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Microbial cell factories, renowned for their economic and environmental benefits, have emerged as a key trend in academic and industrial areas, particularly in the fermentation of natural compounds. Among these, plant-derived terpenes stand out as a significant class of bioactive natural products. The large-scale production of such terpenes, exemplified by artemisinic acid—a crucial precursor to artemisinin—is now feasible through microbial cell factories. In the fermentation of terpenes, two-phase fermentation technology has been widely applied due to its unique advantages. It facilitates in situ product extraction or adsorption, effectively mitigating the detrimental impact of product accumulation on microbial cells, thereby significantly bolstering the efficiency of microbial production of plant-derived terpenes. This paper reviews the latest developments in two-phase fermentation system applications, focusing on microbial fermentation of plant-derived terpenes. It also discusses the mechanisms influencing microbial biosynthesis of terpenes. Moreover, we introduce some new two-phase fermentation techniques, currently unexplored in terpene fermentation, with the aim of providing more thoughts and explorations on the future applications of two-phase fermentation technology. Lastly, we discuss several challenges in the industrial application of two-phase fermentation systems, especially in downstream processing. [ABSTRACT FROM AUTHOR]

Secondary metabolites derived from plants are recognized as valuable products with several successful applications in the pharmaceutical, cosmetic, and food industries. The major limitation to the broader implementation of these compounds is their low manufacturing efficiency. Current efforts to overcome unprofitability depend mainly on biotechnological methods, especially through the application of plant in vitro cultures. This concept allows unprecedented bioengineering opportunities for culture system modifications with in situ product removal. The silica-based xerogels can be used as a novel, porous biomaterial characterized by a large surface area and high affinity to lipophilic secondary metabolites produced by plant tissue. This study aimed to investigate the influence of xerogel-based biomaterials functionalized with methyl, hydroxyl, carboxylic, and amine groups on Rindera graeca transgenic root growth and the production of naphthoquinone derivatives. The application of xerogel-based scaffolds functionalized with the methyl group resulted in more than 1.5 times higher biomass proliferation than for reference untreated culture. The naphthoquinone derivatives' production was noted exclusively in culture systems supplemented with xerogel functionalized with methyl and hydroxyl groups. Applying chemically functionalized xerogels as in situ adsorbents allowed for the enhanced growth and productivity of in vitro cultured R. graeca transgenic roots, facilitating product isolation due to their selective and efficient accumulation. [ABSTRACT FROM AUTHOR]

Microbial cell factories, renowned for their economic and environmental benefits, have emerged as a key trend in academic and industrial areas, particularly in the fermentation of natural compounds. Among these, plant-derived terpenes stand out as a significant class of bioactive natural products. The large-scale production of such terpenes, exemplified by artemisinic acid—a crucial precursor to artemisinin—is now feasible through microbial cell factories. In the fermentation of terpenes, two-phase fermentation technology has been widely applied due to its unique advantages. It facilitates in situ product extraction or adsorption, effectively mitigating the detrimental impact of product accumulation on microbial cells, thereby significantly bolstering the efficiency of microbial production of plant-derived terpenes. This paper reviews the latest developments in two-phase fermentation system applications, focusing on microbial fermentation of plant-derived terpenes. It also discusses the mechanisms influencing microbial biosynthesis of terpenes. Moreover, we introduce some new two-phase fermentation techniques, currently unexplored in terpene fermentation, with the aim of providing more thoughts and explorations on the future applications of two-phase fermentation technology. Lastly, we discuss several challenges in the industrial application of two-phase fermentation systems, especially in downstream processing.

To date, it is still not possible to obtain exhaustive information about organic materials in cultural heritage without sampling. Nonetheless, when studying unique objects with invaluable artistic or historical significance, preserving their integrity is a priority. In particular, organic dye identification is of significant interest for history and conservation research, but it is still hindered by analytes' low concentration and poor fastness. In this work, a minimally invasive approach for dye identification is presented. The procedure is designed to accompany noninvasive analyses of inorganic substances for comprehensive studies of complex cultural heritage matrices, in compliance with their soundness. Liquid extraction of madder, turmeric, and indigo dyes was performed directly from paint layers and textiles. The extraction was supported by hydrogels, which themselves can undergo multitechnique analyses in the place of samples. After extraction, Ag colloid pastes were applied on the gels for SERS analyses, allowing for the identification of the three dyes. For the HPLC-MS/MS analyses, re-extraction of the dyes was followed by a clean-up step that was successfully applied on madder and turmeric. The colour change perceptivity after extraction was measured with colorimetry. The results showed ΔE values mostly below the upper limit of rigorous colour change, confirming the gentleness of the procedure. [ABSTRACT FROM AUTHOR]

Secondary metabolites derived from plants are recognized as valuable products with several successful applications in the pharmaceutical, cosmetic, and food industries. The major limitation to the broader implementation of these compounds is their low manufacturing efficiency. Current efforts to overcome unprofitability depend mainly on biotechnological methods, especially through the application of plant in vitro cultures. This concept allows unprecedented bioengineering opportunities for culture system modifications with in situ product removal. The silica-based xerogels can be used as a novel, porous biomaterial characterized by a large surface area and high affinity to lipophilic secondary metabolites produced by plant tissue. This study aimed to investigate the influence of xerogel-based biomaterials functionalized with methyl, hydroxyl, carboxylic, and amine groups on Rindera graeca transgenic root growth and the production of naphthoquinone derivatives. The application of xerogel-based scaffolds functionalized with the methyl group resulted in more than 1.5 times higher biomass proliferation than for reference untreated culture. The naphthoquinone derivatives’ production was noted exclusively in culture systems supplemented with xerogel functionalized with methyl and hydroxyl groups. Applying chemically functionalized xerogels as in situ adsorbents allowed for the enhanced growth and productivity of in vitro cultured R. graeca transgenic roots, facilitating product isolation due to their selective and efficient accumulation.

