Mixing state of xanthan gum solution in an aerated and agitated fermentor—Effects of impeller size on volumes of mixing regions and circulation time distribution

The effects of the impeller diameter and width on the volumes of the micromixing and macromixing regions, and on the circulation time distribution were investigated at various agitation speeds to formulate the relationships of them in emperical equations. A fermentor was a 10-l capacity, which was equipped with a turbine impeller with six flat balades and aerated at 1 vvm. It was found that the volumes of the micromixing and macromixing regions depended on the tip speed of the impeller, ND, and the discharging performance of the impeller, ND²W, respectively, in the xabthan gum solution with concentrations of 0.9, 1.8, 2.7, and 3.9%. Empirical equations were derived to estimate the volume of each mixing region from the impeller diameter, D, impeller width, W, agitation speed, N, and consistency coefficient of the xanthan gum solution. On the other hand, the circulation time distribution could be estimated empirically from only the impeller diameter and agitation speed, regardless of variation in the impeller width and consistency coefficient of the xanthan gum solution tested.     

Xanthan batch fermentations: compared performances of a bubble column and a stirred tank fermentor

Fermentations of Xanthomonas campestris, NRRL B-1459, were carried out in a bubble column fermentor (BCF) and in a stirred tank fermentor (STF) to allow comparison of representative variables measured during the microbial growth and the gum production. The microbial growth phase was described by a logistic rate equation where maximum cell concentration was provided by nitrogenous compounds balance. The average value of the maximum specific growth rate was higher in the bubble column (μ _M =0.5 h^?1) than in the stirred reactor (μ _M =0.4 h^?1). The upper values of xanthan yield (Y _g-x =0.65 kg xanthan/kg glucose; Y _{O 2?x} xanthan/kg oxygen) and specific production rate (q _x =0.26 kg xanthan/kg biomass · h) were measured when the oxygen transfer coefficient was kept up above 80 h^?1 in the STF fermentor. In the bubble column the fermentation achieved in the same culture medium lasts two times longer than in the stirred aerated tank; this was attributed to the low value of the oxygen transfer coefficient (K _L a =20 h^?1) at the beginning of the gum synthesis phase. The results obtained in the stirred tank were the basis to estimate the optimal biomass concentration which enables to achieve a culture in non-limiting oxygen transfer conditions. Nevertheless, the transfer characteristics were more homogeneous in the bubble column than in the stirred tank where dead stagnant zones were observed. This is of primary importance when establishing fermentation kinetics models.     

Optimization of the aeration and agitation speed of Aeribacillus palidus 418 exopolysaccharide production and the emulsifying properties of the product

The specific properties of exopolysaccharides (EPS) from thermophilic microorganisms have attracted interest in their optimized production. In this study, the ability of Aeribacillus pallidus 418 to grow and produce polysaccharide in a 5-l stirred tank bioreactor was investigated. Agitation rates of 100, 200, 600, 900, and 1100 revolutions per minute (rpm), at an air flow rate of 0.5 gas volumes per unit medium volume per minute (vvm), and aeration rates of 0.25, 0.5, 1.0, and 1.5 vvm, at an agitation rate of 900 rpm, were examined. A maximum EPS yield of 170 μg/ml has been registered in a single impeller bioreactor equipped with an original Narcissus impeller at agitation speed of 900 rpm, with an aeration rate of 0.5 vvm. The bioprocess oxygen uptake rate (OUR) and oxygen mass transfer coefficient (K_La) were evaluated. The emulsifying properties of the specific EPS produced by A. pallidus 418 were determined. Stable oil-in-water emulsions, a low level of separated water phase and high dispersion stability were found, which together demonstrate the prospects for the industrial exploration of EPS production. Enhanced synergism between the A. pallidus 418 synthesized EPS and various commercially used hydrocolloids was observed; superior synergy was achieved in combination with xanthan gum.     

