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Severe butanol toxicity to the metabolism of solventogenic clostridia significantly impede the application of fermentative butanol as a biofuel. Liquid-liquid extraction is an efficient method to reduce the butanol toxicity by in-situ removing it in the extractant phase. Butanol mass transfer into extractant phase in static acetone-butanol-ethanol (ABE) extractive fermentation with biodiesel as the extractant could be enhanced by adding a tiny amount of surfactant such as tween-80. In the case of corn-based ABE extractive fermentation by Clostridium acetobutylicum ATCC 824 using biodiesel originated from waste cooking oil as extractant, addition of 0.14% (w/v) tween-80 could increase butanol production in biodiesel and total solvents production by 21% and 17%, respectively, compared to those of control under non-surfactant existence. Furthermore, a mathematical model was developed to elucidate the mechanism of enhanced ABE extractive fermentation performance. The results indicated that the mass transfer improvement was obtained by effectively altering the physical properties of the self-generated bubbles during ABE extractive fermentation, such as reducing bubble size and extending its retention time in extractant phase, etc. Overall, this study provided an efficient approach for enhancing biobutanol production by integration of bioprocess optimization and model interpretation.

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High broth viscosity due to the accumulation of hyaluronic acid (HA) causes a limited yield of HA. It is a major problem of HA production using Streptococcus zooepidemicus. Extractive fermentation via in situ product recovery (ISPR) was utilized to enhance the HA production. Resins from Amberlite: IRA400 Cl; IRA900 Cl; IRA410 Cl; IRA402 Cl; and IRA67 were tested for the HA adsorption. IRA67 showed high adsorption capacity on HA. The study of the adsorption via a 2 L stirred tank bioreactor of S. zooepidemicus fermentation was investigated to elucidate the adsorption of HA onto IRA67 in dispersed and integrated internal column systems. The application of a dispersed IRA67 improved the HA production compared to the fermentation without resin addition by 1.37-fold. The HA production was further improved by 1.36-fold with an internal column (3.928 g/L) over that obtained with dispersed IRA67. The cultivation with an internal column shows the highest reduction of viscosity value after the addition of IRA67 resin: from 58.8 to 23.7 (mPa·s), suggesting the most effective ISPR of HA. The improved biosynthesis of HA indicated that an extractive fermentation by ISPR adsorption is effective and may streamline the HA purification.

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This study achieved high production of hexanol via gas fermentation using Clostridium carboxidivorans P7 by extracting hexanol from the fermentation broth. The hexanol extraction efficiency and inhibitory effects on C. carboxidivorans P7 of 2-butyl-1-octanol, hexyl hexanoate and oleyl alcohol were examined, and oleyl alcohol was selected as the extraction solvent. Oleyl alcohol was added at the beginning of fermentation and during fermentation or a small volume of oleyl alcohol was repeatedly added during fermentation. The addition of a small volume of oleyl alcohol during fermentation was the most effective for CO consumption and hexanol production (5.06 g/L), yielding the highest known hexanol titer through any type of fermentation including gas fermentation. Hexanol production was further enhanced to 8.45 g/L with the repeated addition of oleyl alcohol and ethanol during gas fermentation. The results of this study will enable sustainable and carbon-neutral hexanol production via gas fermentation.

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Conventional fermentation processes need to be upgraded to produce a wide array of biomolecules to overcome lower product yield. The cost of production of biomolecules using the fermentation method could be reduced by increasing the product yield by various process enhancement methods. In this study, different innovative process enhancement methods were evaluated to increase the co-production of uricase and alkaline protease at the bioreactor level. Ultrasound-assisted fermentation (UAF), Extractive fermentation (ATPS), and Ultrasound-assisted extractive fermentation (UATPS) are the three innovative methods used for process enhancement. Maximum enzyme production was obtained in a combinatorial approach of ultrasound and extractive fermentation, i.e., ultrasound-assisted extractive fermentation where uricase and protease production enhanced by 2.5 fold and 1.9 fold, respectively, as compared to conventional fermentation.