In situ extraction is a method for separating plant secondary metabolites from in vitro systems of plant biomass cultures. The study aimed to investigate the MTMS-based xerogels morphology effect on the growth kinetics and deoxyshikonin productivity in xerogel-supported in vitro culture systems of Rindera graeca hairy root. Cultures were supplemented with three types of xerogel, i.e., mesoporous gel, microporous gel, and agglomerated precipitate, in the disintegrated or monolithic form. Structure, oil sorption capacity, and SEM analyses for xerogel-based additives were performed. Application of monolithic macroporous xerogel resulted in the highest biomass proliferation, i.e., 5.11-fold fresh biomass increase after four weeks of the screening culture. The highest deoxyshikonin production (i.e., 105.03 µg) was noted when hairy roots were maintained with particles of disintegrated mesoporous xerogel. The detailed kinetics investigations (6-week culture) revealed the highest growth of hairy root biomass and secondary metabolite production, equaling 9.46-fold fresh weight biomass and 204.08 µg deoxyshikonin, respectively. MTMS-based xerogels have been recognized as selective biocompatible scaffolds for boosting the proliferation of transgenic roots or for productivity enhancement of naphthoquinones without detrimental effects on biomass growth, and their successful applicability in in situ removal of secondary plant metabolites has been experimentally confirmed. [ABSTRACT FROM AUTHOR]

To date, it is still not possible to obtain exhaustive information about organic materials in cultural heritage without sampling. Nonetheless, when studying unique objects with invaluable artistic or historical significance, preserving their integrity is a priority. In particular, organic dye identification is of significant interest for history and conservation research, but it is still hindered by analytes’ low concentration and poor fastness. In this work, a minimally invasive approach for dye identification is presented. The procedure is designed to accompany noninvasive analyses of inorganic substances for comprehensive studies of complex cultural heritage matrices, in compliance with their soundness. Liquid extraction of madder, turmeric, and indigo dyes was performed directly from paint layers and textiles. The extraction was supported by hydrogels, which themselves can undergo multitechnique analyses in the place of samples. After extraction, Ag colloid pastes were applied on the gels for SERS analyses, allowing for the identification of the three dyes. For the HPLC-MS/MS analyses, re-extraction of the dyes was followed by a clean-up step that was successfully applied on madder and turmeric. The colour change perceptivity after extraction was measured with colorimetry. The results showed ΔE values mostly below the upper limit of rigorous colour change, confirming the gentleness of the procedure.

An in situ formation of ionic liquid was used for preconcentration of four triazole fungicides in food samples. The microextraction method was used for the first time in the literature for preconcentration of triazole fungicides. In the developed method, tributylhexadecylphosphonium bromide ([P44412]Br) and potassium hexafluorophosphate (KPF6) were used for the formation of hydrophobic ionic liquid. After centrifugation, the fine microdroplets were produced in one step, providing the extraction step in a quick and environmentally friendly manner. The functional group of the hydrophobic ionic liquid was investigated using FT-IR. Various extraction parameters were studied and optimized. In the extraction method, 0.01 g of [P44412]Br and 0.01 g of KPF6, centrifugation at 4500 rpm for 10 min were used. The optimized technique provided a good linear range (90–1000 μg L−1) and high extraction recovery, with a low limit of detection (30–50 μg L−1). Methods for the proposed in situ formation of ionic liquid were successfully applied to honey, fruit juice, and egg matrices. The recoveries were obtained in a satisfactory range of 62–112%. The results confirmed the suitability of the proposed microextraction method for selective extraction and quantification of triazole fungicides. [ABSTRACT FROM AUTHOR]

Copyright of Chinese Journal of Bioprocess Engineering is the property of Chinese Journal of Bioprocess Engineering Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

The production of short-chain carboxylates (SCCs) from readily degradable food waste exhibits superior product value compared to biogas production, but SCCs' high solubility poses challenges in their production and recovery. In this study, we applied two novel modules to in-situ extract SCCs from the continuously produced fermentative broth, namely direct coupling of bipolar membrane electrodialysis (BPED) and the integration of upstream membrane permeation (MP) and BPED. The remarkable extraction efficiency of BPED effectively mitigated the inhibitory effects from the accumulation of SCCs products, yielding 475.2 mg gVS−1, which was 20.26% higher than the control phase. The OH− generated by the bipolar membrane was cycled back to regulate the pH of bioreactor, thereby completely eliminating the need for external alkaline additives. The upstream MP effectively prevented ion exchange membrane fouling, and the OH− generated by BPED achieved the in-situ regeneration of MP's alkali extract, reducing the OH− demand by 40% in MP+BPED stage. It was also observed that BPED exhibited higher selectivity towards small-molecule acetic acid, while MP tended to recover larger-molecule caproic acid. Selective extraction of specific SCC drove the product spectrum of the bioreactor to produce more SCC that was selectively extracted. [Display omitted] • In situ BPED extraction increased SCCs yield by 20.26%. • Upstream MP helped to prevent the ion exchange membrane fouling. • The generated OH− by BPED reduced the external base input. • BPED exhibited higher selectivity towards C2 acid while MP towards C6 acid. • Extraction selectivity drove to form a different product spectrum. [ABSTRACT FROM AUTHOR]

Background: β-Caryophyllene, a kind of bicyclic sesquiterpene, is mainly used as a spice in the food and cosmetic industries. Furthermore, it also has significant value in the pharmaceutical industry and is now considered to be used as a new fuel. As a chemical energy heterotrophic microorganism, Escherichia coli can produce a large amount of acetyl-CoA through aerobic respiration, and acetyl-CoA is the common precursor substance in the biosynthesis of all terpenoids. Therefore, E. coli has the potential to be a cell factory to produce terpenoids. Results: A new gene of β-caryophyllene synthase (TPS7) was found by analyzing the genome of Nicotiana tabacum L. using bioinformatics methods. The gene was overexpressed in engineered E. coli with a heterogeneous mevalonate (MVA) pathway to build a recombinant strain CAR1. Subsequent cultivation experiments in shake flask of engineered strain CAR1 verified that 16.1 mg/L β-caryophyllene was detected from the fermentation broth in the shake flask after induction for 24 h with IPTG. The toxic by-product of farnesyl acetate was detected during the process, and CAR1 showed a heavily cellular accumulation of product. We constructed an engineered strain CAR2, in which the downstream genes of the MVA pathway were integrated into the E. coli chromosome, successfully increasing β-caryophyllene production to 100.3 mg/L. The highest production of β-caryophyllene during the fed-batch fermentation was 4319 mg/L. Then we employed in situ extraction fermentation to successfully increase the production of β-caryophyllene by 20% to 5142 mg/L. Conclusion: A new sesquiterpene synthase, TPS7, from tobacco was found to be able to produce β-caryophyllene with high efficiency. Based on this, an engineered E. coli was constructed to produce a much higher concentration of β-caryophyllene than the previous studies. During the fermentation process, we observed that β-caryophyllene tends to accumulate in intracellular space, which will eventually influence the activity of engineered E. coli. As a result, we solved this by metabolism regulation and in situ extractive fermentation. [ABSTRACT FROM AUTHOR]

