Adsorptive recovery and purification of prodigiosin from methanol/water solutions of Serratia marcescens fermentation broth

The use of macroporous polymeric adsorption resins for the recovery and purification of prodigiosin from fermentation broth of Serratia marcescens SMDR was systematically studied, in which the broth was pretreated by coagulation with alum and the resulting precipitate was leached by methanol/water solution. Of the seven resins tested, Diaion HP-20 resin was selected considering the adsorption and desorption abilities for prodigiosin at the same time. The optimal compositions of liquid phases for adsorption and desorption were also examined. Batch experiments were performed at 15 ～ 35°C by varying initial prodigiosin concentration in solution (0.05 ～ 0.5 mmol/L), in which molar fraction of each component in the solution was kept constant. The Freundlich and Langmuir equations were used to describe adsorption isotherms and the related thermodynamic functions were also determined. Fixed-bed experiments were finally conducted to obtain the breakthrough characteristics for the adsorption and desorption of prodigiosin. Under optimized conditions, a purification factor of prodigiosin of 11.4 could be obtained from the pretreated broth after one adsorption-desorption run in fixed bed. The present results had demonstrated the promising potential of HP-20 resins for the recovery and purification of prodigiosin from methanol/water solution of Serratia marcescens fermentation broth.     

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.     

The effect of cerulenin on the production of esperamicin A1 byActinomadura verrucosospora

Summary Addition of cerulenin (0.25–1.0 mM) to cultures ofActinomadura verrucosospora before the onset of esperamicin synthesis inhibited the production of esperamicin A₁ by the microorganism. This result indicates that esperamicin A₁ is biosynthesized in part by the polyketide pathway. Addition of cerulenin to the cultures during the active production phase led to a net decrease in esperamicin A₁ production. The¹⁴C-acetate labeling pattern of esperamicin A₁ in the cultures with or without addition of cerulenin at the active production phase also demonstrated the instability of esperamicin A₁ in the fermentation. This suggests that esperamicin A₁ is unstable and degradation occurs during the active production phase. Addition of the neutral resin Diaion HP-20 (1%) to the fermentation enhanced the production of esperamicin A₁ by 53%.     

大孔树脂提取链霉菌4903菌株除草活性物质的研究

化感活性物质的提取方法是研究化感作用的重要步骤,对化感链霉菌4903的前期研究证明其具有抑菌及除草活性。本文选用了4种大孔吸附树脂(X-5, NKA-II, AB-8和HP-20)对化感链霉菌4903发酵液的除草活性物质进行提取。实验结果显示:树脂AB-8对链霉菌4903菌株发酵液除草活性成分具有较好的吸附性能和较易被解吸的特性,树脂AB-8对链霉菌4903菌株发酵液的静态吸附量约可达到树脂体积的7倍。用树脂2-3倍体积的80%乙醇溶液则可以把吸附的除草活性物质全部从树脂中解吸出来。AB-8适合用于链霉菌4903菌株发酵液除草活性物质的提取和纯化。     

Computer modeling of antibiotic fermentation with on-line product removal

The fermentation of Streptomyces griseus for the production of cycloheximide is similar to other antibiotic fermentations in that product synthesis is subject to feedback regulation and the desired product is degraded in the fermentation broth. The productivity of this fermentation can thus be dramatically increased by removing the antibiotic from the whole broth as it is produced. One means for effecting this on-line product removal is to contact the whole fermentation broth with neutral polymeric resin immobilized in hydrogel beads. The antibiotic adsorbs to the immobilized resin via hydrophobic interactions. In this work, the adsorption of the antibiotic onto the immobilized resin was characterized. A biochemical model of the fermentation was then used to describe the time profiles of biomass, substrate, and antibiotic in a fermentation system in which whole broth is circulated from the fermentor through a continuously stirred extractor containing the adsorbent beads. Various operating conditions were examined to optimize the productivity of the fermentation.     

Improved production of pristinamycin coupled with an adsorbent resin in fermentation by Streptomyces pristinaespiralis

Batch fermentation by Streptomyces pristinaespiralis with the addition of adsorbent resins was used to increase the production of pristinamycin. In consideration of the adsorption capacity and the desorption ability, a polymeric resin, JD-1, was finally selected. The maximum production of pristinamycin in Erlenmeyer flasks went up to 1.13 from 0.4 g l⁻¹, by adding 12% (w/v) resin JD-1 into the culture broth at 20 h after inoculation. In a 3 l bioreactor, pristinamycin fermentation with the addition of 12% (w/v) resin JD-1 at 20 h after inoculation reached 0.8 g l⁻¹, which was a 1.25-fold increase over fermentation without resin.     

