Improved production of teicoplanin using adsorbent resin in fermentations

AIMS: To use adsorbent resins in fermentations to eliminate toxic effects on growth, reduce feedback repression of production and assist in recovery of teicoplanin. METHODS AND RESULTS: An adsorbent resin was added to the culture broth for the adsorption of teicoplanin. Amberlite XAD-16, Diaion HP-20, charcoal and silica gel were investigated as adsorbent resins. The adsorbed teicoplanin was extracted from the resin by 80% methanol after fermentation. Antibiotic activity was quantified by the disc-agar diffusion assay against Bacillus substilis, and qualitative evaluations were based on HPLC using YMC-Pack ODS-A column. Diaion HP-20 was the most effective adsorbent resin when added at a concentration of 5% (w/v) in the inoculation stage. CONCLUSIONS: Addition of Diaion HP-20 in fermentations eliminated toxic effects on growth and reduced feedback repression of teicoplanin by adsorption. There was a 4.2-fold increase in the quantities of teicoplanin. Addition of adsorbent assisted in the recovery of teicoplanin by reducing the recovery steps. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study provide useful information for the production of teicoplanin, a glycopeptide antibiotic produced by Actinoplanes teicomyceticus. Addition of adsorbent in fermentation increased productivity of teicoplanin by more than five times.     

Improvement of the kirromycin fermentation by resin addition

The kirromycin yield ofActinoplanes sp A8924 was improved from 30–50 mg L^–1 to 350 mg L^–1 by mutant selection and medium optimization. The incorporation of polystyrenic resins into the fermentations promoted a further fourfold enhancement of kirromycin productivity to 1500 mg L^–1. The positive effect of resin addition appears to be due to removal of kirromycin from the fermentation broth because kirromycin's minimal inhibitory concentration against the producing strain remained atca 350 mg L^–1.     

Modelling the adsorption kinetics of erythromycin onto neutral and anionic resins

In this study the selective adsorption method was chosen to enable the recovery of erythromycin. The following sorbents were tested: neutral resins (XAD-4, XAD-7 and XAD-16) and an anionic resin (IRA-410). A mathematical kinetic model for the adsorption of erythromycin against time, on XAD-4, XAD-7 and XAD-16 resins, is proposed. Both Freundlich and Langmuir models showed a good fit for the sorbents XAD-7 and IRA-410 resins. The highest adsorption efficiency was observed when synthetic neutral resin, XAD-7 and XAD-16, were used. The estimated affinity and concentration factors show that the neutral resins tested are adequate for the selective adsorption of erythromycin. The estimated values of enthalpy and free energy of adsorption, lower than 12 kJ mol^–1 and –2 kJ mol^–1, respectively, indicate that a physiosorption process occurred.     

Fed-batch hairy root cultures within situ separation

Fed-batch cultures ofL. erythrorhizon hairy root were carried out by controlling sucrose concentration and media conductivity in a shake flask and a modified stirred tank reactor. For the efficient product recovery from the culture,in situ adsorption by XAD-2 was also conducted. When sucrose was used as a carbon source, the highest shikonin production and hairy root growth were obtained. When glucose or fructose was used instead, the growth was severely inhibited. In addition, it was found that alternating feeding of sucrose could be used as an effective strategy for enhancing the productivity of shikonin derivatives., As the XAD-2 amount was increased up to 1.5 g/L, shikonin production was enhanced by removing shikonin produced and other products which might be inhibitory to cell growth. Most amount of shikonin produced was successfully recovered in XAD-2 (Over 99%). Using hairy root culture in a modified stirred tank reactor, the shikonin productivity and hairy root growth rate on the average were 9.34 mg/L day and 0.49 g DCW/L · day, respectively.     

Optimized procedure to generate heavy isotope and selenomethionine-labeled proteins for structure determination using <Emphasis Type="Italic">Escherichia coli</Emphasis>-based expression systems

Generating sufficient quantities of labeled proteins represents a bottleneck in protein structure determination. A simple protocol for producing heavy isotope as well as selenomethionine (Se-Met)-labeled proteins was developed using T7-based Escherichia coli expression systems. The protocol is applicable for generation of single-, double-, and triple-labeled proteins (¹⁵N, ¹³C, and ²H) in shaker flask cultures. Label incorporation into the target protein reached 99% and 97% for ¹⁵N and ¹³C, respectively, and 75% of (non-exchangeable) hydrogen for ²H labeling. The expression yields and final cell densities (OD600 ∼16) were the same as for the production of non-labeled protein. This protocol is also applicable for Se-Met labeling, leading to Se-Met incorporation into the target protein of 70% or 90% using prototrophic or methionine auxotrophic E. coli strains, respectively.     