In situ extraction or "milking" of microalgae is a promising approach to reduce downstream costs in order to produce low-value substances such as lipids from microalgae in an economical way. Due to its ability to secrete high amounts of long-chain hydrocarbons to an extracellular matrix, the green microalga Botryococcus braunii is suitable for the process of in situ extraction as the cost intensive steps of harvesting, dewatering, and cell disruption could be omitted. Based on a previous study investigating various B. braunii strains in terms of growth, lipid accumulation, and solvent compatibility, the B. braunii strains Showa and Bot22 (both B race) were identified as potential candidates for the process of in situ extraction. In order to prove the suitability of these two strains for the process of in situ extraction, this study first determined the optimal extraction time using short-term in situ extraction over 7 days at different starting biomass concentrations of 1.5 and 2.5 g L−1. Furthermore, both strains were treated applying the optimal extraction time in long-term in situ extractions for 30 days to confirm the results from the short-term extractions. The results indicate a strain-dependent optimal extraction time of 300 min day−1 for strain Showa and 200 min day−1 for strain Bot22. During long-term in situ extraction for 30 days, hydrocarbon productivity was 16.99 mg L−1 day−1 (10.53 mg gDW−1 day−1) for strain Showa and 14.53 mg L−1 day−1 (10.48 mg gDW−1 day−1) for strain Bot22. Furthermore, a direct correlation between hydrocarbon productivity achieved by in situ extraction and the hydrocarbon concentration in the biomass of the respective strain could be established. It could be shown that the consideration of the effective extraction time and the phase boundary area is required to calculate an extraction system independent value for the comparison of different extraction setups. [ABSTRACT FROM AUTHOR]

In order to accurately determine the release of moisture and tar in the aerosol of heated tobacco products, an in situ extraction device with a smoking machine trap was designed and tested. The aerosol produced by machine-smoking a heated tobacco product was collected, the Cambridge filter pad inside a bespoke metallic trap was directly extracted and then the moisture was analyzed by GC. The results showed that: 1) The recoveries of the in situ extraction method were in the range of 95.4%-104.6% and the relative standard deviations (RSDs) ranged from 3.72% to 6.09% with the limit of quantification of 0.16 mg/cigarette. 2) Comparing with conventional vibrating extraction, the moisture content in the aerosol determined by the in situ extraction method increased by 15.95%-22.16% under the ISO puffing regime and 38.34%-40.37% under the HCI puffing regime. 3) The main components, such as nicotine, 1,2-propylene glycol and glycerol, in the aerosol of the tested heated tobacco products were not affected by the in situ extraction method. This method was also applicable to the determination of tar release in the smoke of conventional cigarettes. This method could be used for the accurate determination of moisture, tar, nicotine, 1,2-propylene glycol and propanetriol in the aerosol of heated tobacco products. [ABSTRACT FROM AUTHOR]

In situ extraction is a method for separating plant secondary metabolites from in vitro systems of plant biomass cultures. The study aimed to investigate the MTMS-based xerogels morphology effect on the growth kinetics and deoxyshikonin productivity in xerogel-supported in vitro culture systems of Rindera graeca hairy root. Cultures were supplemented with three types of xerogel, i.e., mesoporous gel, microporous gel, and agglomerated precipitate, in the disintegrated or monolithic form. Structure, oil sorption capacity, and SEM analyses for xerogel-based additives were performed. Application of monolithic macroporous xerogel resulted in the highest biomass proliferation, i.e., 5.11-fold fresh biomass increase after four weeks of the screening culture. The highest deoxyshikonin production (i.e., 105.03 µg) was noted when hairy roots were maintained with particles of disintegrated mesoporous xerogel. The detailed kinetics investigations (6-week culture) revealed the highest growth of hairy root biomass and secondary metabolite production, equaling 9.46-fold fresh weight biomass and 204.08 µg deoxyshikonin, respectively. MTMS-based xerogels have been recognized as selective biocompatible scaffolds for boosting the proliferation of transgenic roots or for productivity enhancement of naphthoquinones without detrimental effects on biomass growth, and their successful applicability in in situ removal of secondary plant metabolites has been experimentally confirmed.

Clostridium carboxidivorans converts syngas into industrial alcohols like hexanol, but titers may be limited by product toxicity. Investigation of IC50 at 30 °C (17.5 mM) and 37 °C (11.8 mM) revealed increased hexanol tolerance at lower temperatures. To avoid product toxicity, oleyl alcohol was added as an extraction solvent, increasing hexanol production nearly 2.5-fold to 23.9 mM (2.4 g/L) at 30 °C. This titer exceeds the concentration that is acutely toxic in the absence of a solvent, confirming the hypothesis that current hexanol production is limited by product toxicity. The solvent however had no positive effect at 37 °C. Furthermore, C. carboxidivorans cell membranes adapted to the higher temperature by incorporating more saturated fatty acids, but surprisingly not to hexanol. Corn oil and sunflower seed oil were tested as alternative, inexpensive extraction solvents. Hexanol titers were similar with all solvents, but oleyl alcohol achieved the highest extraction efficiency.

