Use of complex media for the production of scleroglucan by Sclerotium rolfsii MTCC 2156

Submerged fermentation was carried out for the production of scleroglucan by Sclerotium rolfsii MTCC 2156 using complex media, such as coconut water, sugarcane molasses and sugarcane juice at 28+/-2 degrees C and 180 rpm for 72 h. Sugarcane juice gave maximum scleroglucan production of 23.87 g/l as compared to 12.58 and 18.45 g/l with coconut water and sugarcane molasses, respectively. Utilization of these substrates would be ecologically sound and economically advantageous.     

Dissolved oxygen and the scleroglucan fermentation process

The effect of culture dissolved oxygen (D.O.) upon biomass, scleroglucan and oxalate formation bySclerotium glucanicum was examined in a stirred tank fermenter by oxygen enrichment. Controlling culture D.O. at 5 or 10% saturation led to increased biomass formation and decreased scleroglucan formation. The mechanism by which this occurred probably involved a non-specific diversion of C source (sucrose) away from product and towards biomass synthesis. This is at variance with the reported stimulatory effect of limiting D.O. levels upon scleroglucan synthesis. Controlling the culture at higher levels (20 and 30%) involved increases in impeller speed. The results of such changes were distinct from those of D.O., thus demonstrating that attempts to examine D.O. effects by means of impeller speed changes are inappropriate.     

Reactor kinetics for submerged aerobic biofilms

Sclerotium rolfsii ATCC 15205 was cultivated in continuous culture (48 l reactor volume) for scleroglucan production. The maximum volumetric productivity (Q_Pvmax) amounted to 7.2 g/ld and was more than twice as high as in comparable batchwise cultivations. In addition, the relation between (a) volumetric productivity (Q_P [g/ld]) and product yield (Y_{Glucan/Glucose} [1]), (b) volumetric productivity and product quality (M_W [g/mol]), and (c) product quality and impeller tip speed (Nd [m/s]) was studied in continuous culture. It was found, that an increase in volumetric productivity led to a decline in product yield and product quality. Furthermore, an impeller tip speed of >0.7 m/s decreased the attainable product quality considerably. Based on these results, the impact of the operational setpoint of the process in terms of oxygen supply and reactor scale on the economics of scleroglucan production was discussed. In contrast to batchwise cultivation, scleroglucan production in continuous culture proved to be not feasible under non-aseptic conditions.     

Influence of the nitrogen source on the production and rheological properties of scleroglucan produced by Sclerotium rolfsii ATCC 201126

Scleroglucan production by Sclerotium rolfsii ATCC 201126 has been studied using nitrate or ammonium as nitrogen sources at several concentrations. In all the experiments carried out, both growth and production were modelled by an unstructured kinetic model using logistic and Luedeking–Piret equations for describing growth and production, respectively. The kinetic parameters for growth ( and Y_XN) and for production ( and ) were obtained by fitting the data to the model using the single-response non-linear regression technique by means of the algorithm of Marquardt coupled to a fourth-order Runge–Kutta algorithm. Biomass and scleroglucan production were higher when nitrate was the nitrogen source. Rheological properties of scleroglucan produced using nitrate as nitrogen source were studied and rheological parameters calculated, revealing similarity between this biopolymer and commercial scleroglucan.     

The cytokine stimulating activity of (1-->3)-beta-D-glucans is dependent on the triple helix conformation

The immunomodulating properties of comb-like branched (1-->3)-beta-D-glucans scleroglucan, schizophyllan and lentinan depend on branching pattern, molecular weight and higher-order structure. The effect of weight average molecular weight Mw and higher order structure of scleroglucan, on stimulation of human monocytes cultured in vitro to secrete tumor necrosis factor-alpha (TNF-alpha) was investigated. The higher order structures of the scleroglucan samples were determined by electron microscopy. The data showed that the samples with a linear wormlike, triple helical structure with Mw less than 50 x 10(4) g/mol or larger than 110 x 10(4) g/mol stimulated the monocytes more efficiently than samples with Mw in the range (67-110) x 10(4) g/mol. The denaturation of the linear triple helices by NaOH (> 0.25 M), followed by neutralization yielded blends of linear and macrocyclic topologies with concomitant irreversible reduction of the cytokine inducing activity compared with the untreated scleroglucans. The dose-dependent ability to activate monocytes to cytokine production was not restored following annealing of the denatured-renatured samples, despite the fact that electron micrographs revealed similar structures of these annealed samples to the starting material. Pre-incubation of monocytes with antibodies against cluster of differentiation antigens CD14 or CD11b reduced the scleroglucan potency to stimulate TNF-alpha secretion mainly for mAb against CD14 in the presence of serum.     

