This work was supported by the Portuguese Foundation for Science and Technology (FCT) through the Strategic Projects granted to MARE-Marine and Environmental Sciences Centre (UIDP/04292/2020 and UIDB/04292/2020), and Associate Laboratory ARNET (LA/P/0069/2020). FCT also funded the individual doctoral grants awarded to Marta V. Freitas (UI/BD/150957/2021) and Tatiana Pereira (2021. 07791. BD). This work was also financially supported by the project HP4A - HEALTHY PASTA FOR ALL (co-promotion no. 039952), co-funded by ERDF - European Regional Development Fund, under the Portugal 2020 Programme, through COMPETE 2020 - Competitiveness and Internationalisation Operational Programme.

Harnessing Gracilaria gracilis: Bioactive Potential for Sustainable Innovation

The authors have nothing to disclose.

The antimicrobial activity tests in a liquid medium are used to evaluate the effectiveness of antimicrobial substances against microorganisms suspended in a liquid medium and are usually performed to determine the ability of a substance to inhibit growth or kill microorganisms35,36,37,38. They are used to evaluate the sensitivity of microorganisms to antimicrobial agents and are conducted in te...

&#160;Seaweeds can be regarded as an interesting natural raw material to be profited by the pharmaceutical, food, feed, and environmental sectors. They biosynthesize a panoply of molecules, many not found in terrestrial organisms, with relevant biological properties1,2. However, seaweed-optimized cultivation protocols need to be implemented to ensure a large biomass stock.
Cultivation methods must always consider the nature of the seaweed thalli and overall morphology. Gracilaria gracilis is a clonal taxon, meaning the attachment organ produces multiple vegetative axes. Propagation by fragmentation (vegetative reproduction) is thus achieved, as each of these axes is fully able to adopt an independent life from the main thallus3. Clonal taxa can be successfully integrated with simple and fast one-step cultivation methodologies, as large amounts of biomass are obtained by splitting the thallus into small fragments that quickly regenerate and grow into new, genetically identical individuals. Both haplontic and diplontic thalli may be used in this process. Although the genus exhibits a complex haplo-diplontic isomorphic triphasic life cycle, sporulation is rarely necessary except when genetic renewal of the stocks is required to achieve improved crops. In this case, both tetraspores (haplontic spores formed by meiosis) and carpospores (diplontic spores formed by mitosis) give rise to macroscopic thalli that can then be grown and propagated by vegetative reproduction4. Growth cycles are dictated by environmental conditions and the physiological state of the individuals, among other biological factors such as the emergence of epiphytes and the adhesion of other organisms. Therefore, optimizing growing conditions is crucial to ensure high productivity and produce good quality biomass5.
The extraction of bioactive compounds from seaweed, including G. gracilis, can be achieved through various methods6,7. The choice of the extraction method depends on the specific compounds of interest, the target application, and the characteristics of the seaweed. In this study, we focused on solvent extraction, which involves using green solvents, such as water or ethanol, to dissolve and extract bioactive compounds from the seaweed biomass. The extraction can be carried out through maceration in a versatile and effective way and can be used for a wide range of compounds. It is a simple and widely used method involving soaking biomass in a solvent for an extended period, typically at room or slightly elevated temperatures. The solvent is stirred to enhance the extraction process. After the desired extraction time, the solvent is separated from the solid material by filtration or centrifugation.
Water is a commonly used solvent in food applications due to its safety, availability, and compatibility with a wide range of food products. Water extraction is suitable for polar compounds such as polysaccharides, peptides, and certain phenolics. However, it may not effectively extract non-polar compounds. Ethanol is also a widely used solvent in food applications and can be effective for extracting a variety of bioactive molecules, including phenolic compounds, flavonoids, and certain pigments. Ethanol is generally recognized as safe for use in food and can be easily evaporated, leaving behind the extracted compounds. It is worth noting that the choice of extraction method should consider factors such as efficiency, selectivity, cost-effectiveness, and environmental impact. The optimization of extraction parameters, such as solvent concentration, extraction time, temperature, and pressure, is crucial to achieving optimal yields of bioactive compounds from G. gracilis or other seaweeds.