In this work, in situ extraction of essential oil from Pinus koraiensis seed scales using tea saponin-induced ultrasonic pretreatment-microwave-assisted hydrodistillation (UP-MAHD) was studied. By fitting the extraction kinetic curves with different factors, a first-order kinetic model was determined to be more suitable. The key factors were screened and optimized via Plackett Burman Design (PBD) and BOX-Behnken Design (BBD). Under the optimization parameters of a liquid material ratio of 14 mL/g, power of 100 W, time of 15 min, temperature of 50 °C, and tea saponin dose of 0.4% for the ultrasonication and then microwave at 458 W for 18 min, the actual yield was 8.02 μL/g± 0.35% and was highly consistent with the theoretical yield of 8.21 μL/g. UP-MAHD-treated Pinus koraiensis seed scales essential oil (PKSSEO) had good inhibition functional activity of α-amylase. In addition, the chemical composition of the PKSSEO were determined using Gas chromatography mass spectrometry (GC MS) and Fourier transform infrared spectroscopy (FT IR), thermal gravimetry (TG), and differential scanning calorimetry (DSC), and its spatial structure characterization (SSC) obtained via UP-MAHD treatment was enhanced. In summary, this extraction method efficiently extracted PKSSEO with a lower effect on its activity and composition and could have solid future development prospects. Our study provided effective theoretical data support for the reuse and development of abandoned Pinus koraiensis seed scales resources. [Display omitted] • UP-MAHD in situ extraction of essential oil from Pinus koraiensis seed scales induced by tea saponin was performed. • Nonlinear extraction kinetics theories were applied; key factors were screened and optimized via Plackett Burman and Box Behnken Designs, respectively. • The inhibitory enzyme activity, GC MS, FT IR, and thermal stability (TGA, DSC) of the essential oil were examined. • The mechanism of UP-MAHD in situ extraction was elucidated. The spatial structure characteristics of the residue were comparatively studied. [ABSTRACT FROM AUTHOR]

An in situ formation of ionic liquid was used for preconcentration of four triazole fungicides in food samples. The microextraction method was used for the first time in the literature for preconcentration of triazole fungicides. In the developed method, tributylhexadecylphosphonium bromide ([P44412]Br) and potassium hexafluorophosphate (KPF6) were used for the formation of hydrophobic ionic liquid. After centrifugation, the fine microdroplets were produced in one step, providing the extraction step in a quick and environmentally friendly manner. The functional group of the hydrophobic ionic liquid was investigated using FT-IR. Various extraction parameters were studied and optimized. In the extraction method, 0.01 g of [P44412]Br and 0.01 g of KPF6, centrifugation at 4500 rpm for 10 min were used. The optimized technique provided a good linear range (90–1000 μg L−1) and high extraction recovery, with a low limit of detection (30–50 μg L−1). Methods for the proposed in situ formation of ionic liquid were successfully applied to honey, fruit juice, and egg matrices. The recoveries were obtained in a satisfactory range of 62–112%. The results confirmed the suitability of the proposed microextraction method for selective extraction and quantification of triazole fungicides.

Nucleotide excision repair (NER) is the most versatile DNA repair pathway that removes a wide variety of DNA lesions caused by different types of physical and chemical agents, such as ultraviolet radiation (UV), environmental carcinogen benzo[a]pyrene and anti-cancer drug carboplatin. The mammalian NER utilizes more than 30 proteins, in a multi-step process that begins with the lesion recognition within seconds of DNA damage to completion of repair after few hours to several days. The core proteins and their biochemical reactions are known from in vitro DNA repair assays using purified proteins, but challenge was to understand the dynamics of their rapid recruitment and departure from the lesion site and their coordination with other proteins and post-translational modifications to execute the sequential steps of repair. Here, we provide a brief overview of various techniques developed by different groups over last 20 years to overcome these challenges. However, more work is needed for a comprehensive knowledge of all aspects of mammalian NER. With this aim, here we provide detailed protocols of three simple yet innovative methods developed by many teams that range from local UVC irradiation to in situ extraction and sub-cellular fractionation that will permit study of endogenous as well as exogenous NER proteins in any cellular model. These methods do not require unique reagents or specialized instruments, and will allow many more laboratories to explore this repair pathway in different models. These techniques would reveal intracellular movement of these proteins to the DNA lesion site, their interactions with other proteins during repair and the effect of post-translational modifications on their functions. We also describe how these methods led us to identify hitherto unexpected role of poly(ADP-ribose) polymerase-1 (PARP1) in NER. Collectively these three simple techniques can provide an initial assessment of the functions of known and unknown proteins in the core or auxiliary events associated with mammalian NER. The results from these techniques could serve as a solid foundation and a justification for more detailed studies in NER using specialized reagents and more sophisticated tools. They can also be suitably modified to study other cellular processes beyond DNA repair.

Unique phytochemical profile of plants belonging to Boraginaceae family provides a prolific resource of lipophilic pigments from the group of naphthoquinone derivatives. To overcome low compound content, the major obstacle of plant-based production, immobilization of Rindera graeca roots in in vitro cultures was implemented for efficient production of rinderol, novel furanonaphthoquinone derivative with anticancer properties. Chromatographic procedures revealed rinderol presence in extracts of all investigated root lines, derived both from root biomass and post-culture medium. Unexpectedly, in the second stage of the experiment, rinderol production was ceased in control, unmodified culture systems. On the contrary, roots immobilized on PUF rafts uniformly and stably produced rinderol, and its highest amount was noted for transformed root lines after 42 days of cultivation (222.98 ± 10.47 µg/flask). PUF occurred to be the main place of compound accumulation. Moreover, investigation of rinderol biological activity revealed its fast-acting cell death induction in HeLa cervical cancer cells at relatively low concentrations. Presented results revealed successful application of R. graeca roots immobilization on PUF rafts for production and in situ product removal of rinderol, novel lipophilic furanonaphthoquinone with suggested proapoptotic activity.

