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Extractive microbial fermentation of organic compounds in liquid-liquid two-phase systems is a potential strategy to overcome the limitations of microbial fermentation in an aqueous solution, such as low substrate solubility, substrate/product inhibition and product further degradation. A conventional aqueous-organic solvent two-phase system is ina...
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... biotechnology as a sustainable alternative for chemical industry in manufacturing, monitoring and waste management has been recognized. Its benefits include greatly reduced depen- dence on nonrenewable fuels and other resources; reduced potential pollution of industrial processes and products; ability to safely destroy accumulated environmental pollutants; economi- cal and sustainable production of existing and novel products [1]. A basic industrial biotechnology process is schematically represented as shown in Fig. 1 . The reaction and downstream processing in an industrial biotechnology process are very similar to those of chemical engineering. A main difference between the chemical industry and the industrial biotechnology is that the industrial biotechnology utilizes biological agents, such as enzymes, microorganisms, and plant and animal cells, as catalyst. The biocatalyst usually stereoselectively catalyzes chemical reactions at a mild condition. The stereoselectivity of biocatalyst makes industrial biotechnology to have a widespread potential application in synthetic chemistry. However, the stereoselectivity of biocatalyst also leads to a limited substrate spectrum for a given enzyme, i.e., a given enzyme only catalyzes one substrate or a small set of structurally similar building blocks. Protein rational design and directed evo- lution techniques have been managed to solve this problem [2]. Substrate engineering as an alternative strategy also expands the substrate spectrum [3,4]. Biocatalyst working at a mild environment, including in an aqueous solution, at a moderate temperature and pH, etc., makes industrial biotechnology be a friendly environment strategy while compared to that of chemical industry. However, bioreaction in an aqueous solution is usually limited by low substrate solubility, substrate/product inhibition or further degradation of products by whole-cell biocatalyst. Even more, some times there is an adverse thermodynamic equilibrium [5]. All of those lead to a low productivity and a low final product concentration in the industrial biotechnology. An integration of separation unit into a bioreaction process as a strategy for enhancement of productivity and final product concentration in a fermentation process has been developed [6]. For example, extractive fermentation to elimination of 1-butanol inhibition in butanol fermentation process [7] and direct separation of proteins from microbial fermentation broths have been studied extensively [8]. Among them, an addition of nonaqueous reagents into a bioreaction medium for in situ product removal has been attracted extensive attention, which is known as medium engineering [9]. Extractive microbial fermentation in water–organic solvent two-phase system, where the organic solvent auxiliary phase acts as a substrate reservoir and a product extractant to enhance substrate solubility, eliminate substrate/product toxicity and prevent product from further degradation, has been studied extensively [10–15]. However, the toxicity of organic solvent that makes an extractive microbial fermentation of a relatively high polar product is inaccessible to aqueous–organic solvent two-phase system [14]. Next to substrate and product toxicity, product recovery from a fermentative broth and repeated utilization of biocatalyst concern another major problem of microbial fermentation industry. Especially, purification of protein from the product streams of bioreactors and other biological feed streams is widely recognized to be technically and economically challenging. Extractive microbial fermentation in aqueous two-phase system has also been studied extensively [16–19]. Even animal cell growth in an aqueous two-phase system has also been reported [20]. An uneven partitioning of protein in the two phases of aqueous two-phase system provides the possibility of repeated utilization of protein as biocatalyst [17,21,22] or primary purification of proteins [23,24] in an extractive fermentation process. Novel medium engineering methods, such as water immiscible ionic liquids [25–27] and reverse micelle system [28,29], have been exploited continuously for extractive microbial fermentation. Recently, exploitation of cloud point system as a novel two- phase system for extractive microbial fermentation has been carried out in our lab [30]. Extractive microbial fermentation (enzymatic catalysis) in cloud point system has been carried out successfully for the enhancement of substrate solubility [30], extraction of a relatively high polar product [31], concentration and primary purification of protein [32], recycling of microbial cells as biocatalyst [33], and shifting thermodynamic equilibrium of bioreaction process [34]. The present work is a review of the latest progress of extractive fermentation in cloud point system. First, the phase separation of nonionic surfactant aqueous solution to form a cloud point system and its corresponding biocompatibility to microorganisms are detailed. Then the solubilization and bioavailability of an organic compound in cloud point system are discussed. And then some case studies about extractive microbial fermentation in cloud point system in our lab have been presented. Final, microemulsion extraction as a downstream processing strategy for recovery of nonionic surfactant and separation of product is also illustrated. Surfactant, commonly designated as surface-active agent, is an amphiphilic molecule containing an apolar and a polar ...