Modeling and scale-up of the unsterile scleroglucan production process with Sclerotium rolfsii ATCC 15205

An empirical model was applied to describe the growth related formation of scleroglucan in batchwise cultivation of Sclerotium rolfsii. In this case, the level of oxygen supply controls the carbon flux into glucan, biomass, and CO₂ evolution and therefore determines the yield coefficients Y_Glucan/BDM and Y_BDM/O2. It was observed that scleroglucan formation is enhanced under microaerobic conditions. However, as the empirical model and data of actual batch cultivations show, different maxima exist for product end concentration [g/l] and volumetric productivity [g/ld] depending on the total oxygen uptake during cultivation. A sufficient bulk mixing of the highly viscous culture suspension becomes particularly important during large-scale cultivations. In addition, the scleroglucan production process proved to be shear sensitive. A correlation between the attainable molecular weight of the glucan and the stirrer tip velocity in bioreactors of different sizes is presented. For all these reasons, a scale-up of this process is very complex. Large-scale cultivations under microaerobic conditions, aiming for maximum product end concentration, were slowed down by poor bulk mixing leading to a lower carbon flux into glucan formation. On the other hand, a scale-up designed for maximum volumetric productivity using high oxygen supply was successfully conducted up to a reactor volume of 1.500 l. To minimize the loss in product quality (molecular weight of the glucan) due to high stirrer tip velocities, a mixing concept was developed employing reduced agitation combined with maximum aeration to secure a sufficient axial bulk mixing in the reactor.     

Microfibrillar structure of PGG-glucan in aqueous solution as triple-helix aggregates by small angle x-ray scattering.

The conformation of polysaccharide PGG-Glucan, isolated from yeast cell walls, in aqueous solution was investigated by small angle x-ray scattering (SAXS) and multidetector gel permeation chromatography coupled with postcolumn delivery (GPC/PCD) techniques in comparison with scleroglucan. It was shown that both polysaccharides exhibit a rigid rod-like conformation in aqueous solution by SAXS experiments. The mass per unit length (M/L) and radius (R) of rod cross section of PGG-Glucan were measured to be 6300 daltons/nm and 1.89 nm, while those of scleroglucan are 2300 and 0.83, respectively. Utilizing a GPC/light scattering technique, the average aggregation number of PGG-Glucan is 9, while that of scleroglucan is around 3. From the comparison of the M/L and R of the respective rod cross sections as well as their aggregation number data, it is concluded that PGG-Glucan is composed of triple helices, which tend to aggregate as triplets in solution, whereas scleroglucan is composed of a single triple helix. The aggregation number distribution of PGG-Glucan was found to range from 1 to about 25 determined by GPC/PCD. From the observation of a Debye-Scherrer ring type of peak in the macroscopic scattering cross section of PGG-Glucan by SAXS, the existence of a small amount of ordered clusters of PGG-Glucan can be deduced. The "lattice parameter" of these ordered fasces-like clusters is consistent with the radius of the individual triple-helical rods forming a microfibrillar superstructure. These results indicate that higher aggregated forms of PGG-Glucan containing up to 8 triple helices behave as ordered fasces-like clusters. We conclude that PGG-Glucan is triple-helix aggregates formed by rigid rods stacking together side by side. We propose a molecular structural model for PGG-Glucan conformations.     

A single nuclear polymorphism in let-7g binding site affects the doubling time of thyroid nodule by regulating KRAS-induced cell proliferation

As an indicator for the malignancy of thyroid nodules (TN), the doubling time of TN was studied in this study to evaluate the effect of rs712 polymorphism on the progression of TN. In addition, we aimed to study the potential molecular mechanisms underlying the pathological effect of rs712 polymorphism upon TN. A Taqman method was used to genotype the patients according to their rs712 polymorphism. Real-time polymerase chain reaction, western blot, Terminal deoxynucleotidyl transferase dUTP nick end labeling assay and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay was conducted to study the correlation between KRAS expression and the pathological effect of rs712 polymorphism. In-silicon analysis and luciferase assay were utilized to establish the regulatory relationship between let-7g and KRAS. KRAS messenger RNA (mRNA)/protein levels in the GG group were upregulated with a decreased apoptosis index. KRAS mRNA was validated to be a virtual target of let-7g. In addition, the mRNA/protein level of KRAS as well as cell proliferation index was decreased in primary thyroid cancer cells genotyped as TT/TG and transfected with KRAS small interfering RNA (siRNA)/let-7g precursors. The cell apoptosis index was evidently elevated in the KRAS siRNA/let-7g precursors group compared with that in the scramble controls. Moreover, KRAS mRNA/protein only showed slight reduction when GG-genotyped primary thyroid cancer cells were transfected by let-7g precursors. Additionally, let-7g precursors exhibited no significant effect on cell proliferation index or cell apoptosis in GG cells. Rs712 polymorphism T>G in the 3′-untranslated region of KRAS interrupts the interactions between let-7g and KRAS mRNA, leading to a higher cell proliferation index and reduced doubling time of TN.     