Field and mesocosm trials on passive sampling for the study of adsorption and desorption behaviour of lipophilic toxins with a focus on OA and DTX1

It has been demonstrated that polymeric resins can be used as receiving phase in passive samplers designed for the detection of lipophilic marine toxins at sea and was referred to as solid phase adsorption toxin tracking (SPATT). The present study describes the uptake and desorption behaviour of the lipophilic marine toxins okadaic acid (OA) and dinophysistoxin-1 (DTX1) from Prorocentrum lima cultures by five styrene—divinylbenzene based polymeric resins Sepabeads^® SP850, Sepabeads^® SP825L, Amberlite^® XAD4, Dowex^® Optipore^® L-493 and Diaion^® HP-20. All resins accumulated OA and DTX1 from the P. lima culture with differences in adsorption rate and equilibrium rate. Following statistical evaluation, HP-20, SP850 and SP825L demonstrated similar adsorption rates. However, possibly due to its larger pore size, the HP-20 did not seem to reach equilibrium within 72 h exposure as opposed to the SP850 and SP825L. This was confirmed when the resins were immersed at sea for 1 week on the West Coast of Ireland. Furthermore, this work also presents a simple and efficient extraction method suitable to SPATT samplers exposed to artificial or natural culture media.     

Optimization of exopolysaccharide production from Armillaria mellea in submerged cultures

Aims: To study the optimization of submerged culture conditions for exopolysaccharide (EPS) production by Armillaria mellea in shake‐flask cultures and also to evaluate the performance of an optimized culture medium in a 5‐l stirred tank fermenter. Methods and Results: Shake flask cultures for EPS optimal nutritional production contained having the following composition (in g l^?1): glucose 40, yeast extract 3, KH₂PO₄ 4 and MgSO₄ 2 at an optimal temperature of 22°C and an initial of pH 4·0. The optimal culture medium was then cultivated in a 5‐l stirred tank fermenter at 1 vvm (volume of aeration per volume of bioreactor per min) aeration rate, 150 rev min^?1 agitation speed, controlled pH 4·0 and 22°C. In the optimal culture medium, the maximum EPS production in a 5‐l stirred tank fermenter was 588 mg l^?1, c. twice as great as that in the basal medium. The maximum productivity for EPS (Q_p) and product yield (Y_P/S) were 42·02 mg l^?1 d^?1 and 26·89 mg g^?1, respectively. Conclusions: The optimal culture conditions we proposed in this study enhanced the EPS production of A. mellea from submerged cultures. Significance and Impact of the Study: The optimal culturing conditions we have found will be a suitable starting point for a scale‐up of the fermentation process, helping to develop the production of related medicines and health foods from A. mellea.     

Biosynthesis of rubradirin as an ansamycin antibiotic from Streptomyces achromogenes var. rubradiris NRRL3061

The four overlapping cosmids from the rubradirin producer, Streptomyces achromogenes var rubradiris NRRL 3061, have 58 ORFs within a 105.6 kb fragment. These ORFs harbored essential genes responsible for the formation and attachment of four distinct moieties, along with the genes associated with regulatory, resistance, and transport functions. The PKS (rubA) and glycosyltransferase (rubG2) genes were disrupted in order to demonstrate a complete elimination of rubradirin production. The rubradirin biosynthetic pathway was proposed based on the putative functions of the gene products, the functional identification of sugar genes, and the mutant strains. The GeneBank accession number for the sequence reported in this paper is AJ871581.     

Enhanced production of periplasmic interferon alpha-2b by Escherichia coli using ion-exchange resin for in situ removal of acetate in the culture

The possibility of using in situ addition of anion-exchange resin for the removal of acetate in the culture aimed at improving growth of E. coli and expression of periplasmic human interferon-α2b (PrIFN-α2b) was studied in shake flask culture and stirred tank bioreactor. Different types of anion-exchange resin were evaluated and the concentration of anion-exchange resin was optimized using response surface methodology. The addition of anion-exchange resins reduced acetate accumulation in the culture, which in turn, improved growth of E. coli and enhanced PrIFN-α2b expression. The presence of anion-exchange resins did not influence the physiology of the cells. The weak base anion-exchange resins, which have higher affinity towards acetate, yielded higher PrIFN-α2b expression as compared to strong anion-exchange resins. High concentrations of anion-exchange resin showed inhibitory effect towards growth of E. coli as well as the expression of PrIFN-α2b. The maximum yield of PrIFN-α2b in shake flask culture (501.8 μg/L) and stirred tank bioreactor (578.8 μg/L) was obtained at ion exchange resin (WA 30) concentration of 12.2 g/L. The production of PrIFN-α2b in stirred tank bioreactor with the addition of ion exchange resin was about 1.8-fold higher than that obtained in fermentation without ion exchange resin (318.4 μg/L).     