Seaweeds have been found to exhibit antimicrobial activity against a wide range of microorganisms, including bacteria, fungi, and viruses8. This activity is attributed to bioactive components, including phenolics, polysaccharides, peptides, and fatty acids. Several studies have demonstrated their efficacy against pathogens such as Escherichia coli, Staphylococcus aureus, Salmonella sp., and Pseudomonas aeruginosa, among others9. The antimicrobial activity of seaweeds is attributed to the presence of bioactive compounds that can interfere with microbial cell walls, membranes, enzymes, and signaling pathways10. These compounds may disrupt microbial growth, inhibit biofilm formation, and modulate immune responses.
Red seaweeds, also known as rhodophytes, are a group of algae that can exhibit antimicrobial activity against a variety of microorganisms. Within this group, G. gracilis contains various bioactive compounds that may contribute to its reported antimicrobial activity. While the specific molecules can vary, the common classes that have been reported in G. gracilis and may possess antimicrobial properties are polysaccharides, phenolics, terpenoids, and pigments11. However, it is important to note that the presence and amounts of these components can vary depending on factors such as the location of seaweed collection, seasonality, physiological condition of the thalli, and environmental conditions. Therefore, the specific class and concentration of antimicrobial compounds in G. gracilis may vary accordingly.
G. gracilis has also been found to hold antioxidant properties, containing various phenolic compounds, which have been shown to scavenge free radicals and reduce oxidative stress12. Antioxidants help to protect cells from damage caused by reactive oxygen species and have potential health benefits. Antioxidant capacity can be evaluated directly through different methods, including the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity and, indirectly, through the quantification of total polyphenolic content (TPC)13.
Even though an ingredient is reported to have a prominent bioactivity, its cytotoxicity assessment is indispensable in evaluating natural and synthetic substances to be used in contact with living cells or tissues. There are several methods for measuring cytotoxicity, each one with advantages and limitations. Overall, they offer a range of options to evaluate the harmful effects of many substances on cells and, at the same time, to investigate the mechanisms of cell damage and death14.
In this work, we use the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, a colorimetric method introduced by Mosmann (1983)15. This method measures the reduction of tetrazolium salts to a purple formazan product by metabolically active cells. The higher the amount of formazan crystals, the higher the number of viable cells, thus providing an indirect measure of cytotoxicity14. Since in this work, G. gracilis water and ethanol extracts are intended to be incorporated into dermo-cosmetic formulations, the in vitro cytotoxicity evaluation is performed in a keratinocyte (HaCaT) cell line.
Concerning the food application, seaweeds are generally low in calories and nutritionally rich in dietary fibers, essential elements and amino acids, polysaccharides, polyunsaturated fatty acids, polyphenols, and vitamins2,16. G. gracilis is no exception, having an interesting nutritional value. Freitas et al. (2021)4 found that cultivated G. gracilis had higher levels of protein and vitamin C and maintained the level of total lipids compared to wild seaweed. This may represent an economic and environmental advantage, as nutritionally speaking, production is preferable to the exploitation of wild resources. In addition, consumers are increasingly concerned about the type of food they eat, so it is important to introduce new ingredients for food enrichment and use new resources to obtain extracts that can add value to a product and claim a &#34;clean label.&#34; Besides, the current market is very competitive, requiring the development of new products and innovative strategies to differentiate manufacturers from their competitors17.
The enrichment of products with poor nutritional value, such as pasta, with marine resources, including seaweed, is a strategy to introduce this resource as a new food and a market differentiation strategy through a product with distinct nutritional value. On the other hand, G. gracilis is a source of natural red pigments such as phycobiliproteins18, having high potential for applications in the food industry. This seaweed has shown high interest in several areas, and its application can be made using the whole seaweed, extracts and/or the remaining biomass. In this work, we demonstrate some examples of such applications.