Scleroglucan gelation by in situ neutralization of the alkaline solution

Scleroglucan gels were prepared by neutralization of aqueous alkaline solution of scleroglucan by in situ release of acid. Transition of scleroglucan chains in a disordered state to the triple-helical state result in cross-linking of the polysaccharide. The pH and the storage and loss moduli, G' and G' ', were determined as a function of time after initiating the pH reduction. Experiments were performed in the temperature range from 20 to 90 degrees C and in the polymer concentration range from C(p) = 10-200 mg/mL. The concentration dependence of the apparent plateau value of the storage modulus showed a lower critical concentration, C(p,0), in the range 12-15 mg/mL needed for gelation and a second power dependence of G' on concentration in the range 50-200 mg/mL. In situ pH reduction was achieved using formamide (methanoic acid) or ethyl acetate (ethyl ethanoate) which hydrolyze in alkaline solution, yielding carboxylic acids. The more rapid hydrolyses of ethyl acetate compared to formamide yielded a faster decrease of pH in solution and a more rapid increase of the storage modulus. The present gelation process of scleroglucan based on the in situ reduction of pH has advantages in applications where external control of the conditions is difficult to achieve.     

Low-cost method for the preservation of Sclerotium rolfsii Proimi F-6656: Inoculum standardization and its use in scleroglucan production

Summary The Castellani's Method for the preservation of Sclerotium rolfsii in sterile distilled water was tested. Culturing on Potato Dextrose Yeast extract (PDY) slants, the current system used, was also evaluated. Preservation of sclerotia according to the Castellani's method allowed the strain survival for more than two years. Comparing with the strain periodically activated, a critical decrease (about 80%) in -glucan synthesizing capacity was detected for mycelium preserved either on PDY slants or in water. Activation of stored sclerotia followed by subculturing in liquid Production Medium (PM) allowed preparation of homogeneous suspensions for batch fermentations, and scleroglucan concentrations achieved were similar to those with the strain periodically activated.     

Short chain regioselectively hydrolyzed scleroglucans induce maturation of porcine dendritic cells

Branched β-1,3/1,6-glucans (scleroglucan) were produced by cultivation of Sclerotium rolfsii ATCC 15205. Regioselective hydrolysis at the β-1,3-linkage of the cell-free and purified polysaccharide was performed in borosilicate glass bottles at pH 5, 121°C, and 1 bar for 72 h. The mixture was divided into four molar mass fractions by stepwise cross-flow filtration using different cutoffs. In vitro studies revealed that scleroglucan hydrolysates with a low molar mass of less than 5 kDa significantly stimulated the activation and maturation of porcine monocyte derived dendritic cells (MoDC) by upregulation of CD40 and CD80/86 as well as by reduction of antigen uptake. MoDC treated with low molar mass scleroglucan showed a considerable increase in the amounts of secreted proinflammatory cytokine tumor necrosis factor alpha and stimulated the proliferation of lymphocytes. Therefore, scleroglucan molecules of low molecular weight are able to induce activation and maturation of porcine DC, which are key initiators of inflammatory and adaptive immune responses, and could provide improved protection against infectious diseases.     

Enhancement of 5-aminolevulinate production with recombinant Escherichia coli using batch and fed-batch culture system

5-Aminolevulinate (ALA) production with recombinant Escherichia coli Rosetta (DE3)/pET28a(+)-hemA was studied. In batch fermentation, the addition of glucose and glycine was effective to improve ALA production. Then the fed-batch fermentation was conducted with continuous feeding of precursors. When the concentrations of succinic acid and glycine were 7.0 g/l and 4.0 g/l, respectively, in the feeding, the ALA yield reached 4.1g/l. But the molar yield (ALA/glycine) was decreased in the fed-batch fermentation compared to batch fermentation. And it was found that the pH control during fed-batch cultivation was very important for the cell growth and ALA production. A two-stage pH value controlling strategy was suggested, in which, the pH value in the first 6h was regulated at pH 5.9, after then at pH 6.2, and the ALA yield was as high as 6.6g/l via fed-batch fermentation.     

