch studies conformed well to the Langmuir Model. Maximum adsorption capacities (q_max) were obtained for both plant species and each metal. The maximum adsorption capacities (q_max) achieved with M. spicatum were 10.37 mg/g for Cu²⁺, and 15.59 mg/g for Zn²⁺ as well as 46.49 mg/g for Pb²⁺ and with C. demersum they were 6.17 mg/g for Cu²⁺, 13.98 mg/g for Zn²⁺ and 44.8 mg/g for Pb²⁺. It was found that M. spicatum has a better adsorption capacity than C. demersum for each metal tested. Gibbs free energy and the specific surface area based on the q_max values were also determined for each metal.     

Cost-effective copper removal by electrosorption powered by microbial fuel cells

This work studied a cost-effective electrosorption that driven by microbial fuel cells (MFC-sorption) to remove Cu²⁺ from wastewater without an external energy supply. The impact factors, adsorption isotherms and kinetics of the novel process were investigated. It indicated that a low electrolyte concentration and a high solution pH could enhance the Cu²⁺ removal efficiency, while the adsorption capacity increased with the increase of numbers of MFCs in series and the initial Cu²⁺ concentration. The adsorption isotherms study indicated that the monolayer adsorption in MFC-sorption was dominant. The kinetics study suggested the increase of initial Cu²⁺ concentration could enhance the initial adsorption rate. The electrode characterizations verified the existence of Cu₂O and Cu on the electrode surface of active carbon fibers (ACFs), suggesting that MFC-sorption was not only an adsorption process, but also a redox reaction process.