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Citations
... The possibility of exploiting CPE as an extraction method for fermentative systems was already suggested, in particular for the ability of some fermentation products to solubilize within micellar systems, made by non-ionic surfactants, which turn into two easily separable phases (Purkait et al., 2005;Wang and Dai, 2010). ...
... TritonX-114 was chosen in our study for several reasons. First, TritonX-114 is widely used in biotechnological cloud point extraction processes and its efficiency has been widely studied and reported (Wang and Dai, 2010). ...
... In the bacterial cell-stabilized Pickering emulsions, bacterial cells attached on the oil-water interfaces assimilate both hydrophobic nutrients solubilized in the oil phase and hydrophilic ones solubilized in the water phase for microbial growth (Fig. 1). For microbial transformation in a water-nonaqueous two-phase system, it is generally believed that the nonaqueous phase acts as reservoir to supply the hydrophobic chemicals continuously to the water phase where microbial transformation by living whole cells as biocatalyst occurs (Heipieper et al. 2007;Wang and Dai 2010). Figure 1 discloses that the oil-water interface of Pickering emulsions is an exceptional habitat for microbial growth via direct assimilation of hydrophobic chemicals solubilized in the oil phase. ...
Water–oil interface of bacterial cell-stabilized Pickering emulsions is an exceptional habitat for microbial assimilation of both hydrophobic nutrients solubilized in oil phase and hydrophilic ones solubilized in water phase. Crystal substrate inhibition, i.e., decreasing phytosterol degradation with the increase loading of crystal phytosterols, is always observed during microbial transformation of phytosterols into steroid synthons in Mycolicibacterium sp (China Center of Industrial Culture Collection, CICC 21,097) cell-stabilized Pickering emulsions. In the present work, we confirmed that crystal substrate inhibition was attributed to the interaction between M. neoaurum and phytosterol crystals that led to the detachment of bacterial cells from the oil–water interfaces in bacterial cell-stabilized Pickering emulsions. Under the selected operation condition (25 ml BEHP per 40 ml water, 60 g/L glucose, 25 g/L phytosterols), the product androst-4-ene-3, 17-dione (AD) and androsta-1, 4-dien-3, 17-dione (ADD) concentration increased linearly with the progress of microbial transformation and reached almost 6 g/L at the 11th day. This is a paradigm for microbial transformation of crystal substrates as well as in the presence of other surface active additives (such as chitosan and nonionic surfactants) in bacterial cell-stabilized Pickering emulsions.
Key points
• Microbial transformation of crystal phytosterols in Pickering emulsions
• Crystal substrate inhibition occurring during microbial transformation
• Interaction between phytosterol crystals and bacterial cells leading to demulsification
... Cloud point extraction is a liquid-liquid extraction that uses nonionic surfactant solutions as a second phase. In these systems, the aqueous solution of a nonionic surfactant has the temperature increased above the cloud point, from which a twophase system is formed, a surfactant-rich phase (coacervate) and a surfactant-diluted phase [103,104]. In the surfactantrich phase (coacervate), the nonionic surfactant may form lamellar, reverse micelle, etc., which depends on the surfactant molecular structure and the conditions, since the diluted phase is a surfactant micelle solution near or above its CMC (critical micellar concentration) [104]. ...
... In these systems, the aqueous solution of a nonionic surfactant has the temperature increased above the cloud point, from which a twophase system is formed, a surfactant-rich phase (coacervate) and a surfactant-diluted phase [103,104]. In the surfactantrich phase (coacervate), the nonionic surfactant may form lamellar, reverse micelle, etc., which depends on the surfactant molecular structure and the conditions, since the diluted phase is a surfactant micelle solution near or above its CMC (critical micellar concentration) [104]. This type of technique is more suitable for the recovery of hydrophobic and amphiphilic products, due to the nature of the molecule and the variety of surfactants available [14]. ...
