High-value Metabolites Research Articles

A Novel AP2/ERF Transcription Factor CR1 Regulates the Accumulation of Vindoline and Serpentine in Catharanthus roseus.

As one type of the most important alkaloids in the world, terpenoid indole alkaloids (TIAs) show a wide range of pharmaceutical activities that are beneficial for clinical treatments. Catharanthus roseus produces approximately 130 identified TIAs and is considered to be a model plant to study TIA biosynthesis. In order to increase the production of high medical value metabolites whose yields are extremely low in C. roseus, genetic engineering combined with transcriptional regulation has been applied in recent years. By using bioinformatics which is based on RNA sequencing (RNA-seq) data from methyl jasmonate (MeJA)-treated C. roseus as well as phylogenetic analysis, the present work aims to screen candidate genes that may be involved in the regulation of TIA biosynthesis, resulting in a novel AP2/ERF transcription factor, CR1 (Catharanthus roseus 1). Subsequently, virus-induced gene silencing (VIGS) of CR1 was carried out to identify the involvement of CR1 in the accumulations of several TIAs and quantitative real-time PCR (qRT-PCR) was then applied to detect the expression levels of 7 genes in the related biosynthetic pathway in silenced plants. The results show that all the 7 genes were upregulated in CR1-silenced plants. Furthermore, metabolite analyses indicate that silencing CR1 could increase the accumulations of vindoline and serpentine in C. roseus. These results suggest a novel negative regulator which may be involved in the TIAs biosynthetic pathway.

Rapid chemotaxonomic profiling for the identification of high-value carotenoids in microalgae

Microalgae hold considerable promise as biofactories for the mass production of high-value metabolites. In this study, 110 strains isolated from waterbodies of the West of Ireland were successfully brought into culture. Chemotaxonomic pigment profiles were obtained in under 7 min using a C18 monolithic HPLC column. This method provided good reproducibility and baseline resolution for 30 of the 54 pigments detected in the culture collection and mixed pigment extracts. Twelve of the isolated strains exhibiting promising carotenoid profiles were selected to further investigate carotenoid cell contents in a batch culture mode, which typically showed higher yields during the exponential phase. Candidates for future carotenoid biorefinery include Brachiomonas submarina (6.1 mg g−1 DW lutein), Rhodella sp. (0.80 mg g−1 DW zeaxanthin) and cf. Amphora sp. (3.3 mg g−1 DW fucoxanthin). This study demonstrates the monolithic HPLC column as a useful screening tool for the rapid chemotaxonomic characterisation and selection of promising strains from culture collections warranting further investigation for physiological optimisation and up-scaling in photobioreactors.

A Novel Organic Electrochemical Transistor-Based Platform for Monitoring the Senescent Green Vegetative Phase of Haematococcus pluvialis Cells.

The freshwater unicellular microalga Haematococcus pluvialis (H. pluvialis) has gained increasing attention because of its high-value metabolite astaxanthin, a super anti-oxidant. For the maximum astaxanthin production, a key problem is how to determine the senescent green vegetative phase of H. pluvialis cells to apply the astaxanthin production inducers. The conventional methods are time-consuming and laborious. In this study, a novel platform based on organic electrochemical transistor (OECT) was produced. A significant channel current change of OECTs caused by settled H. pluvialis cells on the poly(3,4-ethylenedioxythiophene): polystyrene sulfonate (PEDOT: PSS) film was recorded commencing from 75 min and a stationary stage was achieved at 120 min after the combined treatment of blue light irradiation and sodium bicarbonate solution additives, which indicate the onset and maturation of the senescent green vegetative phase, respectively. Therefore, the appropriate time point (120 min after sample loading) to apply astaxanthin production inducers was determined by as-fabricated OECTs. This work may assist to develop a real-time biosensor to indicate the appropriate time to apply inducers for a maximum astaxanthin production of H. pluvialis cells.

Saccharomyces cerevisiae as Cell Factory for the Production of Plant Natural Products

Background: Plant natural products (PNPs) play a key role in human health and life quality through the treatment of diseases and as supplements in the food and healthcare industry. These high-value metabolites are traditionally extracted from the natural resource, but ongoing research is exploring the possibilities to construct efficient microbial cell factories that can replace current production methods. Keywords: Plant secondary metabolite, enzyme discovery, metabolic engineering, yeast chassis strain, PNPs, biosynthesis pathway.