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Carotenoids are hydrophobic compounds that exhibit excellent bioactivity and can be produced by recombinant S. cerevisiae. Irradiating microorganisms with ultrasonic waves increase the productivity of various useful chemicals. Ultrasonic waves are also used to extract useful chemicals that accumulate in microbial cells. In this study, we aimed to improve the carotenoid production efficiency of a recombinant S. cerevisiae using an ultrasonic-irradiation based two-phase extractive fermentation process. When isopropyl myristate was used as the extraction solvent, a total of 264 mg/L of carotenoid was produced when batches were subjected to ultrasonic-irradiation at 10 W, which was a 1.3-fold increase when compared to the control. Transcriptome analysis suggested that one of the reasons for this improvement was an increase in the number of living cells. In fact, after 96 h of fermentation, the number of living cells increased by 1.4-fold upon irradiation with ultrasonic waves. Consequently, we succeeded in improving the carotenoid production in a recombinant S. cerevisiae strain using a ultrasonic-irradiated two-phase extractive fermentation and isopropyl myristate as the solvent. This fermentation strategy has the potential to be widely applied during the production of hydrophobic chemicals in recombinant yeast, and future research is expected to further develop this process.

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The present study evaluated the influence of the variables polyethylene glycol (PEG) molar mass, pH, PEG concentration and sodium citrate concentration in the integrated production of the protease from Aspergillus tamarii Kita UCP1279 by extractive fermentation, obtaining as a response the partition coefficient (K), activity yield (Y) and concentration factor (CF). The enzyme preferably partitioned to the top phase and obtained in the system formed by variables MPEG = 400 g mol-1, CPEG = 20% (w w-1), and CCIT = 20% (w w-1) and pH 6, in this condition were obtained CF = 1.90 and Y = 79.90%. The protease showed stability at a temperature of 60 °C for 180 min, with optimum temperature 40 °C and pH 8.0. For the ions and inhibitors effects, the protease activity increased when exposed to Fe2+, Ca2+ and Zn2 + and inhibited by EDTA, being classified as metalloprotease. The kinetic parameters Km (35.63 mg mL-1) and Vmax (1.205 mg mL-1 min-1) were also estimated. Thus, the protease showed desirable characteristics that enable future industrial applications, especially, for beer industry.

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There have been many studies on the activities and polysaccharide production of Sanghuangporus vaninii. However, few studies have looked at triterpene production from S. vaninii using liquid-state fermentation. A method for enhancing the production of triterpenes by in situ extractive fermentation (ISEF) was studied. Eight solvents were investigated as extractants for triterpene production in the ISEF system. The results showed that using vegetable oil as an extractant significantly increased the yield of total triterpenes and biomass of S. vaninii YC-1, reaching 18.98 ± 0.71 and 44.67 ± 2.21 g/L, respectively. In 5 L fermenter experiments, the added vegetable oil improved the dissolved oxygen condition of the fermentation broth and promoted the growth of S. vaninii YC-1. Furthermore, adding vegetable oil increased the expression of fatty acid synthesis-related genes such as FAD2 and SCD, thereby increasing the synthesis of unsaturated fatty acids in the cell membrane of S. vaninii YC-1. Therefore, the cell membrane permeability of S. vaninii YC-1 increased by 19%. Our results indicated that vegetable oil increased the permeability of S. vaninii YC-1 cell membranes to promote the production of total triterpenes. The use of vegetable oil as an extractant was thus effective in increasing the yield of triterpenes in the ISEF system.