Biotechnology and Bioengineering techniques have been widely used in the production of biofuels, chemicals, pharmaceuticals and food additives, being considered a "green" form of production because they use renewable and non-polluting energy sources. On the other hand, in the traditional processes of production, the target product obtained by biotechnological routes must undergo several stages of purification, which makes these processes more expensive. In the last few years, some works have focused on processes that integrate fermentation to the recovery and purification steps necessary to obtain the final product required. This type of process is called In Situ Product Recovery or Extractive Fermentation. However, there are some differences in the concepts of the techniques used in these bioprocesses. In this way, this Review sought to compile relevant content on considerations and procedures that are being used in this field, such as evaporation, liquid-liquid extraction, permeation and adsorption techniques. Also, the objective of this review was to approach the different configurations in the recent literature of the processes employed and the main bioproducts obtained, which can be used in the food, pharmaceutical, chemical and/or fuel additives industry. We intended to elucidate concepts of these techniques, considered very recent, but which emerge as a promising alternative for the integration of bioprocesses. This article is protected by copyright. All rights reserved.
... Nonionic surfactant Triton X-100 solution transforms the cell membrane structure of E. coli and enables intracellular components to be released [19]. In categorizing surfactant toxicity, it was follow the order ionic surfactant ˃ nonionic surfactant ˃ polymeric nonionic surfactant [20]. In the earlier studies, there were proposed three different explanations to the surfactant effect on cellulase saccharification. ...
Lignocellulosic are renewable and abundant, and estimate to be produced in10-50 billion tons/year as dry matter. Cellulose component in lignocelluloses can be converted into glucose as a feedstock for ethanol fermentation. This paper contains of research results of sengon waste pulp convertion became glucose and ethanol by enzymatic hydrolysis and the purpose of this study was to obtain information on the optimum conditions of treatment to produce high ethanol content of sengon waste pulp using the Response Surface Methodology (RSM) method and high loading substrate. Response surface methodology based on a three level, three variable Central Composite Rotable Design (CCRD) was used to evaluate the interactive effect of Tween 20 concentration (0-2%), cellulase concentration (10-15 FPU/g substrate-dw) and substrate loading (20-35% dw). The results showed the highest ethanol content (15.2%) with addition 35% of substrate; 15 FPU/g substrate of enzyme and 1% Tween 20 with equations : Y = -66, 551 + 0.491X1 + 10.794 X2 – 0.025 X12 - 0.472 X22 - 1, 549 X32 + 0.065 X1X2 + 0.234 X1X3 – 0.086 X2X3. So that the use of the RSM method can know quickly which combination of treatments can produce high ethanol levels through the resulting equation. Distinctive dosages of each variable within these ranges need to be matched to each other according to resulted mathematical formula from RSM to give optimum condition points. Several optimum condition points had been validated and ethanol concentration higher than 18%v in the fermentation broth had been achieved with acuration of equation (R ² ) 0.97.
... Liu et al. (2016) managed to mutagenize Shiraia spores using cobalt-60 gamma irradiation to obtain mutated strains for higher HA production [10]. Apart from these conventional optimization methods, extractive fermentation in water-organic solvent two-phase system, also known as perstractive fermentation or milking process, is becoming an efficient strategy to enhance fungal products [11]. In extractive fermentation, organic surfactant is added to permeabilize cells for intracellular products across the cell membrane and extract the fungal products consecutively in the surfactant micelle aqueous solution. ...
... Great efforts have been made on the selection of different surfactant, optimization of addition time, effects of surfactant concentration, solubility and bioavailability, and fermentation mode in nonionic surfactant micelle aqueous solution [11]. However, the underlying mechanism on the effects of nonionic surfactant on the production of fungal metabolites is still largely unknown. ...
... The extent of enhancement of productivity and final product concentration of microbial metabolites is closely associated with the type of extractants and their interaction with the microbial cells in an extractive fermentation process [11]. Due to the benefit of using TX100 as elicitor in Shiraia cultures [17,18], we chose TX100 as the addictive to explore the mechanism of extractive fermentation. ...