Biocompatible extraction emerges recently as a means to reduce costs of biotechnology processing of microalgae. In this frame, this study aimed at determining how specific culture conditions and the associated cell morphology impact the biocompatibility and the extraction yield of β-carotene from the green microalga Dunaliella salina using n-decane. The results highlight the relationship between the cell disruption yield and cell volume, the circularity and the relative abundance of naturally permeabilized cells. The disruption rate increased with both the cell volume and circularity. This was particularly obvious for volume and circularity exceeding 1500 µm3 and 0.7, respectively. The extraction of β-carotene was the most biocompatible with small (600 µm3) and circular cells (0.7) stressed in photobioreactor (30% of carotenoids recovery with 15% cell disruption). The naturally permeabilized cells were disrupted first; the remaining cells seems to follow a gradual permeabilization process: reversibility (up to 20 s) then irreversibility and cell disruption. This opens new carotenoid production schemes based on growing robust β-carotene enriched cells to ensure biocompatible extraction.

Copyright of China Brewing is the property of China Brewing Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

As global temperatures continue to increase the move to renewable energy sources is becoming an essential step in achieving a sustainable future. Biofuels from microalgae, while still more expensive to produce than fossil fuels, are of great interest for the high microalgae biomass growth rates, high lipid content, relatively low water requirements, and ability to be grown on non-arable land. Botryococcus braunii is a freshwater green microalga which grows in colonies supported by an extracellular matrix (ECM) of liquid hydrocarbons. Recent studies have investigated the efficiency of non-destructive in situ solvent extraction of lipids from B. braunii with a variety of solvents. These lipids are encapsulated in a retaining wall coated by a sheath of amphiphilic fibrils which have been found to act as a barrier to solvents. By carrying out repeated in situ solvent extraction in a high shear environment, we found that access to the ECM can be significantly improved. Sustainable botryococcene and total lipid productivities respectively of 29.9 ± 8.5 and 71.3 ± 20.3 mg L−1 day−1 were achieved with heptane extraction, a shear rate of 415 s−1, initial culture density of 1.62 ± 0.12 g L−1, contact period of 4 h, and a recovery period of 3 days. Nutrient supplementation to replace those lost in the emulsion entrained biomass will be required to maintain a sustainable process. The combined presence of heptane with high shear proved to be an effective colony disruption method which could be an effective pre-treatment followed by a low shear, high solvent contact, and extraction method. [ABSTRACT FROM AUTHOR]

Product selectivity and yield in chemical reactions can be limited by side product formation or low conversions due to equilibrium limitations. Extractive reaction systems (ERS) employ an in situ liquid‐liquid extraction that separates the product from the reaction phase to overcome these difficulties. The design of ERS requires a broad knowledge of the discipline of process intensification and extraction and reaction engineering. Furthermore, specific knowledge about the interplay of reaction and extraction phenomena is unique to ERS. This review gives an overview of the design of ERS and enables their application to any suitable reaction. [ABSTRACT FROM AUTHOR]

Abstract Background High cost of traditional substrates and formation of by-products (such as acetone and ethanol) in acetone–butanol–ethanol (ABE) fermentation hindered the large-scale production of biobutanol. Here, we comprehensively characterized a newly isolated solventogenic and xylanolytic Clostridium species, which could produce butanol at a high ratio with elimination of ethanol and conversion of acetone to more value-added product, isopropanol. Ultimately, direct butanol production from hemicellulose was achieved with efficient expression of indigenous xylanase by the novel strain via consolidated bioprocessing. Results A novel wild-type Clostridium sp. strain NJP7 was isolated and characterized in this study, which was capable of fermenting monosaccharides, e.g., glucose into butanol via a fermentative acetone–isopropanol–butanol pathway. With enhancement of buffering capacity and alcohol dehydrogenase activities, butanol and isopropanol titer by Clostridium sp. strain NJP7 was improved to 12.21 and 1.92 g/L, respectively, and solvent productivity could be enhanced to 0.44 g/L/h. Furthermore, with in situ extraction with biodiesel, the amount of butanol and isopropanol was finally improved to 25.58 and 5.25 g/L in the fed-batch mode. Meanwhile, Clostridium sp. strain NJP7 shows capability of direct isopropanol–butanol production from hemicelluloses with expression of indigenous xylanase. 2.06 g/L of butanol and 0.54 g/L of isopropanol were finally achieved through the temperature-shift simultaneous saccharification and fermentation, representing the highest butanol production directly from hemicellulose. Conclusion The co-production of isopropanol with butanol by the newly isolated Clostridium sp. strain NJP7 would add on the economical values for butanol fermentation. Furthermore, the high isopropanol-butanol production with in situ extraction would also greatly enhance the economic feasibility for fermentative production of butanol–isopropanol in large scale. Meanwhile, its direct production of butanol–isopropanol from polysaccharides, hemicellulose through secretion of indigenous thermostable xylanase, shows great potential using lignocellulosic wastes for biofuel production.

Unique biosynthetic abilities revealed by plants determine in vitro cultures of hairy roots as a suitable source of pharmaceutically relevant bioactive compounds. The basic aim of the study was to examine the applicability of aerogel composed of methyltrimethoxysilane (MTMS) for immobilization of Rindera graeca hairy roots by identifying quantitative effects of biomass proliferation and naphthoquinones extracellular secretion in the aerogel-supported culture system. R. graeca hairy roots were simultaneously cultured for 28-days, as (i) nonimmobilized biomass (reference system), (ii) biomass immobilized on macroporous polyurethane foam (PUF), (iii) biomass with disintegrated MTMS aerogel, (iv) biomass immobilized on polypropylene (PP) fibers (as control), and (v) biomass immobilized on monolithic PP-reinforced MTMS aerogel. MTMS aerogel exhibited high level of biocompatibility toward R. graeca hairy roots which grew into the structure of monolithic aerogel-based constructs. Monolithic MTMS-based constructs significantly promoted the proliferation of hairy roots, resulting in 55% higher fresh mass than the reference system. The highest level of naphthoquinones productivity, i.e., 653 µg gDW−1, was noted for PUF-supported culture system.