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The aim of this study was to analyse the effect of spontaneous microbial maceration on the release and extraction of the flavonoids and phenolics from olive leaves. Bioprofiling based on thin-layer chromatography effect-directed detection followed by ATR-FTIR spectroscopy proved to be a reliable and convenient method for simultaneous comparison of the extracts. Results show that fermentation significantly enhances the extraction of phenolic compounds and flavonoids. The polyphenolic content was increased from 6.7 µg GAE (gallic acid equivalents) to 25.5 µg GAE, antioxidants from 10.3 µg GAE to 25.3 µg GAE, and flavonoid content from 42 µg RE (rutin equivalents) to 238 µg RE per 20 µL of extract. Increased antioxidant activity of fermented ethyl acetate extracts was attributed to the higher concentration of extracted flavonoids and phenolic terpenoids, while increased antioxidant activity in fermented ethanol extract was due to increased extraction of flavonoids as extraction of phenolic compounds was not improved. Lactic acid that is released during fermentation and glycine present in the olive leaves form a natural deep eutectic solvent (NADES) with significantly increased solubility for flavonoids.

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Extractive fermentation has been proven to be efficient in enhancing the secretion and production of secondary metabolites in submerged fermentation by Monascus spp., owing to increased cell membrane permeability and resolved product inhibition. In this study, we investigated the regulation effect of Triton X-100 on cell growth and secondary metabolite biosynthesis in submerged fermentation of M. purpureus DK. The results show that the maximum monascus pigments (MPs), citrinin (CIT) production, and specific growth rate are 136.86 U/mL, 4.57 mg/L, and 0.04 h-1, respectively, when 3 g/L of Triton X-100 is supplemented after fermentation for 10 d, and the extracellular MPs and CIT increased by 127.48% and 288.57%, respectively. RT-qPCR shows that the expression levels of MPs and CIT biosynthesis gene clusters are significantly upregulated, whereas those of glycolysis, tricarboxylic acid cycle, respiratory chains, and ATP synthase are downregulated. This study provides a vital strategy for extractive fermentation under extreme environmental conditions for further enhancing MP production.

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Pittosporum angustifolium, known as gumbi gumbi, is a native Australian plant, which has traditionally been used as an Aboriginal medicine. This study investigates the effect of different solvents and extractive fermentation on the content and natural products composition of Pittosporum angustifolium extracts, and compares their antioxidant activity, in vitro α-amylase inhibition, and anti-inflammatory properties. Anti-inflammatory activity of the extracts was determined by measuring the inhibition of nitric oxide (NO) production. Extracts were characterised with FTIR-ATR spectroscopy, and screened for antioxidant activities and α-amylase inhibitory activity via High-performance thin-layer chromatography (HPTLC)-Effect-directed analysis (EDA) with direct bioautography. HPTLC combined with chemical derivatization and bioassays was used for EDA screening. The results show that lactic acid fermentation of gumbi gumbi leaves boosts the antioxidant activity in extracts by increasing the total phenolic content, but does not affect (increase or decrease) α-amylase inhibitory activity or nitrogen scavenging/anti-inflammatory activity. Analysis of the ATR-FTIR spectra from the band at RF = 0.85 that inhibits α-amylase, suggests that fatty acid esters are responsible for the enzyme inhibition; both saturated fatty acid esters in unfermented extracts and unsaturated fatty acid esters in fermented extracts. The ATR-FTIR spectra of the polyphenolics in fermented extracts (RF = 0.15-0.20) suggests the presence of soluble lignin fragments (i.e. lignins depolymerized into monomers and oligomers during the fermentation process).

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Fibrinolytic enzymes have been considered promising for treatment and protection of healthy circulation due its ability to dissolve the fibrin in blood clots. Extractive fermentation is a not explored and efficient downstream process which segregates the desired product simultaneously in a fermentation process fast and economically. Extraction of fibrinolytic enzymes by Bacillus stearothermophilus DPUA 1729 employing conventional aqueous two-phase systems (ATPS) and extractive fermentation with ATPS was evaluated. The results of both systems were compared using a factorial design with PEG molar mass, PEG and salt concentrations as independent variables and extraction parameters as a response. In all conditions evaluated it was observed a similar partitioning of fibrinolytic enzymes through the phases, both in conventional ATPS and extractive fermentation. Salt concentration and interaction among PEG and salt concentration influenced in the partition coefficient. The fibrinolytic activity was determined by hydrolysis of fibrin in plate using the extract of one condition from extractive fermentation. The zone degradation presented a diameter of 7.03 ± 0.94 mm. In conclusion, there was no significant difference among the results obtained using conventional ATPS and extractive fermentation, however, the second one presents more advantages and can integrate production and extraction in one single step, reducing the costs.