Shiraia mycelial culture is a promising biotechnological alternative for the production of hypocrellin A (HA), a new photosensitizer for anticancer photodynamic therapy (PDT). The extractive fermentation of intracellular HA in the nonionic surfactant Triton X-100 (TX100) aqueous solution was studied in the present work. The addition of 25 g/L TX100 at 36 h of the fermentation not only enhanced HA exudation to the broth by 15.6-fold, but stimulated HA content in mycelia by 5.1-fold, leading to the higher production 206.2 mg/L, a 5.4-fold of the control on day 9. After the induced cell membrane permeabilization by TX100 addition, a rapid generation of nitric oxide (NO) and hydrogen peroxide (H2O2) was observed. The increase of NO level was suppressed by the scavenger vitamin C (VC) of reactive oxygen species (ROS), whereas the induced H2O2 production could not be prevented by the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO), suggesting that NO production may occur downstream of ROS in the extractive fermentation. Both NO and H2O2 were proved to be involved in the expressions of HA biosynthetic genes (Mono, PKS and Omef) and HA production. NO was found to be able to up-regulate the expression of transporter genes (MFS and ABC) for HA exudation. Our results indicated the integrated role of NO and ROS in the extractive fermentation and provided a practical biotechnological process for HA production.
... Generally, it is widely accepted that solvents with a log p value of higher than four are suitable for ISEF [17]. N-tetradecane is a relatively nontoxic solvent with a log p value of 7.22 [18]. According to our previous reports, alkanes with different chain lengths had different extraction capacities for AQ [19]. ...
The goals of this study were to increase the production of antroquinonol (AQ) and to elucidate the response mechanism of the cell membrane during the in situ extractive fermentation (ISEF) of Antrodia camphorata S-29. Through ISEF, the concentration of AQ reached a maximum of 146.1 ± 2.8 mg/L, which was approximately (7.4 ± 0.1)-fold that of the control (coenzyme Q0-induced fermentation). Transcriptome sequencing showed that four genes (FAD2, fabG, SCD, and FAS1) related to fatty acid biosynthesis were upregulated. FAD2 and SCD may regulate the increase in oleic acid (C18:1) and linoleic acid (C18:2) in the cell membrane of A. camphorata S-29, resulting in an increase in cell membrane permeability. AQ was successfully transferred to the n-tetradecane phase through the cell membrane, reducing product feedback inhibition and improving the production of AQ from A. camphorata S-29.
... Extractive fermentation technology has been successfully applied as an effective method for improving the extraction of fungal intracellular products (Kleinegris et al. 2011;Wang and Dai 2010). With the addition of extractive agents in the fermentation broth, the micellare aqueous solution can separate into two phases, where one is a dilute phase (aqueous solution) while the other is a coacervate phase (extractant-rich phase). ...
Pneumocandin B0 is a hydrophobic secondary metabolite that accumulates in the mycelia of Glarea lozoyensis and inhibits fungal 1,3-β-glucan synthase. Extractive batch fermentation can promote the release of intracellular secondary metabolites into the fermentation broth and is often used in industry. The addition of extractants has been proven as an effective method to attain higher accumulation of hydrophobic secondary metabolites and circumvent troublesome solvent extraction. Various extractants exerted significant but different influences on the biomass and pneumocandin B0 yields. The maximum pneumocandin B0 yield (2528.67 mg/L) and highest extracellular pneumocandin B0 yield (580.33 mg/L) were achieved when 1.0 g/L SDS was added on the 13th day of extractive batch fermentation, corresponding to significant increases of 37.63 and 154% compared with the conventional batch fermentation, respectively. The mechanism behind this phenomenon is partly attributed to the release of intracellular pneumocandin B0 into the fermentation broth and the enhanced biosynthesis of pneumocandin B0 in the mycelia.
... The application of two-liquid phase media has long been established to supply hydrophobic organic [1][2][3][4][5][6] and/or remove organic reaction products in situ [7][8][9][10] to reduce their inhibitory and toxic effects on the biocatalyst. In case of the whole-cell ωoxyfunctionalisation of linear alkanes by the AlkBGT enzyme system, the excess alkane substrate is often used as a second phase for product extraction. ...
Two‐liquid phase reaction media have long been used in bioconversions to supply or remove hydrophobic organic reaction substrates and products to reduce inhibitory and toxic effects on biocatalysts. In case of the terminal oxyfunctionalisation of linear alkanes by the AlkBGT monooxygenase the excess alkane substrate is often used as a second phase to extract the alcohol, aldehyde and acid products. However, the selection of other carrier phases or surfactants is complex due to the large amount of parameters that are involved, such as: biocompatibility, substrate bioavailability and product extraction selectivity. This study combines systematic high‐throughput screening with chemometrics to correlate physicochemical parameters of a range of co‐solvents to product specificity and yield using a multivariate regression model. Partial least square regression showed that the defining factor for product specificity are the solubility properties of reaction substrate and product in the co‐solvent, as measured by Hansen solubility parameters. Thus the polarity of co‐solvents determines the accumulation of either alcohol or acid products. Whereas usually the acid product accumulates during the reaction, by choosing a more polar co‐solvent the 1‐alcohol product can be accumulated. Especially with Tergitol as co‐solvent, a 3.2 fold improvement in 1‐octanol yield to 18.3 mmol l‐1 was achieved relative to the control reaction without co‐solvents. This article is protected by copyright. All rights reserved.