Abstract: Verticillins are a group of epipolythiodioxopiperazine alkaloids that have displayed potent cytotoxicity. To evaluate their potential further, a larger supply of these compounds was needed for both in vivo studies and analogue development via semisynthesis. To optimize the biosynthesis of these secondary metabolites, their production was analyzed in two different fungal strains (MSX59553 and MSX79542) under a suite of fermentation conditions. These studies were facilitated by the use of the droplet-liquid microjunction-surface sampling probe (droplet probe), which enables chemical analysis in situ directly from the surface of the cultures. These experiments showed that the production of verticillins was greatly affected by growth conditions; a significantly higher quantity of these alkaloids was noted when the fungal strains were grown on an oatmeal-based medium. Using these technologies to select the best among the tested growth conditions, the production of the verticillin analogues was increased while concomitantly decreasing the time required for fermentations from 5 weeks to about 11 days. Importantly, where we could previously supply 5-10 mg every 6 weeks, we are now able to supply 50-150 mg quantities of key analogues per month via laboratory scale fermentation.Graphical abstract: [ABSTRACT FROM AUTHOR]

In Acetone-Butanol-Ethanol fermentation, bacteria should tolerate high concentrations of solvent products, which inhibit bacteria growth and limit further increase of solvents to more than 20 g/L. Moreover, this limited solvent concentration significantly increases the cost of solvent separation through traditional approaches. In this study, alginate adsorbent immobilization technique was successfully developed to assist in situ extraction using octanol which is effective in extracting butanol but presents strong toxic effect to bacteria. The adsorbent improved solvent tolerance of Clostridium acetobutylicum under extreme condition of high concentration of organic solvent. Using the developed technique, more than 42% of added bacteria can be adsorbed to the adsorbent. Surface area of the adsorbent was more than 10 times greater than sodium alginate. Scanning electron microscope image shows that an abundant amount of pore structure was successfully developed on adsorbents, promoting bacteria adsorption. In adsorbent assisted ABE fermentation, there was 21.64 g/L butanol in extracting layer compared to negligible butanol produced with only the extractant but without the adsorbent, for the reason that adsorbent can reduce damaging exposure of C. acetobutylicum to octanol. The strategy can improve total butanol production with respect to traditional culture approach by more than 2.5 fold and save energy for subsequent butanol recovery, which effects can potentially make the biobutanol production more economically practical.

Lycopene biosynthesis is frequently hampered by downstream processing hugely due to its inability to be secreted out from the producing chassis. Engineering cell factories can resolve this issue by secreting this hydrophobic compound. A highly permeable E. coli strain was developed for a better release rate of lycopene. Specifically, the heterologous mevalonate pathway and crtEBI genes from Corynebacterium glutamicum were overexpressed in Escherichia coli BL21 (DE3) for lycopene synthesis. To ensure in situ lycopene production, murein lipoprotein, lipoprotein NlpI, inner membrane permease protein, and membrane-anchored protein in TolA-TolQ-TolR were deleted for improved membrane permeability. The final strain, LYC-8, produced 438.44 ± 8.11 and 136.94 ± 1.94 mg/L of extracellular and intracellular lycopene in fed-batch fermentation. Both proteomics and lipidomics analyses of secreted outer membrane vesicles were perfect indicators of hypervesiculation. Changes in the ratio of saturated fatty acids, unsaturated fatty acids, and cyclopropane fatty acids coupled with the branching and acyl chain lengths altered the membrane fatty acid composition. This ensured membrane fluidity and permeability for in situ lycopene release. The combinatorial deletion of these genes altered the cellular morphology. The structural and morphological changes in cell shape, size, and length were associated with changes in the mechanical strength of the cell envelope. The enhanced lycopene production and secretion mediated by improved membrane permeability established a cell lysis-free system for an efficient releasing rate and downstream processing, demonstrating the importance of vesicle-associated membrane permeability in efficient lycopene production.

The biosynthetic potential for six lignans accumulation in two lines of Taxus x media hairy roots was investigated. The cultures of KT and ATMA hairy root lines were supplemented with precursors: coniferyl alcohol (CA 1, 10 or 100 µM) and/or l-phenylalanine (100 µM PHEN) and/or methyl jasmonate (100 µM MeJa). Moreover the two-phase in vitro cultures supported with perfluorodecalin (PFD) as a gas carrier and in situ extrahent were used. The hairy root lines differed in lignan production profiles. In the control untreated cultures KT roots did not accumulate secoisolariciresinol and lariciresinol while ATMA roots did not accumulate matairesinol. In ATMA roots the treatment with CA (1 or 10 µM) resulted in the production of lariciresinol and secoisolariciresinol whereas solely lariciresinol was present after 100 µM CA application. Elicitation with 1 µM CA and MeJa yielded with hydroxymatairesinol aglyca and lariciresinol glucosides with their highest content 37.88 and 3.19 µg/g DW, respectively. The stimulatory effect of simultaneous treatment with 1 µM CA, PHEN and MeJa on lignan production was observed when the cultures were supplemented with PFD-aerated or degassed. In ATMA root cultures these applied conditions were the most favourable for matairesinol content which amounted to 199.86 and 160.25 µg/g DW in PFD-aerated and PFD-degassed supported cultures, respectively. In KT root cultures solely, hydroxymatairesinol and coniferin/CA content was enhanced with their highest yield 59.29 and 134.60 µg/g DW in PFD-aerated and PFD-degassed cultures, respectively. [ABSTRACT FROM AUTHOR]

The presence of heavy metals in water resources directly affects consumer health. The quality of surface water resources in Central America is usually low due to the presence of metals and other pollutants. The lack of analytical instrumentation to perform routine monitoring of water has encouraged the development of easy tools to facilitate the determination of heavy metals in waters in remote sites. In this study, we evaluated the use of different sorbents, such as Adsorbsia As600 (titanium dioxide), Iontosorb Oxin, 8-hydroxyquinoline bearing functional groups, and Duolite GT-73, with thiol functionality, for Cd, Ni, Cu, Zn, and Al extraction. It was found that both Adsorbsia As600 and Iontosorb Oxin allowed the adsorption of all metals, and the recovery was achieved using either HCl or ethylenediaminetetraacetic sodium salt (EDTA) solutions. Hence, Adsorbsia As600 was employed for in situ sampling in the metal contamination evaluation of water samples (from 15 wells and nine storage tanks) from the municipality of Torola, Mozarán, El Salvador. The developed procedure allowed all the metals in the samples to be detected, and Ni and Al were found to be above Salvadoran guidelines for drinking water quality. [ABSTRACT FROM AUTHOR]