Transcriptomes of Podophyllum hexandrum unravel candidate miRNAs and their association with the biosynthesis of secondary metabolites

Podophyllotoxin is a high value metabolite having anticancerous activity and procured from a medicinal plant Podophyllum hexandrum. The escalating demand for podophyllotoxin necessitates development of alternate production platforms. Cell cultures have been tried in the past, however, podophyllotoxin yields were very low (0.3%). Multiple genes of biosynthetic pathway have been correlated with podophyllotoxin content. Understanding regulation of podophyllotoxin is, therefore, essential to design a suitable genetic intervention strategy. Six potential miRNAs were identified in NGS transcriptomes of P. hexandrum for which eight potential mRNA targets were discerned. The identified miRNAs and their mRNA targets (UDP glycosyltransferase, flavonol synthase, glyceraldehyde 3-phosphate dehydrogenase, peroxidase, malate dehydrogenase, phosphoenolpyruvate carboxylase, WRKY 37 and MYBF1 transcription factors) were further tested for any correlation through qRT-PCR. miR396b, miR2673a, miR828b and miR2910 established negative correlation with their mRNA targets through downregulation via complementary miRNA:mRNA base pairing. This is the first report on identification, characterisation, validation and expression analysis of miRNAs and their targets in P. hexandrum.

Development of a counterselectable seamless mutagenesis system in lactic acid bacteria

BackgroundLactic acid bacteria (LAB) are receiving more attention to act as cell factories for the production of high-value metabolites. However, the molecular tools for genetic modifying these strains are mainly vector-based double-crossover strategies, which are laborious and inefficient. To address this problem, several counterselectable markers have been developed, while few of them could be used in the wild-type host cells without pretreatment.ResultsThe pheS gene encoding phenylalanyl-tRNA synthetase alpha subunit was identified in Lactococcus lactis NZ9000 genome. When mutant pheS gene (pheS*) under the control of the Lc. lactis NZ9000 l-lactate dehydrogenase promoter (Pldh) was expressed from a plasmid, the resulted PheS* with an A312G substitution rendered cells sensitive to the phenylalanine analog p-chloro-phenylalanine (p-Cl-Phe). This result suggested pheS* was suitable to be used as a counterselectable marker in Lc. lactis. However, the expression level of pheS* from a chromosomal copy was too low to confer p-Cl-Phe sensitivity. Therefore, a strategy of cascading promoters was attempted for strengthening the expression level of pheS*. Expectedly, a cassette 5Pldh-pheS* with five tandem repetitive promoters Pldh resulted in a sensitivity to 15 mM p-Cl-Phe. Subsequently, a counterselectable seamless mutagenesis system PheS*/pG+host9 based on a temperature-sensitive plasmid pG+host9 harboring a 5Pldh-pheS* cassette was developed in Lc. lactis. We also demonstrated the possibility of applying pheS* to be a counterselectable marker in Lactobacillus casei BL23.ConclusionsAs reported in E. coli, pheS* as a counterselectable marker has been demonstrated to be functional in targeted gene(s) deletion in Lc. lactis as well as in L. casei. Moreover, the efficiency and timesaving counterselectable seamless mutagenesis system PheS*/pG+host9 could be used in the wild-type host cells without pretreatment.

Programming mRNA decay to modulate synthetic circuit resource allocation

Synthetic circuits embedded in host cells compete with cellular processes for limited intracellular resources. Here we show how funnelling of cellular resources, after global transcriptome degradation by the sequence-dependent endoribonuclease MazF, to a synthetic circuit can increase production. Target genes are protected from MazF activity by recoding the gene sequence to eliminate recognition sites, while preserving the amino acid sequence. The expression of a protected fluorescent reporter and flux of a high-value metabolite are significantly enhanced using this genome-scale control strategy. Proteomics measurements discover a host factor in need of protection to improve resource redistribution activity. A computational model demonstrates that the MazF mRNA-decay feedback loop enables proportional control of MazF in an optimal operating regime. Transcriptional profiling of MazF-induced cells elucidates the dynamic shifts in transcript abundance and discovers regulatory design elements. Altogether, our results suggest that manipulation of cellular resource allocation is a key control parameter for synthetic circuit design.

