Modification of microstructure and selected physicochemical properties of bacterial cellulose produced by bacterial isolate using hydrocolloid-fortified Hestrin–Schramm medium

Bacterial cellulose (BC) is a biopolymer with applications in numerous industries such as food and pharmaceutical sectors. In this study, various hydrocolloids including modified starches (oxidized starch—1404 and hydroxypropyl starch—1440), locust bean gum, xanthan gum (XG), guar gum, and carboxymethyl cellulose were added to the Hestrin-Schramm medium to improve the production performance and microstructure of BC by Gluconacetobacter entanii isolated from coconut water. After 14-day fermentation, medium supplemented with 0.1% carboxymethyl cellulose and 0.1% XG resulted in the highest BC yield with dry BC content of 9.82 and 6.06 g/L, respectively. In addition, scanning electron microscopy showed that all modified films have the characteristic three-dimensional network of cellulose nanofibers with dense structure and low porosity as well as larger fiber size compared to control. X-ray diffraction indicated that BC fortified with carboxymethyl cellulose exhibited lower crystallinity while Fourier infrared spectroscopy showed characteristic peaks of both control and modified BC films.     

Cloning of a Gene Cluster from Cellvibrio mixtus which Codes for Cellulase, Chitinase, Amylase, and Pectinase

The soil isolate Cellvibrio mixtus UQM2294 degraded a variety of polysaccharides including microcrystalline cellulose. Among 6,000 cosmid clones carrying C. mixtus DNA, constructed in Escherichia coli with pHC79, 50 expressed the ability to degrade one or more of the following substrates: carboxymethyl cellulose, chitin, pectin (polygalacturonic acid), cellobiose, and starch. These degradative genes are encoded in a single 94.1-kilobase segment of the C. mixtus genome; a preliminary order of the genes is starch hydrolysis, esculin hydrolysis, cellobiose utilization, chitin hydrolysis, carboxymethyl cellulose hydrolysis, and polygalacturonic acid hydrolysis. A restriction endonuclease cleavage map was constructed, and the genes for starch, carboxymethyl cellulose, cellobiose, chitin, and pectin hydrolysis were subcloned.     

Immobilization of the α-amylase of Bacillus amyloliquifaciens TSWK1-1 for the improved biocatalytic properties and solvent tolerance

The α-amylase of Bacillus amyloliquifaciens TSWK1-1 (GenBank Number, GQ121033) was immobilized by various methods, including ionic binding with DEAE cellulose, covalent coupling with gelatin and entrapment in polyacrylamide and agar. The immobilization of the purified enzyme was most effective with the DEAE cellulose followed by gelatin, agar and polyacrylamide. The K _m increased, while V _max decreased upon immobilization on various supports. The temperature and pH profiles broadened, while thermostability and pH stability enhanced after immobilization. The immobilized enzyme exhibited greater activity in various non-ionic surfactants, such as Tween-20, Tween-80 and Triton X-100 and ionic surfactant, SDS. Similarly, the enhanced stability of the immobilized α-amylase in various organic solvents was among the attractive features of the study. The reusability of the immobilized enzyme in terms of operational stability was assessed. The DEAE cellulose immobilized α-amylase retained its initial activity even after 20 consequent cycles. The DEAE cellulose immobilized enzyme hydrolyzed starch with 27 % of efficiency. In summary, the immobilization of B. amyloliquifaciens TSWK1-1 α-amylase with DEAE cellulose appeared most suitable for the improved biocatalytic properties and stability.     

<Emphasis Type="Italic">Rhodococcus</Emphasis> sp. Q5, a novel agarolytic bacterium isolated from printing and dyeing wastewater

An agar-degrading bacterium, Rhodococcus sp. Q5, was isolated from printing and dyeing wastewater using a mineral salts agar plate containing agar as the sole carbon source. The bacterium grew from pH 4.0 to 9.0, from 15 to 35°C, and in NaCl concentrations of 0–5 %; optimal values were pH 6.0, 30°C, and 1 % NaCl. Maximal agarase production was observed at pH 6.0 and 30°C. The bacterium did not require NaCl for growth or agarase production. The agarase secreted by Q5 was inducible by agar and was repressed by all simple sugars tested except lactose. Strain Q5 could hydrolyze starch but not cellulose or carboxymethyl cellulose. Agarase activity could also be detected in the medium when lactose or starch was the sole source of carbon and energy. Strain Q5 could grow in nitrogen-free mineral media; an organic nitrogen source was more effective than inorganic carbon sources for growth and agarase production. Addition of more organic nitrogen (peptone) to the medium corresponded with reduced agarase activity.     