... This phenomenon was also observed in the heterogeneous chemical reaction between water-insoluble OMPs and water-soluble primary amines (Fig. 2). At the same time, nonionic surfactant micelle aqueous solution is also applied as a biocompatible medium for extractive fermentation (Wallace and Balskus 2016;Wang and Dai 2010). In our previous work, it is confirmed that crystal OMPs were produced by cell suspension culture with resting cells as whole cell biocatalyst in an aqueous solution while the crystal OMPs are dispersed into the fermentation broth during extractive fermentation in a nonionic surfactant micelle aqueous solution (Lu et al. 2018). ...
Filamentous fungi Monascus sp. has been utilized for fermentative production of food colorant (Red Yeast Rice) for more than 1000 years in China. The main colorant components of Red Yeast Rice are mixture of red Monascus pigments (RMPs) with various primary amine residues. In the present work, the non-natural primary amine p-aminobenzamide, exhibiting as non-involved in nitrogen microbial metabolism, nontoxicity to microbial cells, and chemical reactivity with orange Monascus pigments (OMPs), was screened. Based on the screened result, RMPs with the single p-aminobenzamide residue were produced by cell suspension culture in a nonionic surfactant micelle aqueous solution via in situ chemical modification of OMPs. Furthermore, in situ chemical modification of OMPs also provided a strategy for maintaining a relatively low OMP concentration and then efficient accumulation of high concentration of RMPs (3.3 g/l).
... [4][5][6][7][8] Hence the key to this extractive fermentation approach is to identify suitable extractants that meet the biocompatibility requirements and have strong extractive ability. [9][10][11][12][13] In recent years, extractive fermentation has been applied to microbial fermentation. For instance, Dhamole et al. 14 found that addition of the nonionic surfactant L62 to the broth increases the butanol yield by 2.25-fold and facilitates subsequent butanol separation. ...
... 1,2,8,12 Moreover, the percentage of extracellular yellow pigment increased from 28 to 31.69% in the presence of Brij 35 (Fig. 1a), indicating that Brij 35 changed the overall distribution of the three pigments, which is consistent with the findings of Zhong et al. 23 The biomass results indicated that M. purpureus NJ1 grew well in the aqueous media in the presence of the nonionic surfactants, except for the surfactants Span 60, Brij 30 and Pluronic L61. With the addition of Brij 35, the dry cell weight (DCW) increased by 10.41% over that of the control group (Fig. 1c). Therefore Brij 35 not only was effective at increasing the yield of pigments but also demonstrated good biocompatibility. ...
BACKGROUND
Different nonionic surfactants in submerged fermentation of Monascus sp. demonstrate significant differences regarding increasing pigment yield. In this study, 15 surfactants from five series were analyzed to investigate the influence of nonionic surfactants on Monascus pigments, with the aim of simultaneously obtaining a novel nonionic surfactant.
RESULTS
Addition of the novel surfactant Brij 35 greatly enhanced pigment excretion and demonstrated good biocompatibility. Extracellular red, orange and yellow pigments increased by 1.47‐, 1.71‐ and 2.07‐fold respectively. Production of extracellular pigments was not only related to the hydrophile–lipophile balance value (HLB) but also affected by the cloud point temperature (CP) of the fermentation medium. It was found that nonionic surfactants can improve cell membrane permeability and cell storage capacity by modifying the cell walls of Monascus mycelium and by increasing lipid droplet levels, enhancing pigment excretion. Different nonionic surfactants modify Monascus mycelium to different degrees.
CONCLUSION
The novel surfactant Brij 35, which has good capacity for pigment extraction and biocompatibility, was identified in the analysis. The effects of nonionic surfactants on the secretion of pigments are related to not only the modification of the cell wall and internal structure but also the CP and HLB. © 2018 Society of Chemical Industry