<table><tbody><tr><td>Absolute Ethanol</td><td>Aga, Portugal</td><td>64-17-5</td><td></td></tr><tr><td>Ammonium Chloride</td><td>PanReac</td><td>12125-02-9</td><td></td></tr><tr><td>Amphotericin B</td><td>Sigma-Aldrich</td><td>1397-89-3</td><td></td></tr><tr><td>Analytical scale balance</td><td>Sartorius, TE124S</td><td>22105307</td><td></td></tr><tr><td>&lt;em&gt;Bacillus subtilis subsp. spizizenii&lt;/em&gt;</td><td>German Collection of Microorganisms and Cell Cultures (DSMZ)</td><td>DSM 347</td><td></td></tr><tr><td>Biotin</td><td>Panreac AppliChem</td><td>58-85-5</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf, 5810R</td><td>5811JH490481</td><td></td></tr><tr><td>Chloramphenicol</td><td>PanReac</td><td>56-75-7</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; Chamber</td><td>Memmert</td><td>N/A</td><td></td></tr><tr><td>Cool White Fluorescent Lamps</td><td>OSRAM Lumilux Skywhite</td><td>N/A</td><td></td></tr><tr><td>Densitometer McFarland</td><td>Grant Instruments</td><td>N/A</td><td></td></tr><tr><td>DMEM medium</td><td>Sigma-Aldrich</td><td>D5796</td><td></td></tr><tr><td>DMSO</td><td>Sigma-Aldrich</td><td>67-68-5</td><td></td></tr><tr><td>DPPH</td><td>Sigma, Steinheim, Germany</td><td>1898-66-4</td><td></td></tr><tr><td>&lt;em&gt;Escherichia coli (DSM 5922)&lt;/em&gt;</td><td>German Collection of Microorganisms and Cell Cultures (DSMZ)</td><td>DSM5922</td><td></td></tr><tr><td>Ethanol 96%</td><td>AGA-Portugal</td><td>64-17-5</td><td></td></tr><tr><td>Ethylenediaminetetraacetic Acid Disodium Salt Dihydrate (Na2EDTA)</td><td>J.T.Baker</td><td>6381-92-6</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Sigma-Aldrich</td><td>F7524</td><td></td></tr><tr><td>Filter Paper (Whatman No.1)</td><td>Whatman</td><td>WHA1001320</td><td></td></tr><tr><td>Flasks</td><td>VWR International, Alcabideche, Portugal&amp;nbsp;</td><td>N/A</td><td></td></tr><tr><td>Folin-Ciocalteu</td><td>VWR Chemicals</td><td>31360.264</td><td></td></tr><tr><td>Gallic Acid&amp;nbsp;</td><td>Merck</td><td>149-91-7</td><td></td></tr><tr><td>Germanium (IV) Oxide, 99.999%</td><td>AlfaAesar</td><td>1310-53-8</td><td></td></tr><tr><td>HaCaT cells &amp;ndash; 300493</td><td>CLS-Cell Lines Services, Germany&amp;nbsp;</td><td>300493</td><td></td></tr><tr><td>Hot Plate Magnetic Stirrer</td><td>IKA, C-MAG HS7</td><td>06.090564</td><td></td></tr><tr><td>Iron Sulfate</td><td>VWR Chemicals</td><td>10124-49-9</td><td></td></tr><tr><td>Laminar flow hood</td><td>TelStar, Portugal</td><td>526013</td><td></td></tr><tr><td>LB Medium&amp;nbsp;</td><td>VWR Chemicals</td><td>J106</td><td></td></tr><tr><td>&lt;em&gt;Listonella anguillarum&lt;/em&gt;</td><td>German Collection of Microorganisms and Cell Cultures (DSMZ)</td><td>&amp;nbsp;DSM 21597</td><td></td></tr><tr><td>Manganese Chloride</td><td>VWR Chemicals</td><td>7773.01.5</td><td></td></tr><tr><td>Micropipettes&amp;nbsp;</td><td>Eppendorf, Portugal</td><td>N/A</td><td></td></tr><tr><td>Microplates</td><td>VWR International, Alcabideche, Portugal&amp;nbsp;</td><td>10861-666</td><td></td></tr><tr><td>Microplates</td><td>Greiner</td><td>738-0168</td><td></td></tr><tr><td>Microplates (sterile)</td><td>Fisher Scientific</td><td>10022403</td><td></td></tr><tr><td>Microplate reader</td><td>&amp;nbsp;Epoch Microplate Spectrophotometer, BioTek, Vermont, USA</td><td>1611151E</td><td></td></tr><tr><td>MTT</td><td>Sigma-Aldrich</td><td>289-93-1</td><td></td></tr><tr><td>Muller-Hinton Broth (MHB)</td><td>VWR Chemicals</td><td>90004-658</td><td></td></tr><tr><td>Oven</td><td>Binder, FD115</td><td>12-04490</td><td></td></tr><tr><td>Oven</td><td>Binder, BD115</td><td>04-62615</td><td></td></tr><tr><td>Penicillin</td><td>Sigma-Aldrich</td><td>1406-05-9</td><td></td></tr><tr><td>pH meter Inolab</td><td>&amp;nbsp;VWR International, Alcabideche, Portugal&amp;nbsp;</td><td>15212099</td><td></td></tr><tr><td>Pippete tips</td><td>Eppendorf, Portugal</td><td>5412307</td><td></td></tr><tr><td>Pyrex Bottles Media Storage</td><td>&amp;nbsp;VWR International, Alcabideche, Portugal&amp;nbsp;</td><td>16157-169</td><td></td></tr><tr><td>Rotary Evaporator</td><td>Heidolph, Laborota 4000</td><td>80409287</td><td></td></tr><tr><td>Rotavapor</td><td>IKA HB10, VWR International, Alcabideche, Portugal</td><td>07.524254</td><td></td></tr><tr><td>Sodium Carbonate (Na&lt;sub&gt;2&lt;/sub&gt;CO&lt;sub&gt;3&lt;/sub&gt;)</td><td>Chem-Lab</td><td>497-19-8</td><td></td></tr><tr><td>Sodium Chloride (NaCl)&amp;nbsp;</td><td>Normax Chem</td><td>7647-14-5</td><td></td></tr><tr><td>Sodium Phosphate Dibasic</td><td>Riedel-de Ha&amp;euml;n</td><td>7558-79-4</td><td></td></tr><tr><td>SpectraMagic NX</td><td>Konica Minolta, Japan</td><td></td><td>color data analysis software</td></tr><tr><td>Spectrophotometer</td><td>Evolution 201, Thermo Scientific, Madison, WI, USA</td><td>5A4T092004</td><td></td></tr><tr><td>Streptomycin</td><td>Sigma-Aldrich</td><td>57-92-1</td><td></td></tr><tr><td>Thiamine</td><td>Panreac AppliChem</td><td>59-43-8</td><td></td></tr><tr><td>Trypsin-EDTA</td><td>Sigma-Aldrich</td><td>T4049</td><td></td></tr><tr><td>Tryptic Soy Agar (TSA)</td><td>VWR Chemicals</td><td>ICNA091010617</td><td></td></tr><tr><td>Tryptic Soy Broth (TSB)&amp;nbsp;</td><td>VWR Chemicals</td><td>22091</td><td></td></tr><tr><td>Ultrapure water&amp;nbsp;</td><td>Advantage A10 Milli-Q lab, Merck, Darmstadt, Germany</td><td>F5HA17360B</td><td></td></tr><tr><td>Vacuum pump</td><td>Buchi, Switzerland</td><td>FIS05-402-103</td><td></td></tr><tr><td>Vitamin B12</td><td>Merck</td><td>68-19-9</td><td></td></tr></tbody></table>