Role of Sodium Carboxymethyl Cellulose and Hydroxyethyl Starch in Hematopoietic Cell Line Cultures

Sodium carboxymethyl cellulose and hydroxyethyl starch were found to support rapid growth of two hematopoietic cell lines. The polymers were not metabolized by the cells. In the presence of these compounds, lower rates of glucose utilization and lactic acid production were observed. The uptake of glucose by the cells decreased as the concentration of the polymer in the medium was increased. These results indicate that sodium carboxymethyl cellulose and hydroxyethyl starch probably protect the cells against physical stress in suspended cultures.     

Identification of a flavobacterium strain virulent against Giardia lamblia cysts

We have isolated from a Kentucky stream a bacterial strain capable of killing the cyst form of Giardia lamblia. This bacterium, designated Sun4, is a Gram-negative, aerobic rod which produces a yellow pigment, but not of the flexirubin-type. Although true gliding motility has not been observed in Sun4, this strain does exhibit a spreading colony morphology when grown on R2A agar. Strain Sun4 has been identified by 16S rRNA sequencing and phylogenetic analysis as belonging to the genus Flavobacterium, and is most closely related to Cytophaga sp. strain Type 0092 and associated Flavobacterium columnare strains. Lipid analysis also identified fatty acids characteristic of the Cytophaga–Flavobacterium group of bacteria. In culture, Sun4 is able to degrade casein and cellulose, but not chitin, gelatin, starch, or agar. Degradation of Giardia cysts by Sun4 appears to require direct cellular contact as neither cell-free extracts nor cells separated from the cysts by dialysis membranes showed any activity against cysts. Activity against Giardia cysts is rapid, with Sun4 killing over 90% of cysts within 48 h. Strain Sun4 requires elevated levels of Ca²⁺ for optimal growth and degradative activity against Giardia cysts. We propose that bacterial strains such as Sun4 could be used as biological control agents against Giardia cysts in drinking water treatment systems.     

The structure of water absorbed in collagen. I. The dielectric properties

The dielectric constant and the loss factor of water in collagen are measured for various water, NaCl, and HCl contents at frequencies ranging from 10² to 10⁵ Hz and at temperatures ranging from ?70° to +23°C. For all measurements, both the dielectric constant and the loss decrease monotonically as the frequency increases, or the temperature decreases; the absence of a maximum in the loss curves as a function of temperature and frequency indicates an extremely broad spectrum of relaxation times. By shifting the curves obtained for the dielectric constant and the loss factor along the log–frequency axis, all data, obtained at different temperatures, can be represented on master curves valid for 23°C. In order to explain these results, water molecules are assumed to be hydrogen bonded to each other in long chains. All water molecules in a chain can, cooperatively, be oriented in two different directions along the channel, resulting in large, reversible, dipole moments. These chains are not rigid, but are flexible liquid-like structures. Diffusion of chains as entities is assumed to be the rate-limiting step for dipole reorientation. Although the rate of diffusion decreases inversely proportional to chain length, the activation energy is independent of chain length. At lower temperatures, diffusion becomes slower, until at the glass point, approximately ?100°C, it ceases.     

Biologically active polymer supports based on cellulosic derivatives: synthesis and kinetic study

Bioactive cellulose derivatives have been synthesised by coupling enzymes/antibiotics on carboxymethyl cellulose acid chloride and cellulose carbonate. The effect of pH and temperature on the enzymatic activity of amyloglucosidase immobilised on cellulose carbonate was studied. Michaelis-Menten kinetics have been obeyed to the first degree of approximation despite the restricted mobility of the attached enzyme on the polymer support. Lineweaver-Burk plots for the amyloglucosidase immobilized on carboxymethyl cellulose acid chloride at ambient pH with cellulose carbonate at pH 8 have also been plotted. The Michaelis-Menten constant for the immobilized amyloglucosidase on cellulose carbonate at pH 8 was 9.1 mM, and the activation energy for starch hydrolysis was 21.8 kcals/mole.     