The synergetic effect from the combination of different adsorption resins in batch and semi-continuous cultivations of S. Cerevisiae cell factories to produce acetylated Taxanes precursors of the anticancer drug Taxol

In situ product recovery is an efficient way to intensify bioprocesses as it can perform adsorption of the desired natural products in the cultivation. However, it is common to use only one adsorbent (liquid or solid) to perform the product recovery. For this study, the use of an in situ product recovery method with three combined commercial resins (HP-20, XAD7HP, and HP-2MG) with different chemical properties was performed. A new yeast strain of Saccharomyces cerevisiae was engineered using CRISPR Cas9 (strain EJ2) to deliver heterologous expression of oxygenated acetylated taxanes that are precursors of the anticancer drug Taxol ® (paclitaxel). Microscale cultivations using a definitive screening design (DSD) were set to get the best resin combinations and concentrations to retrieve high taxane titers. Once the best resin treatment was selected by the DSD, semi-continuous cultivation in high throughput microscale was performed to increase the total taxanes yield up to 783 ± 33 mg/L. The best T5α-yl Acetate yield obtained was up to 95 ± 4 mg/L, the highest titer of this compound ever reported by a heterologous expression. It was also observed that by using a combination of the resins in the cultivation, 8 additional uncharacterized taxanes were found in the gas chromatograms compared to the dodecane overlay method. Lastly, the cell-waste reactive oxygen species concentrations from the yeast were 1.5-fold lower in the resin's treatment compared to the control with no adsorbent aid. The possible future implications of this method could be critical for bioprocess intensification, allowing the transition to a semi-continuous flow bioprocess. Further, this new methodology broadens the use of different organisms for natural product synthesis/discovery benefiting from clear bioprocess intensification advantages.     

Application of solid-phase extraction to agar-supported fermentation

Agar-supported fermentation (Ag-SF), a variant of solid-state fermentation, has recently been improved by the development of a dedicated 2 m² scale pilot facility, Platotex. We investigated the application of solid-phase extraction (SPE) to Ag-SF in order to increase yields and minimize the contamination of the extracts with agar constituents. The selection of the appropriate resin was conducted on liquid-state fermentation and Diaion HP-20 exhibited the highest recovery yield and selectivity for the metabolites of the model fungal strains Phomopsis sp. and Fusarium sp. SPE applied to Ag-SF resulted in a particular compartmentalization of the culture. The mycelium that requires oxygen to grow migrates to the top layer and formed a thick biofilm. The resin beads intercalate between the agar surface and the mycelium layer, and trap directly the compounds secreted by the mycelium through a “solid–solid extraction” (SSE) process. The resin/mycelium layer is easily recovered by scraping the surface and the target metabolites extracted by methanol. Ag-SF associated to SSE represents an ideal compromise for the production of bioactive secondary metabolites with limited economic and environmental impact.     

大孔吸附树脂吸附链霉菌702抗真菌活性物质工艺

研究大孔吸附树脂吸附链霉菌702抗真菌活性物质的工艺条件。采用5种不同大孔吸附树脂吸附链霉菌702发酵液提取液中抗真菌活性物质,选择吸附效果较佳树脂进行吸附条件优化,以桔青霉为指示菌,纳他霉素为对照抗生素．采用“管碟法”测定抗真菌活性物质含量。结果发现,XAD18树脂吸附效果较好,获得优化吸附条件：上样液pH6,NaCl质量浓度10g/L,上样量22．63mg／g湿树脂,吸附流速2．5mL／min,水洗体积180mL,洗脱流速1．5mL／min,洗脱剂为体积分数10％、50％和90％的甲醇,洗脱方式为梯度洗脱。在确定的工艺条件下XAD18对链霉菌702抗真菌活性物质的吸附率可达90％,洗脱率可达75％,回收率可达80％。     

Fermentative production of self-toxic fungal secondary metabolites

Fungi are well known for their vast diversity of secondary metabolites that include many life-saving drugs and highly toxic mycotoxins. In general, fungal cultures producing such metabolites are immune to their toxic effects. However, some are known to produce self-toxic compounds that can pose production optimization challenges if the metabolites are needed in large amounts for chemical modification. One such culture, LV-2841, was identified as the lead for one of our exploratory projects. This culture was found to be a slow grower that produced trace amounts of a known metabolite, cercosporamide, under the standard flask fermentation conditions, and extensive medium optimization studies failed to yield higher titers. Poor growth of the culture in liquid media was attributed to the self-toxicity of cercosporamide to the producing organism, and the minimum inhibitory concentration (MIC) of cercosporamide was estimated to be in the range of 8–16 μg/ml. Fermentations carried out in media containing Diaion^® HP20 resin afforded significantly higher titers of the desired compound. While several examples of resin-based fermentations of soil streptomyces have been published, this approach has rarely been used for fungal fermentations. Over a 100-fold increase in the production titer of cercosporamide, a self-toxic secondary metabolite, was achieved by supplementing the production medium with a commercially available neutral adsorbent resin.     