valorization of the red seaweed gracilaria gracilis through a biorefinery approach

The interest in seaweeds as an abundant feedstock to obtain valuable and multitarget bioactive ingredients is continuously growing. In this work, we explore the potential of Gracilaria gracilis, an edible red seaweed cultivated worldwide for its commercial interest as a source of agar and other ingredients for cosmetic, pharmacological, food, and feed applications.
G. gracilis growth conditions were optimized through vegetative propagation and sporulation while manipulating the physicochemical conditions to achieve a large biomass stock. Green extraction methodologies with ethanol and water were performed over the seaweed biomass. The bioactive potential of extracts was assessed through a set of in vitro assays concerning their cytotoxicity, antioxidant, and antimicrobial properties. Additionally, dried seaweed biomass was incorporated into pasta formulations to increase food&#39;s nutritional value. Pigments extracted from G. gracilis have also been incorporated into yogurt as a natural colorant, and their stability was evaluated. Both products were submitted to the appreciation of a semi-trained sensorial panel aiming to achieve the best final formulation before reaching the market.
Results support the versatility of G. gracilis whether it is applied as a whole biomass, extracts and/or pigments. Through implementing several optimized protocols, this work allows the development of products with the potential to profit the food, cosmetic, and aquaculture markets, promoting environmental sustainability and a blue circular economy.
Moreover, and in line with a biorefinery approach, the residual seaweed biomass will be used as biostimulant for plant growth or converted to carbon materials to be used in water purification of the in-house aquaculture systems of MARE-Polytechnic of Leiria, Portugal.

Antimicrobial activity
When interpreting the results obtained, it should be borne in mind that the higher the percentage of inhibition, the greater the efficacy of the extract in inhibiting the growth of that specific strain and, consequently, the more interesting the extract is as an antimicrobial. Through this methodology, we can rapidly identify which extracts have greater activity on certain bacterial strain...