Production of agarase from a novel <Emphasis Type="Italic">Micrococcus</Emphasis> sp. GNUM-08124 strain isolated from the East Sea of Korea

The agar degrading bacterial strain GNUM-08124 was isolated from Enteromorpha compressa collected in the East Sea of Korea by using a selective artificial sea water (ASW) agar plate containing agar as the sole carbon source. GNUM-08124 grows to produce a circular, smooth, yellow-colored, and raised colony. Its ability to hydrolyze agar was confirmed by staining the ASW agar plate with Lugol’s solution. In liquid culture, the cell density (A₆₀₀) increased exponentially and reached a maximum level on the third day of cultivation. The specific agarase activity also increased in proportion to the cell density and reached maximum agarolytic activity on the third day. The 16S rRNA sequence of GNUM-08124 showed a close relationship to Micrococcus luteus (99.65%) and Micrococcus endophyticus (99.15%), which led us to assign it to the genus Micrococcus. Physiological studies indicated that optimal growth conditions were between 30 and 40°C, pH 4 and 7, using media containing between 5 and 10% NaCl (w/v), respectively. The GNUM-08124 strain was a grampositive, urease-positive, and catalase-positive bacterium. It could not hydrolyze gelatin, cellulose, xylan, or starch, but fermented a broader range of substrates, including Dglucose, D-galactose, D-fructose, D-lactose, D-trehalose, D-mannitol, D-melibiose, D-raffinose, D-xylose, methyl-α-D-glucopyranoside, N-acetyl-glucosamine, and xylitol, than those fermented by M. luteus or M. endophyticus, suggesting GNUM-08124 is a novel agar hydrolyzing microorganism belonging to Genus Micrococcus. Micrococcus sp. GNUM-08124 showed the highest agarase activity when it was cultured in ASW-YP medium supplemented with 0.4% glucose, but demonstrated lower activity in rich media (LB or TSB), in spite of superior cell growth, implying that agarase production is tightly regulated in an agar-dependent manner and repressed in rich conditions.     

Massilia kyonggiensis sp. nov., isolated from forest soil in Korea

A Gram-negative, short, rod-shaped bacterium, TSA1^T, was isolated from forest soil collected at Kyonggi University, South Korea. Assessment of 16S rRNA gene sequence similarity indicated that the strain is related to Massilia niastensis 5516S-1^T (98.3%), M. haematophila CCUG 38318^T (97.9%), M. aerilata 5516S-11^T (97.9%), M. tieshanensis TS3^T (97.6%), and M. varians CCUG 3529^T (97.1%). Colonies grown on Reasoner’s 2A agar at 30°C for 2 days were transparent, white, round, smooth, and glossy. The cells grew at 10–42°C (optimum: 25–37°C) and pH 5–9 (optimum: 5–9) and in 0–2% NaCl (optimum: 0–1%). TSA1^T was able to grow on trypticase soy and nutrient agar, but not on Luria-Bertani or MacConkey agar. The strain was catalase- and oxidasepositive and able to degrade starch and casein, but not carboxymethyl cellulose. The predominant quinone of TSA1^T was Q-8, the major fatty acids were summed feature 3 and C_16:0, and the DNA G+C content was 66.7 mol%. Given these findings, we propose that this strain is a novel species of the genus Massilia. We suggest the name Massilia kyonggiensis sp. nov. (type strain, KACC 17471^T =KEMB 9005-031^T =JCM 19189^T).     

Dielectric saturation of the aqueous boundary layers adjacent to charged bilayer membranes