High scleroglucan production by Sclerotium rolfsii: Influence of medium composition

Scleroglucan production by Sclerotium rolfsii was markedly affected by the C-source concentration, showing a highest value with 150 g sucrose l–1. Production was also influenced by the N-source, being considerably higher in media containing NO3– than in those containing NH4, which had a clear inhibitory effect. Once defined the optimum culture medium composition, the highest exopolysaccharide production (ca. 26 g scleroglucan l–1) was achieved after 72 h of fermentation at shake flask scale. High values of yield (Yp/c = 0.49), productivity (Pr = 0.365 g l–1 h–1) and specific productivity (Pr/x = 0.031 g (g biomass)–1 h–1) were observed, and productivity was 1.5 times further increased by scalling-up to fermenter scale. Addition of L-threonine, sunflower oil and ascorbic acid diminished exopolysaccharide production. © Rapid Science Ltd. 1998     

Oxygen uptake rate optimization with nitrogen regulation for erythromycin production and scale-up from 50 L to 372 m3 scale

Effects of different nitrogen sources on the erythromycin production were investigated in 50 l fermenter with multi-parameter monitoring system firstly. With the increase of soybean flour concentration from 27 g/l to 37 g/l to the culture medium, the erythromycin production had no obvious increase. Whereas adding corn steep liquor 15 g/l in the medium was beneficial for the production of erythromycin, the maximum erythromycin production was 22.2% higher than that of the control. It was found that corn steep liquor can regulate and enhance the oxygen uptake rate (OUR) which characterizes the activity of the microbial metabolism by inter-scale observation and data association. Both Intracellular and extracellular organic acids of central metabolism were analyzed, and it was found that the whole levels of lactic acid, pyruvic acid, citric acid, and propionic acid were higher than those of control before 64th h. The consumption amount of amino acids, which could be transformed into the precursors for erythromycin synthesis (i.e. threonine, serine, alanine, glycine and phenylalanine), were elevated compared with the control in erythromycin biosynthesis phase. The results indicated that corn steep liquor can regulate OUR to certain level in the early phase of fermentation, and enhance the metabolic flux of erythromycin biosynthesis. Erythromycin production was successfully scaled up from a laboratory scale (50 l fermenter) to an industrial scale (132 m(3) and 372 m(3)) using OUR as the scale-up parameter. Erythromycin production on industrial scale was similar to that at laboratory scale.     

Identification and analysis of the metabolic functions of a high-salt-tolerant halophilic aromatic yeast <Emphasis Type="Italic">Candida etchellsii</Emphasis> for soy sauce production

Salt-tolerant yeasts are very important for the flavor formation in soy sauce fermentation production. A halophilic aromatic yeast was isolated on the basis of the molecular biological and metabolic functions from soy sauce. The ITS nucleotide sequence alignment method was used to identify the strain as Candida etchellsii by subjecting the sequence to NCBI-BLAST in comparison with that of the C. etchellsii strain Miso 0208 (a typical high-salt-tolerant halophilic aromatic yeast strain). Organic acids, amino acids and volatile flavor compounds were produced by the yeast strain which were analyzed by HPLC and SPME-GC/MS methods. Tartaric acid (0.979 ± 0.040 g/l), formic acid (0.636 ± 0.030 g/l), lactic acid (2.80 ± 0.10 g/l), α-alkone glutaric acid (0.132 ± 0.015 g/l), citric acid (2.59 ± 0.10 g/l) and succinic acid (3.03 ± 0.20 g/l) were detected at 72 h of fermentation, respectively. Free and acid hydrolyzed amino acids at levels of 3.7355 ± 0.0027 and 11.5604 ± 0.0037 g/l, respectively, 4-ethyl guaiacols as well as other volatile flavor compounds were also detected.     

Structural properties of poly C-scleroglucan complexes

Successive changes of solvent conditions can be used to dissociate and reassociate the triple-helical structure of (1,3)-beta-D-glucans. Ultramicroscopic techniques have revealed a blend of circular and other structures following renaturation. When this solvent exchange process is carried out in the presence of certain polynucleotides, the process creates a novel macromolecular complex. Here, we use size exclusion chromatography (SEC) to study such (1,3)-beta-D-glucan-polynucleotide complexes. Online multi-angle laser-light scattering (MALLS) and refractive index (RI) detectors allowed determination of molecular weight and radius of gyration of the molecules. An ultraviolet (UV) detector allowed specific detection of the polynucleotide. The poly-cytidylic acid (poly C) shifted to coelution with the linear fraction of the scleroglucan following the renaturation of poly C-scleroglucan blends, indicating that poly C is incorporated in linear, but not in circular, structures of scleroglucan. This conclusion was consistent with AFM topographs that revealed a decreased fraction of circular structures upon addition of poly C during the renaturation process. The combined information about radius of gyration (R(g)) and molecular weight (M(w)) allowed us to conclude that the poly C-scleroglucan complexes are more dense and have a higher persistence length than linear scleroglucan triple helixes. The experimentally determined mass per unit length was used as a basis for elucidating possible molecular arrangements within the poly C-scleroglucan complex.     