RETRACTED: Bioflocculation: An alternative strategy for harvesting of microalgae – An overview

Microalgae based research has been extensively progressed for the production of value added products and biofuels. Potential application of microalgae for biofuel is recently gained more attention for possibilities of biodiesel and other high value metabolites. However, high cost of production of biomass associated with harvesting technologies is one of the major bottleneck for commercialization of algae based industrial product. Based on the operation economics, harvesting efficiency, technological possibilities, flocculation of algal biomass is a superior method for harvesting microalgae from the growth medium. In this article, latest trends of microalgal cell harvesting through flocculation are reviewed with emphasis on current progress and prospect in environmental friendly bio-based flocculation approach. Bio-flocculation based microalgae harvesting technologies is a promising strategy for low cost microalgal biomass production for various applications.

Optogenetic switches for light-controlled gene expression in yeast.

Light is increasingly recognized as an efficient means of controlling diverse biological processes with high spatiotemporal resolution. Optogenetic switches are molecular devices for regulating light-controlled gene expression, protein localization, signal transduction and protein-protein interactions. Such molecular components have been mainly developed through the use of photoreceptors, which upon light stimulation undergo conformational changes passing to an active state. The current repertoires of optogenetic switches include red, blue and UV-B light photoreceptors and have been implemented in a broad spectrum of biological platforms. In this review, we revisit different optogenetic switches that have been used in diverse biological platforms, with emphasis on those used for light-controlled gene expression in the budding yeast Saccharomyces cerevisiae. The implementation of these switches overcomes the use of traditional chemical inducers, allowing precise control of gene expression at lower costs, without leaving chemical traces, and positively impacting the production of high-value metabolites and heterologous proteins. Additionally, we highlight the potential of utilizing this technology beyond laboratory strains, by optimizing it for use in yeasts tamed for industrial processes. Finally, we discuss how fungal photoreceptors could serve as a source of biological parts for the development of novel optogenetic switches with improved characteristics. Although optogenetic tools have had a strong impact on basic research, their use in applied sciences is still undervalued. Therefore, the invitation for the future is to utilize this technology in biotechnological and industrial settings.

Plant Breeding and Secondary Metabolite Overviews

Plant breeding is an art of improving economically important plants on a scientific basis. The main purpose of plant breeding is to evolve improved varieties of plants. Its objectives are to improve yield, quality, disease resistance, drought and frost characteristics of the crops. Plant breeding has been crucial in increasing agricultural production. Plant cell cultures are becoming promising potential alternative sources for the production of high-value secondary metabolites of industrial importance. Plants face genetic erosion because of habitat destruction and over exploitation, thus need to be conserved. Plant tissue culture techniques are advantageous over the conventional agricultural practices vis-a-vis the conservation strategies in the plants in question. Plant cells are biosynthetically tot potent, which means that each cell in culture retains complete genetic information and hence is able to produce the range of chemicals found in the parent plant.

Protonema suspension cultures of the medicinal moss Polytrichum juniperinum

The moss Polytrichum juniperinum was widely used by native North Americans and in traditional Chinese medicine to treat several illnesses including burns, wounds, bleeding, fever, kidney stones, and gallstones. This paper reports the efficient establishment of a protonema suspension culture of this moss and evaluation of key factors such as culture medium, trophic condition, initial pH, and inoculum size for increasing biomass production, which has scarcely been studied in this type of biological system. No significant differences were found for the maximum specific growth rate (μmax~0.09 d−1) and the total phenolic content between MS, Knop, and PPNH4 media, although an effect on tissue differentiation was observed. Growth rate in a mixotrophic condition was (μmax 0.27 d−1) three times greater than in autotrophic and heterotrophic conditions, reaching high cell densities (~11 g DW L−1). Moreover, simple sugars were secreted into the medium during the growth phase. P. juniperinum protonema cultures tolerated a wide initial medium pH range (4.5–8). The tissue growth index significantly decreased from ~7.7 to 1.9 with increased inoculum size (45–300 mg DW L−1) under photoautotrophic conditions. Similar responses were obtained under mixotrophic conditions with different sucrose concentrations (15–45 g L−1), but no responses to sucrose concentration and inoculum sizes were seen under heterotrophic growth conditions. Finally, this high cell density culture of P. juniperinum is suitable for further studies aimed at exploring and establishing a production platform for high-value secondary metabolites.

