Effect of ammonium sulphate concentration and agitation speed on the kinetics of alginate production by Azotobacter vinelandii DSM576 in batch fermentation

Growth and alginate production by Azotobacter vinelandii DSM576 as a function of initial ammonium sulphate concentration (0.45–1.05 g l⁻¹) and agitation speed (300–700 rpm) were studied in batch fermentations at controlled pH. The time course of growth, alginate production and substrate consumption and the effect of nitrogen concentration and agitation speed on kinetic parameters and on maximum alginate molecular weight (MW) was modelled using empirical equations. The kinetics of growth, alginate production and polymerization were deeply affected by agitation speed and, to a lesser extent, by inorganic nitrogen concentration. Average and maximum specific growth rate and maximum alginate MW all increased with agitation speed, and were higher at intermediate ammonium sulphate concentration. Maximum alginate MW (>250,000) was obtained at high agitation speed (600–700 rpm) and alginate depolymerization was limited or did not occur at all when the agitation speed was higher than 500 rpm, while at 400 rpm depolymerization significantly reduced the alginate. However, alginate yield was negatively affected by increasing agitation speed. A good compromise between alginate yield (>2 g l⁻¹) and quality (MW>250,000) was obtained with agitation speed of 500–600 rpm and 0.75–0.90 g l⁻¹ of ammonium sulphate. Journal of Industrial Microbiology & Biotechnology (2000) 25, 242–248. Received 23 February 2000/ Accepted in revised form 04 August 2000     

Alginate production by <Emphasis Type="Italic">Pseudomonas mendocina</Emphasis> in a stirred draft fermenter

In this study alginate production by Pseudomonas mendocina in a laboratory-scale fermenter was investigated. In the experiments the effect of temperature (25–31°C) and agitation (500–620 rev min⁻¹) at a constant air flow of 10 v/v/h were evaluated in relation to the rate of glucose bioconversion to alginate using response surface methodology (RSM). The fermenter configuration was also adapted to a system with a screw mixer and draft tube, due to the change in rheological characteristics of the fermentation broth. The adjusted model indicates a temperature of 29.1°C and agitation of 553 rev min⁻¹ for optimum alginate synthesis. In this fermentation system a Y _p/s of 44.8% was achieved. The alginate synthesized by P. mendocina showed a partially acetylated pattern as previously reported for alginates obtained from other Pseudomonas spp and Azotobacter vinelandii.     

Effect of high rates of sulphur on the CaCO3 and CaSO4 content of calcareous soils

Samples of two calcareous soils from central Iraq were amended with 2, 6 and 10 mg g⁻¹ soil of agriculturegrade sulphur and incubated for 4, 8 and 12 weeks at 30°C and 70% water-holding capacity. At the end of each incubation period, soils were analyzed for sulphate, CaCO₃ and CaSO₄. The sulphate content of the soils increased, whereas the CaCO₃ content decreased, with increasing S levels and incubation time. The gypsum formed was in proportion to the sulphur oxidised and the calcium released from CaCO₃.     

Production of Concentrated Lactococcus lactis subsp. cremoris Suspensions in Calcium Alginate Beads

The effect of simultaneous modification of medium composition and growth conditions on the production of Lactococcus lactis subsp. cremoris biomass in calcium alginate beads was studied by the response surface method. Statistical methods of data analysis for unbalanced experiments are illustrated. The media tested were whey, whey supplemented with yeast extract and/or meat extract, milk, and the commercial medium Gold Complete (Nordica). Fermentations were performed at 23°C under pH control (5.6, 6.0, 6.4, or 6.8). In one complete series, 1% CaCO₃ was added to the growth media. There were strong interactions between CaCO₃ and media, CaCO₃ and pH level, and CaCO₃, media, and pH level. In media with CaCO₃, all first-order interactions between media, pH, and sampling time were significant. The addition of CaCO₃ increased cell counts in whey-meat extract medium, but no significant difference was found with the other media. Uncoupling between growth and acidification occurred between 16 and 22 h. Highest counts were obtained on milk and Gold Complete (6 × 10¹⁰/g). In CaCO₃-containing media, pH influenced cell counts only in whey and in Gold Complete (pH 5.6 and 6.0 giving the best results); pH also influenced the bead mass obtained at the end of the fermentation. Biomass production in alginate gels is proposed as a method of obtaining concentrated cell suspensions without centrifugation or filtration.     