Effect of neutral resins on the production of dynemicins byMicromonospora chersina

Addition of Diaion HP-20 or Amberlite XAD-8 resin to the fermentation ofMicromonospora chersina ATCC 53710 enhanced the production of dynemicin A by 4.7- and 6.9-fold, respectively. Addition of resin suppressed the production of other dynemicin analogs, which comprised 65% of the dynemicin complex in the fermentation.     

Adsorption studies for the separation ofl-tryptophan froml-serine and indole in a bioconversion medium

l-tryptophan was produced froml-serine and indole by immobilized Escherichia coli cells in organic-aqueous systems. Selective adsorption was the method chosen to enable both product separation andl-serine reutilization. Amongst various adsorbents tested activated carbons and neutral polymeric resins (XAD-4 and XAD-7) showed good performance. The neutral resins could selectively concentrate thel-tryptophan from dilute aqueous solutions and adsorbed only 5% of the unconvertedl-serine. High separation factors (l-tryptophan/l-serine and indole/l-tryptophan) were obtained with these adsorbents. Despite a lower capacity, the XAD-7 resin had the advantage of desorbingl-tryptophan with basic or acidic solutions, while organic solvents were required to desorb, at the same concentration levels, this compound from XAD-4.In a packed bed column filled with XAD-4 resin or activated carbon, totall-tryptophan adsorption and recovery were achieved at linear velocities up to 5.0 cm/min and 3.2 cm/min respectively. Successive sorbent reutilization, following continuous sorption and elution steps, was carried out in packed bed columns with the neutral resins and activated carbon.Thel-form of tryptophan, after crystallization, was identified by HPTLC.List of Symbols HPLC High Performance Liquid Chromatography - HPTLC High Performance Thin Layer Chromatography - Trp tryptophan - Ser Serine - A amount of sorbent(g) - c equilibrium solute concentration in the aqueous phase (g/dm³) - c _i initial (before adding the sorbent) liquid phase concentration (g/dm³) - C _T tryptophan concentration in the inlet solution (g/dm³) - C _To tryptophan concentration in the outlet solution (g/dm³) - E _z axial dispersion coefficient (m²/s) - k experimental constant (Eq. 1, 2 and 3) - K ₁ rate constant of adsorption (min^–1) - L column length(m) - n experimental constant (eq. 1, 2 and 3) - q equilibrium solid phase concentration (g solute/g sorbent) - q _max maximum capacity of sorbent (g solute/g sorbent) - t time(s) - v liquid velocity (m/s) - V volume of liquid phase(dm³) - V _e eluted volume(dm³) - V _r volume needed to saturate the column (dm³)     

Production of β-galactosidase by Streptococcus salivarius subsp thermophilus 11F

The production of β-galactosidase by an autolytic strain of Streptococcus salivarius subsp thermophilus 11F was investigated in batch and fed-batch 2-L working volume stirred tank bioreactors. β-Galactosidase was released into the medium upon cell lysis within 1–2 h after the maximum biomass quantity was reached. In batch fermentations the highest β-galactosidase activity of 69 U ml⁻¹ was obtained when the temperature was increased to 42°C after a 4-h growth period at 30°C. In fed-batch experiments the highest β-galactosidase activity of 74 U ml⁻¹ was obtained at a constant 37°C. Received 18 December 1997/ Accepted in revised form 03 February 1998     

Production of <Emphasis Type="SmallCaps">l</Emphasis>-phenylalanine from glycerol by a recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis>