Lactate production in an integrated process configuration: reducing cell adsorption by shielding of adsorbent

The problem of binding of microbial cells to an adsorbent matrix during in situ recovery of bioproducts from a fermentation broth has been addressed by shielding the adsorbent with a thin layer of a non-ionic polymer. Extractive bioconversion of lactic acid by integrating ion-exchange adsorption with the fermentation stage was studied. The effect of coating of the ion-exchanger with agarose on product recovery and cell adsorption was evaluated. Extractive fermentation with both uncoated and coated resin resulted in an increase in reactor productivity as compared to the normal fermentation. The free cell density in the system with agarose-coated beads was similar to that in control fermentation, but was significantly lower in the system with the uncoated ion-exchanger. Electron microscopic scanning of the bead surface after passage of the fermentation broth showed cells attached to the native adsorbent but not to the coated one.     

The effect of neutral resins on the fermentation production of rubradirin

Summary Rubradirin is an antibiotic of complex chemical structure which is activevs. methicillin resistant staphylococci. Its development has been limited due to inadequate production yields. The incorporation of neutral resins into fermentations ofStreptomyces achromogenes v.rubradiris, UC^® 8051 resulted in the enhanced production of rubradirin. Resins HP-20, HP-21, XAD-2, XAD-7 and XAD-16 were employed in flask and tank fermentations. The incorporation of these resins promoted 2- to 4-fold enhancements of the rubradirin activity produced in flask fermentations, and the incorporation of XAD-16 and HP-21 into tank fermentations promoted production titer increases >5 fold.     

In situ product recovery of n-butanol using polymeric resins

Polymeric resins with high n-butanol adsorption affinities were identified from a candidate pool of commercially available materials representing a wide array of physical and chemical properties. Resin hydrophobicity, which was dictated by the chemical structure of its constituent monomer units, most greatly influenced the resin-aqueous equilibrium partitioning of n-butanol whereas ionic functionalization appeared to have no effect. In general, those materials derived from poly(styrene-co-divinylbenzene) possessed the greatest n-butanol affinity, while the adsorption potential of these resins was limited by their specific surface area. Resins were tested for their ability to serve as effective in situ product recovery (ISPR) devices in the n-butanol fermentation by Clostridium acetobutylicum ATCC 824. In small-scale batch fermentations, the addition of 0.05 kg/L Dowex Optipore SD-2 facilitated achievement of effective n-butanol titers as high as 2.22% (w/v), well above the inhibitory threshold of C. acetobutylicum ATCC 824, and nearly twice that of traditional, single-phase fermentations. Retrieval of n-butanol from resins via thermal treatment was demonstrated with high efficiency and predicted to be economically favorable. Due to its modular nature, the proposed ISPR design exhibits strong potential for compatibility with future n-butanol fermentation efforts.     

An efficient route from trifluoroacetates to water soluble free amines using Diaion® HP-20

A series of polybasic lysine and ornithine derivatives were synthesised as trifluoroacetate salts. Attempts to prepare their free amines according to standard methodology were not successful due to the excellent water solubility of these compounds. Free amines were however efficiently obtained if the column with Diaion HP-20 adsorbent was loaded with the trifluoroacetate and 1% NaHCO(3) aq was passed, followed by elution of free amine with methanol.     

Ex situ product recovery for enhanced butanol production by <Emphasis Type="Italic">Clostridium beijerinckii</Emphasis>

In situ butanol recovery fermentation has been intensively studied as an effective alternative to conventional butanol production, which is limited due to the cellular toxicity of butanol. However, the low biocompatibility of adsorbents often leads to failure of in situ recovery fermentations. In this study, Clostridium beijerinckii NCIMB 8052 was cultured in flasks without shaking and in situ recovery fermentation was performed by using an adsorbent L493. The amounts of acetone, butanol, and ethanol (ABE) increased by 34.4 % in the presence of the adsorbent. In contrast, cell growth and production of organic acids and ABE were retarded in the 7-L batch fermentations with in situ butanol recovery. Cell damage occurred in the fermentor upon agitation in the presence of the adsorbent, unlike in static flask cultures with in situ recovery. Ex situ recovery fermentation using circulation of fermentation broth after mid-exponential phase of cell growth was developed to avoid adsorbent-cell incompatibility. No apparent cell damage was observed and 25.7 g/L of ABE was produced from 86.2 g/L glucose in the fed-batch mode using 7 L fermentors. Thus, ex situ recovery fermentation with C. beijerinckii is effective for enhancing butanol fermentation.     