Summary The surface charge density resulting from the adsorption of hydrophobic anions of dipicrylamine onto dioleyl-lecithin bilayer membranes has been measured directly using a high field pulse method. The surface charge density increases linearly with adsorbate concentration in the water until electrostatic repulsion of impinging hydrophobic ions by those already adsorbed becomes appreciable. Then Gouy-Chapman theory predicts that surface charge density will increase sublinearly, with the power [z ⁺/(z ⁺+2)] of the adsorbate concentration, wherez ⁺ is the cation valence of the indifferent electrolyte screening the negatively charged membrane surface. The predicted 1/3 and 1/2 power laws for univalent and divalent cations, respectively, have been observed in these experiments using Na⁺, Mg⁺⁺, and Ba⁺⁺ ions. Gouy-Chapman theory predicts further that the change from linear to sublinear dependence takes place at a surface charge density governed by the static dielectric constant of water and the concentration of indifferent electrolyte. Quantitative agreement with experiment is obtained at electrolyte concentrations of 10^–4 m and 10^–3 m, but can be maintained at higher concentrations only if the aqueous dielectric constant is decreased. A transition field model is proposed in which the Gouy-Chapman theory is modified to take account of dielectric saturation of water in the intense electric fields adjacent to charged membrane surfaces.     

Wood biodeterioration control potential of Acalypha hispida leaf phenolic extract in combination with Trichoderma viride culture filtrate

The phenolic extract of Acalypha leaves inhibited growth of Gloeophyllum sepiarium and Pleurotus sp. (test wood-rot fungi) in potato dextrose agar, starch agar, starch glucose agar, carboxyl methyl cellulose agar and carboxyl methyl cellulose glucose agar. Fungicidal or fungistatic concentration of the extract (10–14 mg/ml) depended on the medium. However a lower concentration of the extract (8–10 mg/ml) in combination with Trichoderma viride culture filtrate caused a similar inhibitory pattern. Degradation of obeche (Triplochiton scleroxylon), mahogany (Khaya ivorensis) and walnut (Lovoa trichilioides) by the test fungi was limited or prevented by extract treatment of 8–10 mg/g wood. A similar inhibitory effect again occurred when a combination of T. viride filtrate and lower extract concentration (6–8 mg extract per gram of wood) was used. On-going wood decay was limited or halted by a combined treatment involving 8–12 mg extract per gram of wood depending on the fungal residence period. Treated stakes exposed to 6 months of tropical wet season retained resistance to fungal attack including soft rot. The phenolic extract of A. hispida may prove useful in an integrated chemical and biological approach to wood treatment.     

Genome Sequence of <Emphasis Type="Italic">Microbulbifer mangrovi</Emphasis> DD-13<Superscript>T</Superscript> Reveals Its Versatility to Degrade Multiple Polysaccharides

Microbulbifer mangrovi strain DD-13^T is a novel-type species isolated from the mangroves of Goa, India. The draft genome sequence of strain DD-13 comprised 4,528,106 bp with G+C content of 57.15%. Out of 3479 open reading frames, functions for 3488 protein coding sequences were predicted on the basis of similarity with the cluster of orthologous groups. In addition to protein coding sequences, 34 tRNA genes and 3 rRNA genes were detected. Analysis of nucleotide sequence of predicted gene using a Carbohydrate-Active Enzymes (CAZymes) Analysis Toolkit indicates that strain DD-13 encodes a large set of CAZymes including 255 glycoside hydrolases, 76 carbohydrate esterases, 17 polysaccharide lyases, and 113 carbohydrate-binding modules (CBMs). Many genes from strain DD-13 were annotated as carbohydrases specific for degradation of agar, alginate, carrageenan, chitin, xylan, pullulan, cellulose, starch, β-glucan, pectin, etc. Some of polysaccharide-degrading genes were highly modular and were appended at least with one CBM indicating the versatility of strain DD-13 to degrade complex polysaccharides. The cell growth of strain DD-13 was validated using pure polysaccharides such as agarose or alginate as carbon source as well as by using red and brown seaweed powder as substrate. The homologous carbohydrase produced by strain DD-13 during growth degraded the polysaccharide, ensuring the production of metabolizable reducing sugars. Additionally, several other polysaccharides such as carrageenan, xylan, pullulan, pectin, starch, and carboxymethyl cellulose were also corroborated as growth substrate for strain DD-13 and were associated with concomitant production of homologous carbohydrase.     

Detection and production condition of acetylxylan esterase from a wood-rotting fungus,Coriolus versicolor

Acetyl esterase production was detected in a wood-rotting fungus,Coriolus versicolor, by the formation of a clear zone on a double layer agar plate containing glucose β-d-pentaacetate. Two polysaccharide acetates, carboxymethyl cellulose acetate and xylan acetate, also served as detectable substrates in place of glucose acetate to form clear zone. In an esterase assay, this fungal esterase showed a higher specificity to acetylxylan than did porcine liver esterase, indicating that it is an acetylxylan esterase.     