Agitation,aeration and shear stress as key factors in inulinase production by Kluyveromyces marxianus

Factorial design and response surface analyses were used to optimize the production of inulinase (2,1-β-d-fructan fructanohydrolase, EC 3.2.1.7) by Kluyveromyces marxianus ATCC 16045, using sucrose as carbon source. Effects of aeration, agitation and type of impeller (disk turbine, marine, pitched blade) were studied in a batch stirred reactor. Two factorial designs 2² were carried out. Agitation speed varied from 50 to 550 rpm (revolution per minute), aeration rate from 0.5 to 2.0 vvm (air volume/broth volume·minute). It has been shown that the enzyme production was strongly influenced by mixing conditions, while aeration rate was shown to be less significant. Additionally, the increase in the agitation speed is limited by the death rate, which increases drastically at high speeds, lowering the enzyme production. Also, the impeller type has significant influence in the production, the disk impeller at 450 rpm and aeration at 1.0 vvm led to an activity of 121 UI/mL, while the pitched blade was shown to be the best impeller for this process, leading to the best production, 176 UI/mL, at 450 rpm and 1.0 vvm. The maximum shear stress for inulinase production was about 0.22 Pa, since higher values cause higher cell death rates, affecting the enzyme production. The same results were confirmed with another microorganism, which was also sensible to shear stress. Therefore, it has been concluded that in some cases, mainly when the microorganism is sensible to shear stress, the interaction between mass transfer and mechanical stress should be considered in scale up processes.     

The effect of hydrodynamic stress on the growth of Xanthomonas campestris cultures in a stirred and sparged tank bioreactor

The specific growth and the xanthan production rates by the bacterium Xanthomonas campestris under different shear levels in shake flasks and in a stirred and sparged tank bioreactor have been studied. The shake flask has been used as a reference for studying the shear effects. An effectiveness factor expressed by the ratio of the observed growth rate and the growth rate without oxygen limitation or cell damage was calculated in both modes of cultures. It was observed that the effectiveness factor was strongly dependent on the operational conditions. A strong oxygen transfer limitation at low stirring rates, indicated by a 54 % decrease in the effectiveness factor was observed. In contrast, at higher stirrer speed, cell damage was caused by hydrodynamic stress in the turbulent bulk of the broth, yielding again a decrease in the effectiveness factor values for stirrer speeds higher than 500 rpm. Cell morphological changes were also observed depending on the agitation conditions, differences in morphology being evident at high shear stress.     

Valorisation of chicken feathers for xanthan gum production using Xanthomonas campestris MO-03

Xanthan gum is an important commercial polysaccharide produced by Xanthomonas species. In this study, xanthan production was investigated using a local isolate of Xanthomonas campestris MO-03 in medium containing various concentrations of chicken feather peptone (CFP) as an enhancer substrate. CFP was produced with a chemical process and its chemical composition was determined. The addition of CFP (1–8?g/l) increased the conversion of sugar to xanthan gum in comparison with the control medium, which did not contain additional supplements. The highest xanthan production (24.45?g/l) was found at the 6?g/l CFP containing control medium in 54?h. This value was 1.73 fold higher than that of control medium (14.12?g/l). Moreover, addition of CFP improved the composition of xanthan gum; the pyruvate content of xanthan was 3.86% (w/w), higher than that of the control (2.2%, w/w). The xanthan gum yield was also influenced by the type of organic nitrogen sources. As a conclusion, CFP was found to be a suitable substrate for xanthan gum production.     

Effect of glucose concentrations on xanthan gum production by xanthomonas campestris

The effect of the glucose concentration on xantham gum production by Xanthomonas campestris ATCC 13951 was studied resulting that the glucose concentration between 30 and 40 g/kg broth was best for xanthan gum production. Controlling the glucose concentration at between 30 and 40 g/kg broth by intermittent addition of glucose prevented the inhibition of cell growth and the cessation of xanthan gum production, which were observation with a higher glucose concentration. By means of a glucose feeding strategy, the xanthan gum concentration reached 43 g/kg broth after 96-h cultivation.     

Shear effects on production of lignin peroxidase byPhanerochaete chrysosporium

Since biosynthesis of lignin peroxidase fromPhanerochaete chrysosporium was known to be sensitive to shear, it is interesting to understand the effects of the shear sensitivity for the over production of lignin peroxidase. In stirred-tank fermentor, the shear-sensitivity in lignin peroxidase biosynthesis was quantified by using Kolmogorov length scale. It was found that agitation at 80 μm Kolmogorov length scale is advantageous for the production of lignin peroxidase fromP. chrysosporium. To overcome the shear sensitivity in lignin peroxidase biosynthesis caused by the agitation,P. chrysosporium was immobilized on various solid carriers. The nylon-immobilizedP. chrysosporium was chosen in the present study as a way to overcome the shear sensitivity at the ranges of above 50 μm Kolmogorov length scale. The adhesion force between immobilized cell and carrier can be predicted by thermodynamic approach and used as a criteria to select an adequate carrier materials for immobilization.     