The production of l-phenylalanine is conventionally carried out by fermentations that use glucose or sucrose as the carbon source. This work reports on the use of glycerol as an inexpensive and abundant sole carbon source for producing l-phenylalanine using the genetically modified bacterium Escherichia coli BL21(DE3). Fermentations were carried out at 37°C, pH 7.4, using a defined medium in a stirred tank bioreactor at various intensities of impeller agitation speeds (300–500 rpm corresponding to 0.97–1.62 m s⁻¹ impeller tip speed) and aeration rates (2–8 L min⁻¹, or 1–4 vvm). This highly aerobic fermentation required a good supply of oxygen, but intense agitation (impeller tip speed ~1.62 m s⁻¹) reduced the biomass and l-phenylalanine productivity, possibly because of shear sensitivity of the recombinant bacterium. Production of l-phenylalanine was apparently strongly associated with growth. Under the best operating conditions (1.30 m s⁻¹ impeller tip speed, 4 vvm aeration rate), the yield of l-phenylalanine on glycerol was 0.58 g g⁻¹, or more than twice the best yield attainable on sucrose (0.25 g g⁻¹). In the best case, the peak concentration of l-phenylalanine was 5.6 g L⁻¹, or comparable to values attained in batch fermentations that use glucose or sucrose. The use of glycerol for the commercial production of l-phenylalanine with E. coli BL21(DE3) has the potential to substantially reduce the cost of production compared to sucrose- and glucose-based fermentations.     

不同甜叶菊品种叶中绿原酸类成分的比较研究

该文以14个扦插培育的甜叶菊品种叶为材料,从8种不同型号的树脂中筛选出一种合适的大孔吸附树脂对甜叶菊叶中绿原酸类成分进行纯化前处理,采用HPLC法对不同甜叶菊品种叶中所含绿原酸类成分进行比较分析。结果表明:(1)在8种不同型号的树脂中,XAD~(-1)6对甜叶菊叶中绿原酸类成分吸附-解析性能最佳。(2)经优化,上样液浓度1.20 mg·mL~(-1)、样品溶液pH 3、解析液乙醇体积分数70%时XAD~(-1)6树脂对甜叶菊叶中绿原酸类成分具有较好的纯化效果。(3) HPLC检测分析表明,在14个品种中共检测出新绿原酸、绿原酸、隐绿原酸、异绿原酸B、异绿原酸A、异绿原酸C六种绿原酸类成分,其中主要成分均为异绿原酸A、绿原酸、异绿原酸C,而在品种3、5、13、14中没有检测出异绿原酸B。(4) 14个品种中6个绿原酸类成分的含量分别为异绿原酸A 20.55~54.3 mg·g~(-1)、绿原酸17.96~32.93 mg·g~(-1)、异绿原酸C 4.15~19.49 mg·g~(-1)、新原酸0.61~4.61 mg·g~(-1)、隐绿原酸0.52~3.11 mg·g~(-1)、异绿原酸B 0.0~3.17 mg·g~(-1),6种绿原酸类成分总量为43.9~97.8 mg·g~(-1)。可见,不同品种甜叶菊叶中绿原酸类成分含量有明显差异,富含绿原酸类成分的甜叶菊品种可用于开发获取绿原酸类物质。     

Effect of agitation and aeration on the production of nitrile hydratase by Rhodococcus erythropolis MTCC 1526 in a stirred tank reactor

Aims: To evaluate the effect of different physicochemical parameters such as agitation, aeration and pH on the growth and nitrile hydratase production by Rhodococcus erythropolis MTCC 1526 in a stirred tank reactor. Methods and Results: Rhodococcus erythropolis MTCC 1526 was grown in 7‐l reactor at different agitation, aeration and controlled pH. The optimum conditions for batch cultivation in the reactor were an agitation rate of 200 rev min^?1, aeration 0·5 v/v/m at controlled pH 8. In this condition, the increase in nitrile hydratase activity was almost threefold compared to that in the shake flask. Conclusion: Agitation and aeration rate affected the dissolved‐oxygen concentration in the reactor which in turn affected the growth and enzyme production. Significance and Impact of the Study: Cultivation of R. erythropolis MTCC 1526 in the reactor was found to have significant effect on the growth and nitrile hydratase production when compared to the shake flask.     

Energy-efficient recovery of butanol from model solutions and fermentation broth by adsorption

This article discusses the separation of butanol from aqueous solutions and/or fermentation broth by adsorption. Butanol fermentation is also known as acetone butanol ethanol (ABE) or solvent fermentation. Adsorbents such as silicalite, resins (XAD-2, XAD-4, XAD-7, XAD-8, XAD-16), bone charcoal, activated charcoal, bonopore, and polyvinylpyridine have been studied. Use of silicalite appears to be the more attractive as it can be used to concentrate butanol from dilute solutions (5 to 790–810 g L⁻¹) and results in complete desorption of butanol (or ABE). In addition, silicalite can be regenerated by heat treatment. The energy requirement for butanol recovery by adsorption–desorption processes has been calculated to be 1,948 kcal kg⁻¹ butanol as compared to 5,789 kcal kg⁻¹ butanol by steam stripping distillation. Other techniques such as gas stripping and pervaporation require 5,220 and 3,295 kcal kg⁻¹ butanol, respectively. Mention of trade names of commercial products in this article/publication is solely for the purpose of providing scientific information and does not imply recommendation or endorsement by the United States Department of Agriculture.     