两步发酵过程中有机酸对产1，3-丙二醇的影响

考察了基因工程菌发酵生产1.3 丙二醇过程中,有机酸对发酵过程的影响,并选用了不同的离子交换树脂对甘油发酵液进行处理。发现有机酸、特别是乳酸对1.3丙二醇生产的抑制作用最明显。在使用离子交换树脂处理有机酸的过程中,确定了使用005号离子交换树脂处理效果最好,005号离子交换树脂可除去大部分的有机酸,处理后的发酵液发酵产1.3丙二醇产量比未处理的发酵液产量提高166%,转化率提高34%。     

In situ butanol recovery from Clostridium acetobutylicum fermentations by expanded bed adsorption

Although butanol is a promising biofuel, its fermentative production suffers from inhibition caused by end product toxicity. The in situ removal of butanol from cultures via expanded bed adsorption offers an effective strategy for mitigating the effects of product toxicity while eliminating the need to clarify cultures via microfiltration. The hydrophobic polymer resin Dowex Optipore L‐493 was found to be both an effective butanol adsorbent and suitable for use in expanded bed adsorption. Recirculation rates through the adsorption column were strongly correlated with and ultimately controlled rates of butanol uptake from the media which, reaching as high as 41.1 g/L h, easily exceed those of its production in a typical fermentation. Vacuum application with vapor collection was found to be an effective means of adsorbent regeneration, with an average of 81% butanol recovery possible, with butanol concentrations in the cold trap reaching as high as 85.8 g/L. Integration of expanded bed adsorption with a fed‐batch Clostridium acetobutylicum ATCC 824 fermentation and its continuous operation for 38.5 h enabled the net production (i.e., in solution and adsorbed) of butanol and total solvent products at up to 27.2 and 40.7 g/L of culture, respectively, representing 2.2‐ and 2.3‐fold improvements over conventional batch culture. While adsorbent biofouling was found to be minimal, further investigation of biofouling in longer‐term studies will provide useful and further insight regarding the robustness of the process strategy. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:68–78, 2014     

Enhanced biotransformation of l-phenylalanine to 2-phenylethanol using an in situ product adsorption technique

An in situ product adsorption technique was used to enhance the biotransformation of l-phenylalanine to 2-phenylethanol by Saccharomyces cerevisiae BD. As a suitable adsorbent, the non-polar macroporous resin D101, selected from several resins tested, showed high adsorption capacity for 2-phenylethanol but not l-phenylalanine. Product inhibition was effectively alleviated by the addition of macroporous resin D101 to the biotransformation medium. When 2 g of hydrated resin D101 was added to 30 mL of the biotransformation medium, the total 2-phenylethanol concentration achieved was 6.17 g/L, of which 3.15 g/L remained in the aqueous phase and 3.02 g/L was adsorbed onto the resin. The molar yield of 2-phenylethanol reached 0.70 after 24 h cultivation. Addition of the macroporous resin greatly increased the volumetric productivity of 2-phenylethanol, and made the downstream processing more feasible and easier to perform in an industrial application.     

HP-20树脂分离与纯化荷叶黄酮的技术研究

荷叶黄酮是荷叶中的主要活性成分之一,主要为黄酮醇和黄酮苷类,成为现代健康产品研究开发的热点。试验以高含量的荷叶黄酮为样品,比较X-5、HP-20,NKA、AB-8及HPD-100几种大孔吸附树脂的吸附效果,结果显示HP-20分离效果最好,静态饱和吸附量可达68.90 mg/g树脂,解吸率达87.23%,动态饱和吸附7 mg/mL的荷叶黄酮的料液可达15 BV。系统优化了HP-20树脂柱分离纯化用70%的乙醇提取的荷叶醇提物,得到荷叶黄酮的分离纯化的技术参数。结果表明:上样浓度为200 mg/mL,流速为2 BV/h的洗脱速度时,40%的乙醇对荷叶黄酮的洗脱能力较强,经过一次分离黄酮含量可达62.06%,此梯度黄酮的得率可达81.40%,荷叶黄酮的总得率为98.17%,经过再分离,荷叶黄酮含量可提高到98.87%。