Screening of oleaginous yeast for lipid production using rice residue from food waste as a carbon source

Rice residue from food waste contained of starch as a mainly component which could be either hydrolyzed to be fermentable sugars or directly used as a carbon source for the growth and high value metabolites production by various microorganisms. Therefore, this study focused on the utilization of rice residue and rice residue hydrolysate from food waste as a carbon source for the growth and lipids production of oleaginous yeast. Rice residue obtained from canteen of the Faculty of Agro-Industry, Chiang Mai University, Thailand. It composed of moisture content (76.68±0.55%), crude fat (1.76±0.47%), crude protein (3.04±0.06%), ash content (0.46±0.07%), and carbohydrate content (18.05±0.01%), respectively. Rice residue was then subjected to enzymatic hydrolysis using α-amylase and amyloglucosidase (AMG), resulting the maximal reducing sugars of 168.02±0.02 g/L. The screening of oleaginous yeast from flowers and leaves samples from Doi-Inthanon National Park, Faculty of Agro-Industry, Chiang Mai University, the culture collection of the Thailand Institute of Scientific and Technological Research (TISTR) and the Division of Biotechnology, Faculty of Agro-Industry, Chiang Mai University were investigated. Sixty-seven isolates were obtained, and only four isolates were identified as oleaginous yeast because of containing high lipids content more than 20% (w/w), when glucose or rice residue hydrolysate was used as a carbon source. Those oleaginous yeasts were identified as Rhodotorula sp. C7, Rhodosporidium paludigenum C10, and the new isolate TC32, respectively. Their growths and lipid productions were compared with Diozegia sp. TISTR5792. The results showed that, C7, C10, TISTR5792 and TC32 produced the maximal lipids content of 24.26±0.56, 23.69±0.91, 22.43±1.09 and 23.07±0.80% (w/w) when cultivated in the basal medium supplemented with enzymatic-rice residue hydrolysate. Surprisingly, we found that TISTR5792 and TC32 could grow well in the medium supplemented with rice residue > (without hydrolysis) and showed lipids content of 18.41±0.10 and 21.67±0.02% (w/w), respectively. These results indicated that rice residue from food waste shows a high potential to be an effective carbon source for the growth and lipid production of the selected oleaginous yeasts.

Thiol Probes To Detect Electrophilic Natural Products Based on Their Mechanism of Action.

New methods are urgently needed to find novel natural products as structural leads for the development of new drugs against emerging diseases such as cancer and multiresistant bacterial infections. Here we introduce a reactivity-guided drug discovery approach for electrophilic natural products, a therapeutically relevant class of natural products that covalently modify their cellular targets, in crude extracts. Using carefully designed halogenated aromatic reagents, the process furnishes derivatives that are UV-active and highly conspicuous via mass spectrometry by virtue of an isotopically unique bromine or chlorine tag. In addition to the identification of high-value metabolites, the process facilitates the difficult task of structure elucidation by providing derivatives that are primed for X-ray crystallographic analysis. We show that a cysteine probe efficiently and chemoselectively labels enone-, β-lactam-, and β-lactone-based electrophilic natural products (parthenolide, andrographolide, wortmannin, penicillin G, salinosporamide), while a thiophenol probe preferentially labels epoxide-based electrophilic natural products (triptolide, epoxomicin, eponemycin, cyclomarin, salinamide). Using the optimized method, we were able to detect and isolate the epoxide-bearing natural product tirandalydigin from Salinispora and thereby link an orphan gene cluster to its gene product.

A Review on the Assessment of Stress Conditions for Simultaneous Production of Microalgal Lipids and Carotenoids.

Microalgal species are potential resource of both biofuels and high-value metabolites, and their production is growth dependent. Growth parameters can be screened for the selection of novel microalgal species that produce molecules of interest. In this context our review confirms that, autotrophic and heterotrophic organisms have demonstrated a dual potential, namely the ability to produce lipids as well as value-added products (particularly carotenoids) under influence of various physico-chemical stresses on microalgae. Some species of microalgae can synthesize, besides some pigments, very-long-chain polyunsaturated fatty acids (VL-PUFA,>20C) such as docosahexaenoic acid and eicosapentaenoic acid, those have significant applications in food and health. Producing value-added by-products in addition to biofuels, fatty acid methyl esters (FAME), and lipids has the potential to improve microalgae-based biorefineries by employing either the autotrophic or the heterotrophic mode, which could be an offshoot of biotechnology. The review considers the potential of microalgae to produce a range of products and indicates future directions for developing suitable criteria for choosing novel isolates through bioprospecting large gene pool of microalga obtained from various habitats and climatic conditions.