Influence of agitation and aeration on growth and antibiotic production by <Emphasis Type="Italic">Xenorhabdus nematophila</Emphasis>

The effect of agitation and aeration on the growth and antibiotic production by Xenorhabdus nematophila YL001 grown in batch cultures were investigated. Efficiency of aeration and agitation was evaluated through the oxygen mass transfer coefficient (K _L a). With increase in K _L a, the biomass and antibiotic activity increased. Activity units of antibiotic and dry cell weight were increased to 232 U ml⁻¹ and 19.58 g l⁻¹, respectively, productivity in cell and antibiotic was up more than 30% when K _L a increased from 115.9 h⁻¹ to 185.7 h⁻¹. During the exponential growth phase, DO concentration was zero, the oxygen supply was not sufficient. So, based on process analysis, a three-stage oxygen supply control strategy was used to improved the DO concentration above 30% by controlling the agitation speed and aeration rate. The dry cell weight and activity units of antibiotic were further increased to 24.22 g l⁻¹ and 249 U ml⁻¹, and were improved by 24.0% and 7.0%, compared with fermentation at a constant agitation speed and a constant aeration rate (300 rev min⁻¹, 2.5 l min⁻¹).     

The oxygen transfer rate influences the molecular mass of the alginate produced by Azotobacter vinelandii

The influence of oxygen transfer rate (OTR) on the molecular mass of alginate was studied. In batch cultures without dissolved oxygen tension (DOT) control and at different agitation rates, the DOT was nearly zero and the OTR was constant during biomass growth, hence the cultures were oxygen-limited. The OTR reached different maximum levels (OTR_max) and enabled to establish various relative respiration rates. Overall, the findings showed that OTR influences alginate molecular mass. The mean molecular mass (MMM) of the alginate increased as OTR_max decreased. The molecular mass obtained at 3.0 mmol l⁻¹ h⁻¹ was 7.0 times higher (1,560 kDa) than at 9.0 mmol l⁻¹ h⁻¹ (220 kDa). An increase in molecular mass can be a bacterial response to adverse nutritional conditions such as oxygen limitation.     

Alginate molecular mass produced by <Emphasis Type="Italic">Azotobacter vinelandii</Emphasis> in response to changes of the O<Subscript>2</Subscript> transfer rate in chemostat cultures

Alginate production by Azotobacter vinelandii growing in chemostat cultures was evaluated under different O₂ transfer rates (OTR). As a result of modifying the culture’s agitation rate from 300 to 500 rpm, the OTR increased from 9 to 15.1 mmol l⁻¹ h⁻¹ and a slight variation in the alginate production (1.7–2.2 g l⁻¹) was observed. At a constant growth rate (0.1 h⁻¹), the mean molecular mass of the alginate was strongly influenced by changes in the OTR, varying from 860 to 1,690 kDa. These results support a possible relationship between alginate polymerization-depolymerization process and the O₂ uptake rate.     