Periodic-peristole agitation for process enhancement of butanol fermentation

Background

Mass transfer plays an important role in determining the efficiency of the biofuel conversion. However, adverse effect of shear stress from traditional agitation inhibits the cell growth and production of biofuels. How to enhance the mass transfer with less adverse effect is considered as one of the important bioengineering issues.

Results

In this study, a novel agitation type, named periodic-peristole was applied to butanol fermentation with Clostridium acetobutylicum ATCC 824. Meanwhile, the enhancement mechanism was studied. Initially, the fermentation performance of periodic-peristole agitation was compared with the traditional Rushton impeller and stationary cultivation. Result showed that the biomass, butanol and total solvent in periodic-peristole group (PPG) was enhanced to 1.92-, 2.06-, and 2.4-fold of those in the traditional Rushton impeller group (TIG), as well as 1.64-, 1.19- and 1.41-fold of those in the stationary group (SG). Subsequently, to get in-depth insight into enhancement mechanism, hydromechanics analysis and metabolic flux analysis (MFA) were carried out. The periodic-peristole agitation exhibits significant difference on velocity distribution, shear force, and mixing efficiency from the traditional Rushton impeller agitation. And the shear force in PPG is only 74 % of that in TIG. According to MFA result, fructose 6-phosphate, pyruvate, acetyl-CoA, oxaloacetate and α-ketoglutarate were determined the key nodes of cells in response to hydrodynamic mechanical stress. Based on such key information, rational enhancement strategies were proposed and butanol production was further improved.

Conclusion

The agitation associated with three issues which resulted in significant changes in cell metabolic behaviors: first, a rebalanced redox status; second, the energy (ATP) acquirement and consumption; third, the tolerance mechanism of the cell for survival of solvent. Periodic-peristole agitation provides an answer to address a long-standing problem of biofuel engineering. Key information derived from current study deepens the understanding of agitation, which can guide the designment of new bioreactors and development of enhancement strategies for biofuel refinery.

     

Effects of agitator configuration and rotational speed on the production of extracellular polysaccharide by Xanthomonas cucurbitae PCSIR B-52

A strain of Xanthomonas cucurbitae PCSIR B-52, efficiently produced extracellular polysaccharide using partially deproteinized low-acid cheese whey without hydrolysis. The effects of the agitator configuration and rotational speed on the viscosity of the fermented broth and the productivity of extracellular polysaccharide based on bacterial growth were evaluated in a batch process. Agitation was performed by a six-bladed disc turbine impeller and by a similar agitator, equipped with six vertically attached stabilizing fins. Comparatively, the magnitude of the decrease in the DO tension resulting from increased viscosity of the broth was less with the stabilizing-fin agitator due to increased system damping. A drastic increase in the mechanical agitation speed after 30-h fermentation, however, increased the broth viscosity and the accumulation of polysaccharide. Moreover, the volume of the macromixing region decreased with increasing rotational speed of agitator.     

High production of xanthan gum by a glycerol-tolerant strain Xanthomonas campestris WXLB-006

The superior properties of xanthan gum make it an industrial aginomoto used in many industries, especially in oil recovery. In the present work, xanthan production from glycerol by a mutant strain Xanthomonas campestris WXLB-006 reached as high as 17.8?g/L in flask culture. With the adoption of pH control, varied aeration and agitation, and varied glycerol feeding strategy, xanthan production reached 33.9?g/L in a 7-L fermenter and fermentation time decreased to 60?hr. Instead of difficultly and costly purifying glycerol, this research provides a very good case for glycerol utilization. At the same time, this is the first report on a high glycerol-tolerant strain for microbial polysaccharide production and 33.9?g/L is the highest production of xanthan gum produced from glycerol so far.     

Incomplete mixing in large bioreactors — a study of its role in the fermentative production of streptokinase

The production of streptokinase in a batch fermentation has been analysed for the role of incomplete macromixing of the broth. The analysis is based on a kinetic model exhibiting inhibition by the substrate and a primary metabolite (lactic acid), and a mixing model comprising two continuous flow reactors (CFRs) with closed-loop recycle. The inoculum is introduced into one region (one CFR) and the mixing process determines its distribution, growth and reactivity. By varying the dilution rates of the CFRs, any degree of macromixing can be simulated. For dilution rates larger than 1.0 h^?1 almost complete macromixing is achieved, for which an analogy has been drawn with micromixing. Increasing the volume of the inoculated region relative to the noninoculated region improves the maximum attainable activity of streptokinase and shortens the time for this. In such a situation an imperfectly mixed bioreactor is superior to a perfectly mixed one, implying that good productivity requires a large inoculated region and incomplete macromixing. These inferences are supported by earlier studies of fluid mixing and relaxation times in bioreactors.     