Watch this Scientific Journal Video about Exploring Seaweed's Bioactive Compounds for Sustainable Innovations in Industries at JoVE.com

Author Spotlight: Exploring Seaweed's Bioactive Compounds for Sustainable Innovations in Industries 

Here, we describe several protocols aiming at an integrated valorization of Gracilaria gracilis: wild species harvesting, in-house growth, and extraction of bioactive ingredients. The extracts&#39; antioxidant, antimicrobial, and cytotoxic effects are evaluated, along with the nutritional and stability assessment of food enriched with whole seaweed biomass and pigments.

Valorization of the Red Seaweed Gracilaria gracilis Through a Biorefinery Approach

The interest in seaweeds as an abundant feedstock to obtain valuable and multitarget bioactive ingredients is continuously growing. In this work, we ...

valorization-red-seaweed-gracilaria-gracilis-through-biorefinery

Research

JoVE Journal

Biology

2.4K Views.  Polytechnic University of Leiria. Ons onderzoek is gericht op de prospectie van nieuwe bioactieve stoffen uit zeewier die nuttig kunnen zijn in verschillende sectoren, zoals de voedings-, cosmetische en farmaceutische industrie. Er is een groeiend bewustzijn in de samenleving over het potentieel van mariene organismen om bioactieve stoffen te leveren. Deze verbindingen kunnen worden gebruikt om duurzame en veiligere functionele cosmetica te produceren om de kwaliteit van voedingsproducten te verbeteren en dienen als bron van ...

Video: Auteur in de schijnwerpers: onderzoek naar de bioactieve stoffen van zeewier voor duurzame innovaties in industrieën 

Experimental Protocol for Biodiesel Production with Isolation of Alkenones as Coproducts from Commercial Isochrysis Algal Biomass

The need to replace petroleum fuels with alternatives from renewable and more environmentally sustainable sources is of growing importance. Biomass-derived biofuels have gained considerable attention in this regard, however first generation biofuels from edible crops like corn ethanol or soybean biodiesel have generally fallen out of favor. There is thus great interest in the development of methods for the production of liquid fuels from domestic and superior non-edible sources. Here we describe a detailed procedure for the production of a purified biodiesel from the marine microalgae Isochrysis. Additionally, a unique suite of lipids known as polyunsaturated long-chain alkenones are isolated in parallel as potentially valuable coproducts to offset the cost of biodiesel production. Multi-kilogram quantities of Isochrysis are purchased from two commercial sources, one as a wet paste (80% water) that is first dried prior to processing, and the other a dry milled powder (95% dry). Lipids are extracted with hexanes in a Soxhlet apparatus to produce an algal oil (&#34;hexane algal oil&#34;) containing both traditional fats (i.e., triglycerides, 46-60% w/w) and alkenones (16-25% w/w). Saponification of the triglycerides in the algal oil allows for separation of the resulting free fatty acids (FFAs) from alkenone-containing neutral lipids. FFAs are then converted to biodiesel (i.e., fatty acid methyl esters, FAMEs) by acid-catalyzed esterification while alkenones are isolated and purified from the neutral lipids by crystallization. We demonstrate that biodiesel from both commercial Isochrysis biomasses have similar but not identical FAME profiles, characterized by elevated polyunsaturated fatty acid contents (approximately 40% w/w). Yields of biodiesel were consistently higher when starting from the Isochrysis wet paste (12% w/w vs. 7% w/w), which can be traced to lower amounts of hexane algal oil obtained from the powdered Isochrysis product.

Experimental Protocol for Biodiesel Production with Isolation of Alkenones as Coproducts from Commercial Isochrysis Algal Biomass

Detailed methods are presented for the production of biodiesel along with the co-isolation of alkenones as valuable coproducts from commercial Isochrysis microalgae.

The need to replace petroleum fuels with alternatives from renewable and more environmentally sustainable sources is of growing importance. ...