Optimization of nutrient components for enhanced phenazine-1-carboxylic acid production by <Emphasis Type="Italic">gacA</Emphasis>-inactivated <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. M18G using response surface method

The nutritional requirements for phenazine-1-carboxylic acid (PCA) production using Pseudomonas sp. M18G, a gacA chromosomal-inactivated mutant of the strain M18, with a high PCA yield, were optimized statistically in shake flask experiments. Based on a single-factor experiment design, we implemented the two-level Plackett–Burman (PB) design with 11 variables to screen medium components that significantly influence PCA production. Soybean meal, glucose, soy peptone, and ethanol were identified as the most important significant factors (P < 0.05). Response surface methodology based on the Center Composite Design (CCD) was applied to determine these factors’ optimal levels and their mutual interactions between components for PCA production. The predicted results showed that 1.89 g l⁻¹ of PCA production was obtained after a 60-h fermentation period, with optimal concentrations of soybean meal powder (33.4 g l⁻¹), glucose (12.7 g l⁻¹), soy peptone (10.9 g l⁻¹), and ethanol (13.8 ml l⁻¹) in the flask fermentations. The validity of the model developed was verified, and the optimum medium led to a maximum PCA concentration of 2.0 g l⁻¹, a nearly threefold increase compared to that in the basal medium. Furthermore, the experiment was scaled up in the 10 l fermentor and 2 g l⁻¹ PCA productions were achieved in 48 h based on optimization mediums which further verified the practicability of this optimum strategy.     

Accumulation of magnesium ions during fermentative metabolism in Saccharomyces cerevisiae

When cells of Saccharomyces cerevisiae were grown aerobically under glucose-repressed conditions, ethanol production displayed a hyperbolic relationship over a limited range of magnesium concentrations up to around 0.5 mM. A similar relationship existed between available Mg²⁺ and ethanol yield, but over a narrower range of Mg²⁺ concentrations. Cellular demand for Mg²⁺ during fermentation was reflected in the accumulation patterns of Mg²⁺ by yeast cells from the growth medium. Entry of cells into the stationary growth phase and the time of maximum ethanol and minimum sugar concentration correlated with a period of maximum Mg²⁺ transport by yeast cells. The timing of Mg²⁺ transport fluxes by S. cerevisiae is potentially useful when conditioning yeast seed inocula prior to alcohol fermentations. Received 04 March 1996/ Accepted in revised form 21 August 1996     

Production of an indolizidine alkaloid,swainsonine by the filamentous fungus,Metarhizium anisopliae

Summary The physiology of Metarhizium anisopliae has been studied with a view to improving swainsonine production. In preliminary work, the stability of pure swainsonine in aqueous solution has been investigated under varying conditions of pH and temperature and in the presence of oxidizing agents. The effects of oxygen on the growth and morphology of M.anisopliae and the production of swainsonine were also examined. Shaken flask fermentations have been carried out in order to develop a medium suitable for production of swainsonine.     

Stimulation of bioluminescence in Noctiluca sp. using controlled temperature changes

Bioluminescence induced by multifarious stimuli has long been observed and is remains under investigation because of its great complexity. In particular, the exact mechanism underlying bioluminescence is not yet fully understood. This work presents a new experimental method for studying Noctiluca sp. bioluminescence under temperature change stimulation. It is a study of Noctiluca sp. bioluminescence using controlled temperature changes in a tank. A characteristic of this experiment is the large volume of water used (1 m³ in a tank of 2 × 1 × 1 m). Temperature changes were controlled by two methods. In the first, a flask filled with hot water was introduced into the tank and in the second, a water heater was used in the tank. Temperature changes were recorded using sensors. Noctiluca sp. bioluminescence was recorded using a Canon 5D Mark II and this allowed the characteristics of Noctiluca sp. bioluminescence under temperature change stimulation to be monitored. Copyright © 2012 John Wiley & Sons, Ltd.