Study of Bound Water of Poly-Adenine Using High Frequency Dielectric Measurements

The high frequency dielectric constant of poly-adenine (poly-A) was measured between 1 MHz and 1 GHz. The purpose of these experiments was to investigate the state of water molecules that are bound to the charged groups of the poly-A molecule. Analysis of the data using the Maxwell's mixture equation revealed the dielectric constant of bound water higher than we expected. Using Onsager's internal field in Debye's equation, we calculated the dielectric constant of water in the vicinity of a charged ion. The result of this computation demonstrates that the dielectric constant of bound water is much smaller than the normal value only in the immediate proximity of charged ions (within 2 Å). The dielectric constant increases rapidly to the normal value as the distance increases from 2 to 4 Å. This observation indicates that charged sites of polyions have only short range interactions with the surrounding water molecules. However, this conclusion pertains only to rotary diffusion of bound water since dielectric measurement is unable to detect translational diffusion.     

Impact of Physicochemical Environment on the Super Disintegrant Functionality of Cross-Linked Carboxymethyl Sodium Starch: Insight on Formulation Precautions

The aim of this work is to improve the understanding of the physicochemical mechanisms involved in the functionality of cross-linked carboxymethyl sodium starch (CCSS) as a tablet super disintegrant (SD). The behavior and properties of this SD (medium uptake, disintegration times, particle size, and rheology) was investigated in a wetting medium of different physicochemical properties. In particular, the relative permittivity (dielectric constant) of these media was intentionally modified for evaluating its effect on CCSS properties. Results showed different swelling behaviors of CCSS particles according to the relative permittivity of the tested media and allow to propose two underlying mechanisms that explain CCSS functionality. Both the intra-particular swelling and the inter-particular repulsion are affected by the relative permittivity of the media. Finally, disintegration test performed on tablets specially formulated with mannitol (used commonly as an excipient and known to modify relative permittivity) confirmed that the functionality of CCSS and therefore the disintegration of the tablet can be altered according to the mannitol content.KEY WORDS: cross-linked carboxymethyl sodium starch, disintegrant functionality, orally disintegrating tablets, relative permittivity, repulsive layers     

福寿螺和田螺消化酶活性比较

为比较福寿螺(Pomacea canaliculata(Lamarck,1828))和当地中国圆田螺(Cipangopaludina chinensis(Gray,1832))消化能力的差异,探索福寿螺成功入侵的机制,以田螺为对照,测定了1—4龄的福寿螺和田螺的胃和肝脏的消化酶——纤维素酶(羧甲基纤维素法)、淀粉酶(3,5-二硝基水杨酸法)和脂肪酶(滴定法)的活性。结果表明:1)相同年龄的福寿螺胃和肝脏中的消化酶活性明显高于田螺。其中,纤维素酶活性分别高出1.00—2.11倍、1.66—2.84倍;淀粉酶活性分别高出1.53—3.47倍、1.47—1.80倍;脂肪酶活性分别高出2.07—4.73倍、6.13—9.93倍。2)在生长发育过程中,福寿螺胃和肝脏中的消化酶活性变化幅度(51.2%—131.2%)明显高于田螺(23.3%—47.1%)。3)福寿螺的各种消化酶之间存在协同作用。如福寿螺的淀粉酶活性与脂肪酶活性呈极显著正相关(胃中r=0.736**、肝脏中r=0.867**)。此外,胃中的淀粉酶活性还与纤维素酶活性呈显著正相关关系(r=0.696*)。相应地,田螺胃中的淀粉酶和脂肪酶之间也存在显著的正相关关系(r=0.706*),而肝脏中的纤维素酶与脂肪酶活性呈显著负相关(r=-0.593*)。4)福寿螺对纤维素类和淀粉类物质都有较强的消化能力,且能较好地消化脂肪类物质,而田螺能消化纤维素类和淀粉类物质,对脂肪的消化能力却很弱。福寿螺的纤维素酶和淀粉酶活性分别是田螺的2.42和1.88倍,脂肪酶活性达到了5.66倍。可见,福寿螺具有较高的消化酶活性,且各消化酶之间存在正协同性。这可能是导致福寿螺食量大、食性杂,使其能快速生长和成功入侵的重要原因之一。     