Background: (-)-α-Bisabolol, also known as levomenol, is an unsaturated sesquiterpene alcohol that has mainly been used in pharmaceutical and cosmetic products due to its anti-inflammatory and skin-soothing properties. (-)-α-Bisabolol is currently manufactured mainly by steam-distillation of the essential oils extracted from the Brazilian candeia tree that is under threat because its natural habitat is constantly shrinking. Therefore, microbial production of (-)-α-bisabolol plays a key role in the development of its sustainable production from renewable feedstock. Results: Here, we created an Escherichia coli strain producing (-)-α-bisabolol at high titer and developed an in situ extraction method of (-)-α-bisabolol, using natural vegetable oils. We expressed a recently identified (-)-α-bisabolol synthase isolated from German chamomile (Matricaria recutita) (titer: 3 mg/L), converted the acetyl-CoA to mevalonate, using the biosynthetic mevalonate pathway (12.8 mg/L), and overexpressed farnesyl diphosphate synthase to efficiently supply the (-)-α-bisabolol precursor farnesyl diphosphate. Combinatorial expression of the exogenous mevalonate pathway and farnesyl diphosphate synthase enabled a dramatic increase in (-)-α-bisabolol production in the shake flask culture (80 mg/L) and 5 L bioreactor culture (342 mg/L) of engineered E. coli harboring (-)-α-bisabolol synthase. Fed-batch fermentation using a 50 L fermenter was conducted after optimizing culture conditions, resulting in efficient (-)-α-bisabolol production with a titer of 9.1 g/L. Moreover, a green, downstream extraction process using vegetable oils was developed for in situ extraction of (-)-α-bisabolol during fermentation and showed high yield recovery (>98%). Conclusions: The engineered E. coli strains and economically viable extraction process developed in this study will serve as promising platforms for further development of microbial production of (-)-α-bisabolol at large scale. [ABSTRACT FROM AUTHOR]

Suspension cell cultures of Corylus avellana were challenged with salicylic acid and its combined use with dibutyl phthalate solvent. Salicylic acid with concentrations of 12.5, 25 and 50 mg L–1 and 10% (v/v) dibutyl phthalate were used and added on day 8 and 10 of subculture, respectively. The results showed that growth, viability and protein content of cells were decreased by the treatments, compared to control. In all treatments, hydrogen peroxide content and lipid peroxidation rate of cells increased, compared to those of the control cells. Activity of phenylalanine ammonia-lyase increased by salicylic acid and, dibutyl phthalate exaggerated effect of salicylic acid. While flavonoids content decreased by the treatments, paclitaxel content increased significantly. The extracellular paclitaxel was more affected, compared to cell-associated paclitaxel and all treatments increased paclitaxel release and specific yield compared to that of the control. The most production of paclitaxel and specific yield of it were observed under effect of combined use of salicylic acid (50 mg L–1) and dibutyl phthalate, suggesting a synergistic accumulative effect.

Given the large amount of crude glycerol formed as a by-product in the biodiesel industries and the concomitant decrease in its overall market price, there is a need to add extra value to this biorefinery side stream. Upgrading can be achieved by new biotechnologies dealing with recovery and conversion of glycerol present in wastewaters into value-added products, aiming at a zero-waste policy and developing an economically viable process. In microbial bioelectrochemical systems (BESs), the mixed microbial community growing on the cathode can convert glycerol reductively to 1,3-propanediol (1,3-PDO). However, the product yield is rather limited in BESs compared to classic fermentation processes, and the synthesis of side-products, resulting from oxidation of glycerol, such as organic acids represents a major burden for recovery of 1,3-PDO. Here, we show that the use of an enriched mixed microbial community of glycerol degraders and in situ extraction of organic acids positively impacts 1,3-PDO yield and allows additional recovery of propionate from glycerol. We report the highest production yield achieved (0.72 mol1,3-PDO mol-1glycerol) in electricity driven 1,3-PDO biosynthesis from raw glycerol, which is very close to the 1,3-PDO yield reported thus far for a mixed microbial culture based glycerol fermentation process. We also present a combined approach for 1,3-PDO production and propionate extraction in a single three chamber reactor system, which leads to recovery of additional 3-carbon compounds in BESs. This opens up further opportunities for an economical upgrading of biodiesel refinery side or waste streams.

The presence of heavy metals in water resources directly affects consumer health. The quality of surface water resources in Central America is usually low due to the presence of metals and other pollutants. The lack of analytical instrumentation to perform routine monitoring of water has encouraged the development of easy tools to facilitate the determination of heavy metals in waters in remote sites. In this study, we evaluated the use of different sorbents, such as Adsorbsia As600 (titanium dioxide), Iontosorb Oxin, 8-hydroxyquinoline bearing functional groups, and Duolite GT-73, with thiol functionality, for Cd, Ni, Cu, Zn, and Al extraction. It was found that both Adsorbsia As600 and Iontosorb Oxin allowed the adsorption of all metals, and the recovery was achieved using either HCl or ethylenediaminetetraacetic sodium salt (EDTA) solutions. Hence, Adsorbsia As600 was employed for in situ sampling in the metal contamination evaluation of water samples (from 15 wells and nine storage tanks) from the municipality of Torola, Mozarán, El Salvador. The developed procedure allowed all the metals in the samples to be detected, and Ni and Al were found to be above Salvadoran guidelines for drinking water quality.