Significance of agitation-induced shear stress on mycelium morphology and lavendamycin production by engineered Streptomyces flocculus

Lavendamycin methyl ester (LME) is a derivative of a highly functionalized aminoquinone alkaloid lavendamycin and could be used as a scaffold for novel anticancer agent development. This work demonstrated LME production by cultivation of an engineered strain of Streptomyces flocculus CGMCC4.1223 ΔstnB1, while the wild-type strain did not produce. To enhance its production, the effect of shear stress and oxygen supply on ΔstnB1 strain cultivation was investigated in detail. In flask culture, when the shaking speed increased from 150 to 220 rpm, the mycelium was altered from a large pellet to a filamentous hypha, and the LME production was almost doubled, while no significant differences were observed among varied filling volumes, which implied a crucial role of shear stress in the morphology and LME production. To confirm this suggestion, experiments with agitation speed ranging from 400 to 1,000 rpm at a fixed aeration rate of 1.0 vvm were conducted in a stirred tank bioreactor. It was found that the morphology became more hairy with reduced pellet size, and the LME production was enhanced threefolds when the agitation speed increased from 400 to 800 rpm. Further experiments by varying initial k _L a value at the same agitation speed indicated that oxygen supply only slightly affected the physiological status of ΔstnB1 strain. Altogether, shear stress was identified as a major factor affecting the cell morphology and LME production. The work would be helpful to the production of LME and other secondary metabolites by filamentous microorganism cultivation.     

Hydrogen peroxide (H<Subscript>2</Subscript>O<Subscript>2</Subscript>) supply significantly improves xanthan gum production mediated by <Emphasis Type="Italic">Xanthomonas campestris</Emphasis> in vitro

To improve xanthan gum productivity, a strategy of adding hydrogen peroxide (H₂O₂) was studied. The method could intensify oxygen supply through degradation of H₂O₂ to oxygen (O₂). In shake flask testing, the xanthan gum yield reached 2.8% (improved by 39.4%) when adding 12.5 mM H₂O₂ after 24 h of fermentation. In fermentor testing, it was obvious that the oxygen conditions varied with the H₂O₂ addition time. Eventually, gum yield of 4.2% (w/w) was achieved (increased by 27.3%). Compared with the method of intense mixing and increasing the air flow rate, adding H₂O₂ to improve the dissolved oxygen concentration was more effective and much better. Moreover, addition of H₂O₂ improved the quality of xanthan gum; the pyruvate content of xanthan was 4.4% (w/w), higher than that of the control (3.2%).     

Evaluation of mixing effect and shear stress of different impeller combinations on nemadectin fermentation

Mass transfer and shear force have significant effects on nemadectin production by Streptomyces cyaneogriseus ssp. noncyanogenus. They are always the conflict-ridden problems in nemadectin fermentation process. In this study, the flow field characteristics under different impeller combinations were quantitatively evaluated in 5 L stirred-tank bioreactor through the laser particle image velocimetry (PIV) system. Results demonstrated that the radial-axial impeller combinations with the time average velocity at 0.38-0.54 U_tip, the turbulent kinetic energy dissipation rate at 6.4–10.6 ε/N³D², and the shear stress rate was 40-150 s⁻¹, were more conductive to cell growth, nemadectin biosynthesis, cell’s activity, respiratory metabolism than other combinations. The highest nemadectin yield was evaluated up to 1543.3 ± 18.5 μg/mL, which was 31.68 % higher than that of the radial flow impeller combinations. This study provided the important guideline for the selection impeller combinations’ on large-scale nemadectin production.     

Lactose consuming strains of Xanthomonas citri subsp. citri (Xcc) insight into the emergence of natural field resources for xanthan gum production

Xanthomonas genus possesses a low level of β-galactosidase gene expression and is therefore unable to produce xanthan gum in lactose-based media. In this study, we report the emergence of some natural field strains of Xanthomonas citri subsp. citri (Xcc) capable to use lactose as a sole carbon source to produce xanthan gum. From 210 Xcc strains isolated from key lime (C. aurantifolia), 27 showed the capacity to grow on lactose containing medium. Xcc lactose consuming strains demonstrated a good level of xanthan production. Amongst all, NIGEBK37 produced the greatest (14.62 g/l) amount of xanthan gum in experimental laboratory conditions. By evaluating the viscosity of the biopolymer at 25 °C, it was demonstrated that xanthan synthesized by strain NIGEBK37 has the highest viscosity (44,170.66 cP). Our results were indicative for the weakness of a commercial strain of Xanthomonas campestris pv. Campestris DSM1706 (Xcc/DSM1706) to produce xanthan in lactose containing medium.     