Carotenoid accumulation in bacteria with enhanced supply of isoprenoid precursors by upregulation of exogenous or endogenous pathways

Carotenoids are isoprenoid pigments of industrial and nutritional interest. Although they are produced in non-carotenogenic Escherichia coli engineered with the appropriate biosynthetic genes, only a limited pool of their metabolic precursors is available in these bacteria. We have compared the production of carotenoids (lycopene) in strains in which the supply of precursors was enhanced either by upregulating the endogenous pathway via overexpression of deoxyxylulose 5-phosphate synthase (DXS) or by incorporating an exogenous MVA+ operon. In strains expressing DXS under the control of a leaky IPTG-inducible promoter, lycopene accumulation was increased up to 8-fold in the absence of inducer. Addition of IPTG, however, negatively affected lycopene production. Although induction of too high levels of the MVA+ operon enzymes also appeared to cause interference with cell metabolism, supplementation with mevalonate (to be metabolized into carotenoid precursors) resulted in a 10-fold increase in lycopene levels in cells with a near wild-type background. An additional 2-fold increase (up to 228 mg/l) was obtained using an engineered BL21 strain. These results confirm that the MVA+ pathway is most convenient to upregulate the production of carotenoids (lycopene) production in E. coli but that factors other than precursor supply should be considered for high pigment accumulation levels.     

Metabolic engineering of Clostridium acetobutylicum for enhanced production of butyric acid

Clostridium acetobutylicum has been considered as an attractive platform host for biorefinery due to its metabolic diversity. Considering its capability to overproduce butanol through butyrate, it was thought that butyric acid can also be efficiently produced by this bacterium through metabolic engineering. The pta-ctfB-deficient C. acetobutylicum CEKW, in which genes encoding phosphotransacetylase and CoA-transferase were knocked out, was assessed for its potential as a butyric acid producer in fermentations with four controlled pH values at 5.0, 5.5, 6.0, and 6.4. Butyric acid could be best produced by fermentation of the CEKW at pH 6.0, resulting in the highest titer of 26.6 g/l, which is 6.4 times higher than that obtained with the wild type. However, due to the remaining solventogenic ability of the CEKW, 3.6 g/l solvents were also produced. Thus, the CEKW was further engineered by knocking out the adhE1-encoding aldehyde/alcohol dehydrogenase to prevent solvent production. Batch fermentation of the resulting C. acetobutylicum HCEKW at pH 6.0 showed increased butyric acid production to 30.8 g/l with a ratio of butyric-to-acetic acid (BA/AA) of 6.6 g/g and a productivity of 0.72 g/l/h from 86.9 g/l glucose, while negligible solvent (0.8 g/l ethanol only) was produced. The butyric acid titer, BA/AA ratio, and productivity obtained in this study were the highest values reported for C. acetobutylicum, and the BA/AA ratio and productivity were also comparable to those of native butyric acid producer Clostridium tyrobutyricum. These results suggested that the simultaneous deletion of the pta-ctfB-adhE1 in C. acetobutylicum resulted in metabolic switch from biphasic to acidogenic fermentation, which enhanced butyric acid production.     

Enhanced rhamnolipid production by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> under phosphate limitation

Rhamnolipid biosurfactants are effective antimicrobial agents and provide a promising alternative to synthetic medicine. Rhamnolipid accumulation by Pseudomonas aeruginosa ATCC 9027, and associated antimicrobial activity, was quantified during phosphate limited culture. The onset of rhamnolipid production occurred below 0.35 mg phosphate/l. Thereafter rhamnolipid accumulated during phosphate exhaustion where nitrogen remained above 0.9 g/l. A maximum 4.261 g rhamnolipid/l (measured as 1.333 g rhamnose/l) was attained at a productivity of 0.013 g rhamnose/l/h. Rhamnolipid accumulation under conditions of phosphate exhaustion and nitrogen excess suggests a non-specificity of the limiting nutrient, and that rhamnolipids will be synthesised provided carbon is in excess of the metabolic capacity. Antimicrobial activity was demonstrated against Mycobacterium aurum, a surrogate for M. tuberculosis, the causal agent of most forms of tuberculosis, by a 45 mm zone of M. aurum inhibition around a well of supernatant containing 3.954 g rhamnolipid/l.