Starch conversion by soluble and immobilized α-amylase

B. subtilis α-amylase was immobilized on cyanogen bromide activated carboxymethyl cellulose. The conversion of wheat starchwas carried out at 72°C in a stirred tank by soluble and immobilized α-amylase. The initial reaction rate with immobilized α-amylase was lower than with the soluble enzyme, but after 1 hr immobilized α-amylase produced a higher quantity of reducing sugars than the soluble enzyme. The action pattern of immobilized α-amylase was different from that of the soluble enzyme: immobilized α-amylase produced relatively more glucose and maltose, except at the beginning of conversion. Immobilized α- readily hydrolyze G₆. The starch conversion by immobilized α-amylase was not diffusion controlled at a stirring rate of 100-300 rpm.     

The influence of the medium dielectric strength upon trypsin kinetics

The use of aqueous alkali for the titration of esterolytic activity when the esters are dissolved in alcoholic solutions, results in an error due to changes in the ionization of the buffer. This is corrected by titrating with alkali in the same solvent as the substrate. Alcohols and other substances which change the dielectric strength of water modify the rate of hydrolysis of BAEE¹ and TSAME by trypsin to an extent proportionate to their effect on the dielectric strength. The reaction rate increases with diminished dielectric strength and vice versa. At low concentrations of substance there seems to be no specific effect other than that derived of the variation in dielectric strength. At higher concentrations, the enzyme might be denatured. In addition, it is probable that specific effects of each substance might intervene. The Coulombic and thermic energies of activation were calculated for the two esters in various solvents. The plot of the logarithm of rate constant vs. reciprocal of dielectric constant yields a straight line with positive slope. This behavior is similar to that of a non-enzymatic positive ion-dipole reaction. Trypsin reacts like a positive ion. The possible influence of the dielectric strength on the regulation of the equilibria involved in the interconversion of the various forms of trypsin in solution (active, inactive, denatured) is discussed.     

The effect of carbohydrate on the development of a Cattleya hybrid in association with its mycorrhizal fungus

To investigate beneficial effects of mycorrhizal fungi to advanced leafy orchids, growth studies on the development of symbiotic seedlings of the orchid Cattleya (aclandiae x schoeffeldiana) x aclandiae were conducted in vitro over a period of 18 months using split plates with minerals and carbohydrates on one side and water agar on the other. Mycorrhizal infection and shoot and root growth of seedlings on the nutrient side were compared to growth on the water agar side with nutrient uptake by the orchid only possible via external mycorrhizal hyphae. Seed germination was followed by mycorrhizal infection and rapid development of protocorms on both nutrient and non-nutrient sides of the plates. With 0.5% starch, development of protocorms was sustained for a least 12 weeks, compared to only 6 weeks with 0.1% starch. Advanced protocorms with two small leaves and a smoll root were transferred at week 22 to new fungal plates. When harvested at week 43, plantlets on 0.5% starch (both nutrient and water agar sides) had 2.7 times the dry weight of plantlets on 0.1% starch. Shoot-root ratios were higher on the lower level of carbon. In all plantlets, mycorrhizal infection involved less than 5% of the root length. With zero, 0.1% or 0.5% starch, the roots were re-infected on transfer to fresh fungal plates but young roots that developed following the transfer stayed free of infection, Plantlets on 0.5% starch (nutrient and water agar side) after 18 months had longer roots than plantlets grown in the absence of starch or on 0.1% starch. Shoots were small but significantly larger on the nutrient side than on the water agar side, independent of the carbohydrate level. The shoot-root ratio was highest on the nutrient side with no starch present. In this latter case, plantlet development was steady but plantlets on the non-nutrient side developed slowly; thus there was little evidence of nutrient translocation by the mycorrhizal fungus from the nutrient to the non-nutrient side in the absence of carbohydrates. Mycorrhizal infection is discussed as a mechanism for heterotrophic carbon assimilation. In advanced leafy orchids of Cattleya, external carbon resulted in increased root growth, decreased shoot/root ratio and sometimes yellowish-green plantlets.