Robust and Biocompatible Hybrid Matrix with Controllable Permeability for Microalgae Encapsulation.

Hybrid beads with entrapped microalgae Chlamydomonas reinhardtii were synthesized for the sustainable production of high value metabolites via photosynthesis. Encapsulating the microalgae requires an exquisite control of material properties, which has been achieved by modifying the composition (alginate, polycation, and silica). A coating of PDADMAC precluded cell leakage as indicated by the OD750 value of the culture medium, and the homogeneous distribution of silica prevented bead shrinkage from the strong electronic force of PDADMAC, resulting in a robust and biocompatible matrix for the cells. Besides fabricating suitable porous beads for the diffusion of expected metabolites, the permeability can be controlled to a certain degree by applying different molecular weights of PDADMAC. The hybrid alginate+silica/CaCl2+PDADMAC beads possessed sufficient mechanical rigidity to sheer force under constant stirring and good chemical stability to chelating agents such as sodium citrate. Moreover, the encapsulated cells exhibited excellent long-term viability and cellular functionality, which retained about 81.5% of the original value after a 120 day encapsulation as observed by microscopy and oximetry measurement. This study is not only significant for understanding the critical role of polycations and silica involved in the synthesis of hybrid beads but also important for real-scale bioengineering applications.

Microalgae-based unsaponifiable matter as source of natural antioxidants and metal chelators to enhance the value of wet Tetraselmis chuii biomass

AbstractThe present work aimed to determine the antioxidant, metal chelating and neuroprotective potential of the unsaponifiable matter (UM) of Tetraselmis chuii to be applied to a biorefinery setting. The UM obtained via saponification from crude lipids extracted from microalgal wet biomass showed a radical scavenging activity (RSA) towards the DPPH radical of 90.7±1.3% and 57.1±1.2% at a concentration of 10 and 5 mg/mL, respectively. The UM fraction also displayed metal chelating capacity at a concentration of 5 mg/mL: 58.5±1.4% and 50.9±4.0% for copper and iron, respectively. The chemical characterization of the UM revealed significant levels of total phenolics (TPC, 13.61 mg GAE/g) and carotenoids (2.45 mg/g of β-carotene, lutein and violaxanthin). Overall, the separation of the UM containing high value metabolites might significantly upgrade the total wet biomass value in a biorefinery, allowing the exploitation of a stream with relevant antioxidant and metal chelating activities.

New taxane biosynthetic gene encoding a β-phenylalanine-CoA ligase identified in jasmonate-elicited Taxus cells

The data-generating power of “omics” technologies is increasingly being harnessed in studies of plant biology to shed light on aspects such as metabolite production patterns, gene sequences and protein expression. Taxol and related taxanes, produced by several Taxus species, are high-value secondary metabolites widely used in cancer therapy due to their antineoplastic activity. To date, several biosynthetic steps of these compounds remain undefined. After carrying out a genome-wide expression analysis of jasmonate-elicited Taxus baccata cell cultures using complementary DNA-amplified fragment length polymorphism (cDNA-AFLP), we found that the increased taxane production in the target cultures was correlated with an extensive elicitor-induced genetic reprogramming. Subsequent in silico analysis allowed us to identify 15 genes with jasmonate-induced differential expression putatively involved in five unknown steps of taxane biosynthesis. Among them, the TB768 gene and its predicted 3D structure showed a strong homology with other genes previously reported to encode acyl-CoA ligases, suggesting it was involved in the formation of the taxol lateral chain. In vitro analysis confirmed that the TB768 gene encodes an acyl-CoA ligase, localized in the cytoplasm and able to convert b-phenylalanine into its CoA ester derivative. The latter is then attached to baccatin III in one of the last steps of the taxol biosynthetic pathway. In determining that the TB768 gene encodes a cytosolic CoA ligase with an important role in the taxol biosynthetic pathway, we have corroborated that the combination of a cDNA-AFLP transcriptome profiling approach with bioinformatics analysis constitutes an efficient set of tools for the identification of new genes involved in the metabolism of valuable bioactive compounds. The newly described gene will contribute to the establishment of sustainable taxol production systems through metabolic engineering or synthetic biology approaches.