Improvement of poly(γ-glutamic acid) biosynthesis and quantitative metabolic flux analysis of a two-stage strategy for agitation speed control in the culture of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> NX-2

In this study, the production of poly(γ-glutamic acid) by Bacillus subtilis NX-2 (PGA) at different agitation speeds was investigated. Based on the analysis of specific cell growth rate (μ) and specific PGA formation rate (q _p ), a two-stage strategy for agitation speed control was proposed. During the first 24 h, an agitation speed of 600 rpm was used to maintain a high μ for better cell growth, which then reduced to 400 rpm after 24 h to maintain a high q _p to enhance PGA production. Using this method, the maximum concentration of PGA reached 40.5 ± 0.91 g/L and the PGA productivity was 0.56 ± 0.012 g/L/h, which was 17.7 and 9.8% higher, respectively, than the best results obtained when a constant agitation speed was used. The flux distributions and the related enzymes of 2-oxoglutarate could be affected by this two-stage strategy for agitation speed. The activity of isocitrate dehydrogenase and glutamate dehydrogenase at the key node of 2-oxoglutarate increased, and more flux distribution was directed to glutamate. The flux distribution from extracellular to intracellular glutamate also increased and improved PGA production as the glutamate uptake rates increased using the agitation-shift control method.     

Fermentation and microencapsulation of the nematophagous fungus <Emphasis Type="Italic">Hirsutella rhossiliensis</Emphasis> in a novel type of hollow beads

In this work, fermentation and formulation aspects of the nematophagous fungus Hirsutella rhossiliensis BBA were investigated. When incubated in 2% (w/w) glucose and 0.5% (w/w) yeast extract medium in a 1-L Erlenmeyer flask without baffles, heavy pellet formation was observed. Only 40% of the mycelium had a size less than 500 μm. When a flask with three baffles was used, the portion of mycelium <500 μm rose to 95%. In the next step, the influence of aeration rate and stirrer speed on production of finely dispersed mycelium in a stirred tank reactor was investigated. The best fermentation results were obtained at 0.4 vvm and 400 rpm stirrer speed with 90% mycelium <500 μm and 5 g/L biomass. Then, mycelium was microencapsulated in hollow beads based on sulfoethylcellulose (SEC). Experiments on the capsule nutrient reservoir showed that 15% (w/w) corn gluten and 0.5% (w/w) yeast extract could be replaced with 3% (w/w) autoclaved baker's yeast which was never used as capsule additive before. Radial growth of mycelium out of dried hollow beads containing 1% (w/w) biomass and 3% (w/w) baker's yeast was faster than for alginate beads containing equivalent amounts of biomass and yeast indicating a higher bio-control potential.     

Morphology,cell growth,and polysaccharide production of <Emphasis Type="Italic">Nostoc flagelliforme</Emphasis> in liquid suspension culture at different agitation rates

Noscoc flagelliforme is a terrestrial macroscopic cyanobacterium with high economic value. Free-living cells that were separated from a natural colony of N. flagelliforme were cultivated in a 20-L photobioreactor for 16 days at five agitation rates with impeller tip speeds at 0.3, 0., 0.8, 1.0, and 1.5 m·s⁻¹. With different impeller tip speeds there were significant differences in the cell growth and polysaccharide production, and different types of cell colonies appeared because of different shear forces caused by agitation. At harvest time, cell concentrations with tip speeds of 0.8 and 1.0 m·s⁻¹ were clearly higher than those with the other three tip speeds, but dry cell weights with the tip speeds of 0.3, 0.5, 0.8, and 1.0 m·s⁻¹ were almost the same. The highest RPS (polysaccharide that released into liquid medium) production was obtained with the tip speeds of 0.8 and 1.0 m·s⁻¹, while the highest EPS (polysaccharide that formed capsule or slime layer) production was obtained with the tip speed of 0.5 m·s⁻¹. The tip speed of 1.5 m·s⁻¹ was harmful for both cell growth and polysaccharide production, indicating that an appropriate shear force was needed in the liquid suspension culture of N. flagelliforme.     

Microencapsulation of yeast cells in the calcium alginate membrane

Summary Cells of Saccharomyces cerevisiae (ATCC 24858) were encapsulated in the calcium alginate membrane and cultured. Swelling of the capsule was prevented by adding 0.2 g CaCl₂ to 1 L growth medium. The dry cell concentration based on the inner volume of the capsule reached 309 g/L, which was much higher than could be obtained by cell entrapment. All the cells remained inside the capsule during the cultivation. The flux of CO₂ through the capsule membrane increased approximately twice by adding a nonionic surfactant to the CaCl₂ solution during the step of capsule formation.     