Integrative sampling enables the collection of analyte mass from environmental liquids over extended timeframes from hours to months. While the incentives to complement or replace conventional, time-discrete sampling have been widely discussed, the data quality implications of employing alternative, integrative methods have not yet been systematically studied. A critical analysis of contemporary literature reports showed the data quality of integrative samplers, whether active-advection or passive-diffusion, to be governed by uncertainty in both sampling rate and analyte recovery. Derivation of two lumped parameters, representing the coefficient of accumulation ( α ) of a contaminant from an environmental fluid and the coefficient of subsequent recovery ( ρ ) of its mass from the sampler, produced a conceptual framework for quantifying error sources in concentration data derived from accumulative samplers. Whereas the precision associated with recovery was found to be fairly consistent across eight passive-diffusion and active-advection devices (averaging 5–16% relative standard deviation, RSD), active-advection samplers effectively improve precision in sampling rate (analyte uptake), as determined for two active-advection devices (2–7% average RSD) and five passive devices (12–42% average RSD). In summary, an approach is presented whereby the data quality implications of integrative sampler design can be compared, which can inform the selection, optimization, and development of sampling systems to complement the state of the art. [ABSTRACT FROM AUTHOR]

BACKGROUND: The application of controlled postharvest abiotic stresses in carrots induces the production of phenolic compounds, mainly chlorogenic acids, which present diverse biological functions. Aqueous two-phase systems (ATPS) is a liquid-liquid extraction technique that has demonstrated its potential for the fractionation and recovery of bioactive compounds. ATPS have the advantages of using environmentally friendly constituents, ease of scale-up and process integration capability. This experimental work addresses for the first time the recovery of bioactive compounds (mainly hydroxycinnamic acid derivatives) from abiotically stressed carrot tissue using ATPS strategies, including in situ extraction, in alcohol-salt and ionic liquid-salt systems. RESULTS: The partitioning of chlorogenic acid was favored in the ethanol-potassium phosphate system achieving a recovery yield of 81.9%. Hydroxycinnamic acid derivatives fractioning was favored in 1-ethyl-3-methylimidazolium acetate (EMIM-Ac)-potassium phosphate ATPS, where recovery yields up to 85.4% were achieved. The in situ extraction strategy not only demonstrated potential for merging extraction and recovery in the same system but also, depending on ATPS used, allowed differential phenolic profiles to be obtained. CONCLUSION: ATPS strategies (either solid-liquid extraction followed by ATPS or in situ extraction) were shown to have great potential for the recovery of bioactives from stressed carrot tissue. [ABSTRACT FROM AUTHOR]

The ban on using processed animal proteins in feedstuffs led the feed sector to look for other sources of protein. Dried distillers grains with solubles (DDGS) could be considered as an important source in this regard. They are imported into Europe mainly for livestock feed. Identifying their origin is essential when labelling is missing and for feed safety, particularly in a crisis situation resulting from contamination. This study investigated applying attenuated total reflection Fourier transform mid-infrared spectroscopy (ATR-FT-MIR) to the oil fraction extracted from samples in situ in order to identify the origin of DDGS. The use of spectroscopic and chemometric tools enabled the botanical and geographical origins of DDGS, as well as the industrial process used to produce them, to be identified. The models developed during the study provided a classification higher than 95% using an external validation set. [ABSTRACT FROM AUTHOR]

Suspension cultures of Arnebia euchroma supported with liquid perfluorodecalin (PFD) degassed, aerated, or ethylene-saturated were investigated as a novel in situ extraction system for enhanced alkannin/shikonin production. Simultaneously, the effect of PFD applied as the liquid gas carrier on the growth of A. euchroma biomass was studied. The similar dry (4-fold) and fresh (7-fold) biomass increase was observed in the control (without PFD addition) and supplemented with PFD-degassed or PFD-aerated cultures while PFD-ethylene application impeded cell growth. The highest total of alkannin/shikonin production (23.23 mg flask) was observed when PFD-aerated has been used and it resulted in about 50 % higher yield of alkannin/shikonin compared with the control culture. Chiral HPLC analysis revealed that in cultures supported with PFD, both alkannin and shikonin were produced. Their mutual ratio varied depending on culture conditions, and the accumulation of alkannin prevailed under almost all culture conditions. PFD has proved to be exceptionally efficient and cell-safe solvent for the in situ extraction of naphthoquinone red pigments without exerting any detrimental effects on cell growth. Extracellularly secreted red naphthoquinones were easily dissolved and extracted from the PFD phase, which can be regenerated and reused (e.g., in continuous culture system). [ABSTRACT FROM AUTHOR]

To prevent degradation of intracellular retinoids through in situ extraction from the cells, a two-phase culture system was performed. Several organic solvents, including n-alkanes, mineral oils and cosmetic raw materials, were applied as the extraction phase. Of the n-alkanes, n-decane had the highest retinoid production as 134 mg/l after 72 h. For mineral oil, light and heavy mineral oil gave retinoid productions of 158 and 174 mg/l after 96 h, respectively. Of other materials, isopropyl myristate gave the highest retinoid production of 181 mg/l. These results indicate that many types of oils can be applied for retinoid production, and optimization of the in situ extraction process will lead to further improve of economical production for the industrial purpose. [ABSTRACT FROM AUTHOR]

In situ extraction is important for highly productive and cost-efficient processes in industrial biotechnology, but it is difficult to establish for intracellularly accumulating carotenoids like β-carotene. In this study, the organic solvent used in aqueous-organic two-phase media exerted a strong effect on the release of β-carotene from recombinant yeast cells. The carotenoid-synthesizing Saccharomyces cerevisiae strain YB/I/E was cultivated in two-liquid-phase media with 20% dodecane or 20% sunflower oil. Up to 0.6 µg/ml β-carotene was released into sunflower oil, but less than 0.1 µg/ml into dodecane, although biocompatibility and solubility of β-carotene is appropriate for both solvents. Addition of linoleic acid, the main component of sunflower oil, to the dodecane phase increased the amount of β-carotene released, indicating that linoleic acid is the component responsible for the β-carotene release into sunflower oil. These findings demonstrate that the effect of the organic solvent should be taken into consideration for further research on in situ extraction of carotenoids. Copyright © 2013 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]