Highly efficient, long life, reusable and robust photosynthetic hybrid core–shell beads for the sustainable production of high value compounds

An efficient one-step process to synthesize highly porous (Ca-alginate–SiO2–polycation) shell: (Na-alginate–SiO2) core hybrid beads for cell encapsulation, yielding a reusable long-life photosynthetically active material for a sustainable manufacture of high-value metabolites is presented. Bead formation is based on crosslinking of an alginate biopolymer and mineralisation of silicic acid in combination with a coacervation process between a polycation and the silica sol, forming a semi-permeable external membrane. The excellent mechanical strength and durability of the monodispersed beads and the control of their porosity and textural properties is achieved by tailoring the silica and alginate loading, polycation concentration and incubation time during coacervation. This process has led to the formation of a remarkably robust hybrid material that confers exceptional protection to live cells against sheer stresses and contamination in a diverse range of applications. Dunaliella tertiolecta encapsulated within this hybrid core–shell system display high photosynthetic activity over a long duration (>1year). This sustainable biotechnology could find use in high value chemical harvests and biofuel cells to photosynthetic solar cells (energy transformation, electricity production, water splitting technologies). Furthermore the material can be engineered into various forms from spheres to variable thickness films, broadening its potential applications.

Enhanced production of fatty acids and astaxanthin in Aurantiochytrium sp. by the expression of Vitreoscilla hemoglobin.

Dissolved oxygen is a critical factor for heterotrophic cell growth and metabolite production. The aim of this study was to investigate the effects of an oxygen-involved protein on cell growth and fatty acid and astaxanthin production in the biologically important thraustochytrid Aurantiochytrium sp. The hemoglobin of the Vitreoscilla stercoraria (VHb) gene was fused upstream with a zeocin resistance gene (ble) and driven by the Aurantiochytrium tubulin promoter. The expression construct was introduced into two strains of Aurantiochytrium sp. by electroporation. Transgenic Aurantiochytrium sp. strains MP4 and SK4 expressing the heterologous VHb achieved significantly higher maximum biomass than their corresponding controls in microaerobic conditions. Furthermore, the transformants of Aurantiochytrium sp. SK4 produced 44% higher total fatty acid and 9-fold higher astaxanthin contents than the wild type control in aerobic conditions. The present study highlights the biotechnological application of VHb in high-cell density fermentation for enhanced biomass production as well as high-value metabolites.

Green and sustainable production of high value compounds via a microalgae encapsulation technology that relies on CO2as a principle reactant

A very promising and facile one-pot synthesis pathway is presented for the microencapsulation of live cells in a very porous core–shell system based upon a robust matrix. (Alginate–SiO2–polycation) shell@(alginate–SiO2) core hybrid beads, on the millimeter scale, containing live cells are obtained through cross-linking chemistry and the polycondensation of silicic acid in conjunction with the use of a polycation to negate the surface charge on silica. Very interestingly it is revealed that the polycation used (PDADMAC) plays a very important role in the formation of highly robust core–shell beads. PDADMAC acts as a catalyst in the polycondensation of silicic acid, leading to the formation of a resistant double layer shell comprising of an interior layer of alginate–SiO2 with a very homogeneous distribution of porous SiO2 and an external layer of porous PDADMAC that confines SiO2 within the bead. The photosynthetic chlorophyta Dunaliella tertiolecta, which produces high value metabolites (such as anti-oxidants, pharmacologically active compounds, neutraceuticals etc.) via photosynthesis, has been encapsulated within this core–shell system. Oximetry and fluorescence measurements highlight how this algal culture can remain photosynthetically active over an extraordinarily long period of 13 months for high value compound production, whilst entrapped within a highly porous, mechanically and chemically stable, optically transparent matrix, with no observable leaching of the cells from the core of the beads. HPLC has been employed to highlight the presence of excreted metabolites, based on neutral sugar building blocks such as arabinose, galactose and xylose, in the surrounding media. These results reveal how this kind of high performance, low-cost, and easily scaleable core–shell living material could be employed in large scale photobioreactors (PBRs), to potentially facilitate metabolite harvesting whilst protecting the culture from external contamination and for green energy production and environmental (CO2) remediation.