A novel approach for the extraction of herbicides and pesticides from water using liquid-core microcapsules

A novel perstraction system using liquid-core microcapsules for pesticide and herbicide removal from aqueous environments is proposed. The microcapsules contain an oil, dibutyl sebacate, surrounded by a hydrogel membrane. The extraction efficiency of the capsules was demonstrated with atrazine, methylparathion, ethylparathion, and 2,4-dichloro-phenoxyacetic acid. The results show that all of the tested compounds could be rapidly extracted, typically 75% extraction within 10 minutes using a capsule: liquid volume ratio of only 3.5% for ethylparathion, and that the rate of extraction increased with increasing hydrophobicity of the compound to be extracted. Higher rates of extraction could be achieved by changing the capsule: liquid volume ratio. The effect of different liquid core solvents, size of capsules, agitation rate, and treatment with complexing agents on the properties of the microcapsules and extraction rate were studied. Capsules of a diameter smaller than 0.800 mm show little external resistance to mass transfer. The main resistance to mass transfer of the pesticides/herbicides was found to reside in the hydrogel membrane composed of cross-linked alginate/polyacrylamide. Removal of divalent cations from the membrane by the addition of citrate, resulted in a 50% increase in the mass transfer coefficient, probably as a result of solubilization and exo-diffusion of alginate.     

Production of a collagenase from Candida albicans URM3622

Culture conditions (pH, time, temperature, inoculum size, orbital agitation speed and substrate concentration) for an extracellular collagenase produced by Candida albicans URM3622 were studied using three experimental designs (one 2⁶⁻² fractionary factorial and two 2³ full factorial). The analysis of the 2⁶⁻² fractionary design data indicated that agitation speed and substrate concentration had the most significant effect on collagenase production. Based on these results, two successive 2³ full factorial design experiments were run in which the effects of substrate concentration, orbital agitation speed and pH were further studied. These two sets of experiments showed that all variables chosen were significant for the enzyme production, with the maximum collagenolytic activity of 6.8 ± 0.4 U achieved at pH 7.0 with an orbital agitation speed of 160 rpm and 2% substrate concentration. Maximum collagenolytic activity was observed at pH 8.2 and 45 °C. The collagenase was stable within a pH range of 7.2–8.2 and over a temperature range of 28–45 °C. These results clearly indicate that C. albicans URM3622 is a potential resource for collagenase production and could be of interest for pharmaceutical, cosmetic and food industry.     

Changes in the cell ultrastructure of the haloalkaliphilic endoevaporite cyanobacterium <Emphasis Type="Italic">‘Euhalothece natronophila’</Emphasis> during fossilization

The ultrastructure of the haloalkaliphilic endoevaporite cyanobacterium ‘Euhalothece natronophila’ Z-M001 from the soda Lake Magadi was investigated during the initial stages of fossilization in a model experimental system. The cyanobacterium was cultivated in concentrated carbonate solution supplemented with calcium chloride. It was revealed that the amorphous CaCO₃ formed under these conditions could interact with the cell wall during the first stages of ‘E. natronophila’ calcification. Evidence is presented that the surface layer of the ‘E. natronophila’ envelope, presumably containing polysaccharide and/or (glyco)protein components, can be involved in the adsorption and subsequent crystallization of CaCO₃ with the formation of a massive “shell” embedding the morphologically intact cells. It was established that the ultrastructure of the cell wall and the intrathylakoid space changed during CaCO₃ mineralization. During the later fossilization stages, cells covered by the calcium-containing “shell” were apparently mummified, and mostly retained their original shape. The encapsulation of cyanobacteria in the trona globule was characterized by a different pattern. It probably involved tight binding of the growing crystal to the glycocalyx components that are anchored in the outer membrane. This may result in its detachment from the underlying peptidoglycan layer. The peptidoglycan was retained, and the protoplasts were ultrastructurally similar to the intact ones. Cyanobacteria incorporated in large trona crystals underwent degradation, deformation, and destruction. This accounts for the fact that massive trona deposits of Lake Magadi lack cyanobacterial fossils that are abundant in calcium-containing strata.     