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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 nonpolluting 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 past 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.

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The use of fed-batch extractive fermentation can overcome inhibitory effects caused by the substrate and ethanol to the yeast cells, since it allows regulate the substrate concentration and remove the product as it is produced. The present study describes the modelling and experimental validation of ethanol production in fed-batch extractive fermentation with in situ ethanol removal by oleic acid in a non-conventional drop column bioreactor (DCB) operated under industrial conditions. The model developed using the hybrid Andrews-Levenspiel equation and ethanol distribution coefficient parameter (KDE) provided an excellent description of the fed-batch extractive ethanol fermentation process with oleic acid. Furthermore, extractive fed-batch fermentation allowed the feed up to 306.6 kg m-3 of substrate (total reducing sugars), with total ethanol concentration in extractive fermentation in the ranging 100.3-139.8 kg m-3 (12.7-17.7 ºGL), 19.9-67.2% higher when compared with the conventional process without ethanol removal. Moreover, this process has the advantage of less effluent generated and energy consumption for ethanol recovery when compared to the conventional process.

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This study aimed to develop a bioprocess using plant oil as the carbon source for lipid-assimilating yeast to produce high-value astaxanthin. Using high-oleic safflower oil as a model, efficient cell growth and astaxanthin production by the engineered Yarrowia lipolytica strain ST7403 was demonstrated, and a considerable portion of astaxanthin was found excreted into the spent oil. Astaxanthin was the predominant carotenoid in the extracellular oil phase that allowed facile in situ recovery of astaxanthin without cell lysis. Autoclaving the safflower oil medium elevated the peroxide level but it declined quickly during fermentation (reduced by 84% by day 3) and did not inhibit cell growth or astaxanthin production. In a 1.5-L fed-batch bioreactor culture with a YnB-based medium containing 20% safflower oil, and with the feeding of casamino acids, astaxanthin production reached 54 mg/L (53% excreted) in 28 days. Further improvement in astaxanthin titer and productivity was achieved by restoring leucine biosynthesis in the host, and running fed-batch fermentation using a high carbon-to-nitrogen ratio yeast extract/peptone medium containing 70% safflower oil, with feeding of additional yeast extract/peptone, to attain 167 mg/L astaxanthin (48% excreted) in 9.5 days of culture. These findings facilitate industrial microbial biorefinery development that utilizes renewable lipids as feedstocks to not only produce high-value products but also effectively extract and recover the products, including non-native ones.Key Points• Yarrowia lipolytica can use plant oil as a C-source for astaxanthin production.• Astaxanthin is excreted and accumulated in the extracellular oil phase.• Astaxanthin is the predominant carotenoid in the extracellular oil phase.• Plant oil serves as a biocompatible solvent for in situ astaxanthin extraction. Graphical abstract.

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Fatty alcohols are important industrial oleochemicals with broad applications and a growing market. Here, we sought to engineer Yarrowia lipolytica to serve as a renewable source of fatty alcohols (specifically hexadecanol, heptadecanol, octadecanol, and oleyl alcohol) directly from glucose. Through screening four fatty acyl-CoA reductase (FAR) enzyme variants across two engineered background strains, we identified that MhFAR enabled the highest production. Further strain engineering, fed-batch flask cultivation, and extractive fermentation improved the fatty alcohol titer to 1.5 g/L. Scale-up of this strain in a 2L bioreactor led to 5.8 g/L total fatty alcohols at an average yield of 36 mg/g glucose with a maximum productivity of 39 mg/L hr. Finally, we utilized this fatty alcohol reductase to generate a customized fatty alcohol, linolenyl alcohol, from α-linolenic acid. Overall, this work demonstrates Y. lipolytica is a robust chassis for diverse fatty alcohol production and highlights the capacity to obtain high titers and yields from a purely minimal media formulation directly from glucose without the need for complex additives.