A Novel Gene Encoding Xanthan Lyase of Paenibacillus alginolyticus Strain XL-1

Xanthan-modifying enzymes are powerful tools in studying structure-function relationships of this polysaccharide. One of these modifying enzymes is xanthan lyase, which removes the terminal side chain residue of xanthan. In this paper, the cloning and sequencing of the first xanthan lyase-encoding gene is described, i.e., the xalA gene, encoding pyruvated mannose-specific xanthan lyase of Paenibacillus alginolyticus XL-1. The xalA gene encoded a 100,823-Da protein, including a 36-amino-acid signal sequence. The 96,887-Da mature enzyme could be expressed functionally in Escherichia coli. Like the native enzyme, the recombinant enzyme showed no activity on depyruvated xanthan. Compared to production by P. alginolyticus, a 30-fold increase in volumetric productivity of soluble xanthan lyase was achieved by heterologous production in E. coli. The recombinant xanthan lyase was used to produce modified xanthan, which showed a dramatic loss of the capacity to form gels with locust bean gum.     

Production of a high viscosity polysaccharide, methylan, in a novel bioreactor

The effect of shear stress on the production of a high viscosity polysaccharide, methylan, from methanol by Methylobacterium organophilum was investigated by using a multidisk mixer. It was observed in the multidisk mixer with defined shear stresses that the specific production rate of methylan increased gradually with increasing shear stress up to 30 Pa, and the production rate was constant beyond 30 Pa. This result suggested that the limited mass transfer from the medium into cells reduced methylan production. A novel bioreactor that provided the large volume of a high shear region was used to increase methylan production. Fed-batch cultures in the novel bioreactor were performed by the dissolved oxygen-stat method of methanol. When 1.13 g/L ammonium ion was added, the concentrations of cells of methylan were 31 and 20.6 g/L, respectively. The productions of cells and methylan in our designed bioreactor were 20 and 50% higher than those obtained in a conventional fermentor. The methylan content reached a maximum of 20.7 g/L in the bioreactor and the viscosity of the fermentation broth was 127 Pa . s, which corresponds to 68 g/L as a xanthan. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 115-121, 1997.     

Formulation variables affecting drug release from xanthan gum matrices at laboratory scale and pilot scale

The purpose of this research was to study processing variables at the laboratory and pilot scales that can affect hydration rates of xanthan gum matrices containing diclofenac sodium and the rate of drug release. Tablets from the laboratory scale and pilot scale proceedings were made by wet granulation. Swelling indices of xanthan gum formulations prepared with different amounts of water were measured in water under a magnifying lens. Granules were thermally treated in an oven at 60°C, 70°C, and 80°C to studythe effects of elevated temperatures on drug release from xanthan gum matrices. Granules from the pilot scale formulations were bulkier compared to their laboratory scale counterparts, resulting in more porous, softer tablets. Drug release was linear from xanthan gum matrices prepared at the laboratory scale and pilot scales, however, release was faster from the pilot scales. Thermal treatment of the granules did not affect the swelling index and rate of drug release from tablets in both the pilot and laboratory scale proceedings. On the other hand, the release from both proceedings was affected by the amount of water used for granulation and the speed of the impeller during granulation. The data suggest that processing variables that affect the degree of wetness during granulation, such as increase in impeller speed and increase in amount of water used for granulation, also may affect the swelling index of xanthan gum matrices and therefore the rate of drug release.     

Glutamate transport and xanthan gum production in the plant pathogen Xanthomonas axonopodis pv. citri

l-glutamate plays a central role in nitrogen metabolism in all living organisms. In the genus Xanthomonas, the nitrogen nutrition is an important factor involved in the xanthan gum production, an important exopolysaccharide with various industrial and biotechnological applications. In this report, we demonstrate that the use of l-glutamate by the phytopathogen Xanthomonas axonopodis pv. citri as a nitrogen source in defined medium significantly increases the production of xanthan gum. This increase is dependent on the l-glutamate concentration. In addition, we have also characterized a glutamate transport system that is dependent on a proton gradient and on ATP and is modulated by amino acids that are structurally related to glutamate. This is the first biochemical characterization of an energy substrate transport system observed in a bacterial phytopathogen with a broad economic and industrial impact due to xanthan gum production.