Evaluation of Chitosan/Alginate Beads Using Experimental Design: Formulation and <Emphasis Type="Italic">In Vitro</Emphasis> Characterization

Bovine serum albumin-loaded beads were prepared by ionotropic gelation of alginate with calcium chloride and chitosan. The effect of sodium alginate concentration and chitosan concentration on the particle size and loading efficacy was studied. The diameter of the beads formed is dependent on the size of the needle used. The optimum condition for preparation alginate–chitosan beads was alginate concentration of 3% and chitosan concentration of 0.25% at pH 5. The resulting bead formulation had a loading efficacy of 98.5% and average size of 1,501 μm, and scanning electron microscopy images showed spherical and smooth particles. Chitosan concentration significantly influenced particle size and encapsulation efficiency of chitosan–alginate beads (p < 0.05). Decreasing the alginate concentration resulted in an increased release of albumin in acidic media. The rapid dissolution of chitosan–alginate matrices in the higher pH resulted in burst release of protein drug.     

Relevance of Rheological Properties of Sodium Alginate in Solution to Calcium Alginate Gel Properties

The purpose of this study is to determine whether sodium alginate solutions’ rheological parameters are meaningful relative to sodium alginate’s use in the formulation of calcium alginate gels. Calcium alginate gels were prepared from six different grades of sodium alginate (FMC Biopolymer), one of which was available in ten batches. Cylindrical gel samples were prepared from each of the gels and subjected to compression to fracture on an Instron Universal Testing Machine, equipped with a 1-kN load cell, at a cross-head speed of 120 mm/min. Among the grades with similar % G, (grades 1, 3, and 4), there is a significant correlation between deformation work (L _E) and apparent viscosity (η _app). However, the results for the partial correlation analysis for all six grades of sodium alginate show that L _E is significantly correlated with % G, but not with the rheological properties of the sodium alginate solutions. Studies of the ten batches of one grade of sodium alginate show that η _app of their solutions did not correlate with L _E while tan δ was significantly, but minimally, correlated to L _E. These results suggest that other factors—polydispersity and the randomness of guluronic acid sequencing—are likely to influence the mechanical properties of the resultant gels. In summary, the rheological properties of solutions for different grades of sodium alginate are not indicative of the resultant gel properties. Inter-batch differences in the rheological behavior for one specific grade of sodium alginate were insufficient to predict the corresponding calcium alginate gel’s mechanical properties.     

Enhanced welan gum production using a two-stage agitation speed control strategy in Alcaligenes sp. CGMCC2428

Batch fermentative production of welan gum by Alcaligenes sp. CGMCC2428 was investigated under various oxygen supply conditions using regulating agitation speed. Based on a three kinetic parameters analysis that includes specific cell growth rate (μ), specific glucose consumption rate (q _s), and specific welan formation rate (q _p), a two-stage agitation speed control strategy was proposed to achieve high concentration, high yield, and high viscosity of welan. During the first 22 h, the agitation speed in 7.5 L fermenter was controlled at 800 rpm to maintain high μ for cell growth. The agitation was then reduced step-wise to 600 rpm to maintain a changing profile with stable dissolved oxygen levels and obtain high qp for high welan accumulation. Finally, the maximum concentration of welan was reached at 26.3 ± 0.89 g L⁻¹ with a yield of 0.53 ± 0.003 g g⁻¹ and the welan gum viscosity of 3.05 ± 0.10 Pa s, which increased by an average of 15.4, 15.2, and 20.1% over the best results controlled by constant agitation speeds.     