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BACKGROUND: In situ extractive fermentation (ISEF) is an important technique for improving metabolite productivity. The different extractants can induce the synthesis of different bioactive metabolites of Antrodia camphorata during ISEF. However, a lack of research on the molecular genetics of A. camphorata during ISEF currently hinders such studies on metabolite biosynthetic mechanisms. RESULTS: To clarify the differentially expressed genes during ISEF, the gene transcriptional expression features of A. camphorata S-29 were analysed. The addition of n-tetradecane as an extractant during ISEF showed more pronounced up-regulation of ubiquinone and other terpenoid-quinone biosynthesis pathway genes (CoQ2, wrbA and ARO8). When oleic acid was used as an extractant, the terpenoid backbone biosynthesis and ubiquinone and other terpenoid-quinone biosynthesis pathways were significantly enriched, and genes (IDI, E2.3.3.10, HMGCR atoB, and CoQ2) related to these two pathways were also significantly up-regulated. The CoQ2 genes encode puru-hydroxybenzoate:polyprenyltransferase, playing an important role in antroquinonol synthesis. The IDI, E2.3.3.10, HMGCR and atoB genes of the terpenoid backbone biosynthesis pathway might play an important role in the synthesis of the triquine-type sesquiterpene antrodin C. CONCLUSION: This investigation advances our understanding of how two different extractants of n-tetradecane and oleic acid affect the biosynthesis of metabolites in A. camphorata. It is beneficial to provide potential strategies for improving antrodin C and antroquinonol production by genetic means. © 2020 Society of Chemical Industry.

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BACKGROUND: Nonionic surfactant Brij 35 in submerged fermentation of Monascus can significantly increase Monascus pigment yield. Here, the effects of nonionic surfactant Brij 35 on Monascus pigment secretion in extractive fermentation are discussed in terms of cell morphology, cloud point change, and pigment stability. RESULTS: At Brij 35 concentrations up to 32 g L-1 , the higher concentrations led to the loosening of the network structure on the surface of the fungal wall, enhanced cell wall permeability, and increased abundance of lipid droplets. Alternatively, when the concentration of Brij 35 exceeded 32 g L-1 , a large amount of substances accumulated on the surface of the fungal wall, permeability reduced, and the degree of oil droplet dispersion in cells decreased. Further, during extractive fermentation, Brij 35 induced formation of a grid structure on the fungal wall surface beginning on day 2, increased the number of intracellular lipid droplets, and promoted intracellular pigment secretion into the extracellular environment. When the cloud point temperature in the fermentation system approached that of fermentation, the nonionic surfactant exhibited stronger Monascus pigment extraction capacity, thereby enhancing pigment yield. Hence, Brij 35 can improve pigment stability and effectively reduce damage caused by natural factors, such as light and temperature. CONCLUSION: Brij 35 promotes the secretion of pigment by changing the fungal wall structure and cloud point, as well as by improving pigment stability. © 2020 Society of Chemical Industry.

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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.

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Caproic acid (CA) was produced by Megasphaera elsdenii T81 with Jerusalem artichoke tubers (JA) as a feedstock. More CA was produced under the medium with the acid hydrolysate of JA than the comparative medium with a carbon composition similar to that of JA. CA was produced up to 13.0 g/L and 0.52 g/L/h with extractive fermentation using a mixed solvent of alamine 336 in oleyl alcohol at 37 °C. The JA cost to produce 1 ton of CA is only 505 USD, which is much lower than that required for purchasing sucrose (860 USD) in CA production. As a result of the analysis performed using SuperPro Designer, including the cost of distillation to obtain pure CA, the estimated production cost for CA from dry JA is 1869 USD/ton CA at the production scale of 2000 ton/year, which is lower than the current market price for petroleum-derived CA (~2500 USD/ton).

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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.

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