Bioerosion caused by foraging of the tropical chiton Acanthopleura gemmata at One Tree Reef, southern Great Barrier Reef

The bioerosive potential of the intertidal chiton Acanthopleura gemmata on One Tree Reef was determined by quantification of CaCO₃ in daily faecal pellet production of individuals transplanted into mesocosms after nocturnal-feeding forays. Mean bioerosive potential was estimated at 0.16 kg CaCO₃ chiton⁻¹ yr⁻¹. Bioerosion rates were estimated for populations on two distinct chiton habitats, reef margin (0.013 kg CaCO₃ m⁻² yr⁻¹) and beachrock platform (0.25 kg CaCO₃ m⁻² yr⁻¹). Chiton density on the platform was orders of magnitude greater than on the reef margin. The surface-lowering rate (0.16 mm m⁻² yr) due to bioerosion by the beachrock population is a substantial contribution to the total surface-lowering rate of 2 mm m⁻² yr⁻¹ previously reported for One Tree Reef across all erosive agents. At high densities, the contribution of A. gemmata to coral reef bioerosion budgets may be comparable to other important bioeroders such as echinoids and fish.     

Calcium alginate entrapped preparation of α-galactosidase: its stability and application in hydrolysis of soymilk galactooligosaccharides

Thermostable α-galactosidase from Aspergillus terreus _GR was insolubilized using concanavalin A obtained from jack bean extract and in order to maintain the integrity of complex in the presence of its substrate or products, this complex was crosslinked with glutaraldehyde. Soluble α-galactosidase entrapped in calcium alginate retained 82% of enzyme activity whereas, Con A-α-galactosidase complex entrapped in calcium alginate and crosslinked Con A-α-galactosidase complex entrapped calcium alginate retained 74 and 61% activity, respectively. A fluidized bed reactor was constructed for continuous hydrolysis of galactooligosaccharides in soymilk using crosslinked Con A-α-galactosidase complex entrapped calcium alginate. Optimum conditions such as pH (5.0) and temperature (65°C) were the same for all immobilized enzyme preparations and soluble enzyme. Crosslinked Con A-α-galactosidase entrapped complex exhibited enhanced thermostability and showed 62% of activity (38%) after 360 min at 65°C. Entrapped crosslinked Con A-α-galactosidase complex preparation was superior in the continuous hydrolysis of oligosaccharides in soymilk by batch processes compared to the other entrapped preparations. The entrapped crosslinked concanavalin A-α-galactosidase complex retained 95% activity after eight cycles of use.     

Calcium carbonate budgets for two coral reefs affected by different terrestrial runoff regimes, Rio Bueno, Jamaica

A process-based carbonate budget was used to compare carbonate framework production at two reef sites subject to varying degrees of fluvial influence in Rio Bueno, Jamaica. The turbid, central embayment was subjected to high rates of fluvial sediment input, framework accretion was restricted to ≤30 m, and net carbonate production was 1,887 g CaCO₃ m⁻² year⁻¹. Gross carbonate production (GCP) was dominated by scleractinians (97%), particularly by sediment-resistant species, e.g. Diploria strigosa on the reef flat (<2 m). Calcareous encrusters contributed very little carbonate. Total bioerosion removed 265 g CaCO₃ m⁻² year⁻¹ and was dominated by microborers. At the clear-water site, net carbonate production was 1,236 g CaCO₃ m⁻² year⁻¹; the most productive zone was on the fore-reef (10 m). Corals accounted for 82% of GCP, and encrusting organisms 16%. Bioerosion removed 126 g CaCO₃ m⁻² year⁻¹ and was dominated by macroborers. Total fish and urchin grazing was limited throughout (≤20 g CaCO₃ m⁻² year⁻¹). The study demonstrates that: (1) carbonate production and net reef accretion can occur where environmental conditions approach or exceed perceived threshold levels for coral survival; and (2) although live coral cover (and carbonate production rates) were reduced on reef-front sites along the North Jamaican coast, low population densities of grazing fish and echinoids to some extent offset this, thus maintaining positive carbonate budgets.