Environment

A Method for 2-Photon Imaging of Blood Flow in the Neocortex through a Cranial Window

The ability to image the cerebral vasculature (from large vessels to capillaries) and record blood flow dynamics in the intact brain of living rodents is a powerful technique. Using in vivo 2-photon microscopy through a cranial window it is possible to image fluorescent dyes injected intravenously. This permits one to image the cortical vasculature and also to obtain measurements of blood flow. This technique was originally developed by David Kleinfeld and Winfried Denk. The method can be used to study blood flow dynamics during or after cerebral ischemia, in neurodegenerative disorders, in brain tumors, or in normal brain physiology. For example, it has been used to study how stroke causes shifts in blood flow direction and changes in red blood cell velocity or flux in and around the infarct. Here we demonstrate how to use 2-photon microscopy to image blood flow dynamics in the neocortex of living mice using fluorescent dyes injected into the tail vein.

Cortical blood flow dynamics can be studied in vivo by imaging fluorescent dextran dyes injected into the tail vein of rodents with 2-photon microscopy. This video shows how to image blood flow dynamics in neocortex of mice through a glass-covered cranial window preparation.

The ability to image the cerebral vasculature (from large vessels to capillaries) and record blood flow dynamics in the intact brain of living rodents is ...

Immobilization of Multi-biocatalysts in Alginate Beads for Cofactor Regeneration and Improved Reusability

We have recently developed a simple, reusable and coupled whole-cell biocatalytic system with the capability of cofactor regeneration and biocatalyst immobilization for improved production yield and sustained synthesis. Described herewith is the experimental procedure for the development of such a system consisting of two E. coli strains that express functionally complementary enzymes. Together, these two enzymes can function co-operatively to mediate the regeneration of expensive cofactors for improving the product yield of the bioreaction. In addition, the method of synthesizing an immobilized form of the coupled biocatalytic system by encapsulation of whole cells in calcium alginate beads is reported. As an example, we present the improved biosynthesis of L-xylulose from L-arabinitol by coupling E. coli cells expressing the enzymes L-arabinitol dehydrogenase or NADH oxidase. Under optimal conditions and using an initial concentration of 150 mM L-arabinitol, the maximal L-xylulose yield reached 96%, which is higher than those reported in the literature. The immobilized form of the coupled whole-cell biocatalysts demonstrated good operational stability, maintaining 65% of the yield obtained in the first cycle after 7 cycles of successive re-use, while the free cell system almost completely lost the catalytic activity. Therefore, the methods reported here provides two strategies that could help improve the industrial production of L-xylulose, as well as other value-added compounds requiring the use of cofactors in general.

Presented is the protocol for co-immobilizing whole-cell biocatalysts for cofactor regeneration and improved reusability, using the production of L-xylulose as an example. The cofactor regeneration is achieved by coupling two Escherichia coli strains expressing functionally complementary enzymes; the whole-cell biocatalyst immobilization is achieved by cell encapsulation in calcium alginate beads.

We have recently developed a simple, reusable and coupled whole-cell biocatalytic system with the capability of cofactor regeneration and biocatalyst ...

Chemistry

Cultivation of Green Microalgae in Bubble Column Photobioreactors and an Assay for Neutral Lipids

There is significant interest in the study of microalgae for engineering applications such as the production of biofuels, high value products, and for the treatment of wastes. As most new research efforts begin at laboratory scale, there is a need for cost-effective methods for culturing microalgae in a reproducible manner. Here, we communicate an effective approach to culture microalgae in laboratory-scale photobioreactors, and to measure the growth and neutral lipid content of that algae. Instructions are also included on how to set up the photobioreactor system. Although the example organisms are species of Chlorella and Auxenochlorella, this system can be adapted to cultivate a wide range of microalgae, including co-cultures of algae with non-algae species. Stock cultures are first grown in bottles to produce inoculum for the photobioreactor system. Algae inoculum is concentrated and transferred to photobioreactors for cultivation in batch mode. Samples are collected daily for the optical density readings. At the end of the batch culture, cells are harvested by centrifuge, washed, and freeze dried to obtain a final dry weight concentration. The final dry weight concentration is used to create a correlation between the optical density and the dry weight concentration. A modified Folch method is subsequently used to extract total lipids from the freeze-dried biomass and the extract is assayed for its neutral lipid content using a microplate assay. This assay has been published previously but protocol steps were included here to highlight critical steps in the procedure where errors frequently occur. The bioreactor system described here fills a niche between simple flask cultivation and fully-controlled commercial bioreactors. Even with only 3-4 biological replicates per treatment, our approach to culturing algae leads to tight standard deviations in the growth and lipid assays.

Here, we present a protocol to construct lab-scale bubble column photobioreactors and use them to culture microalgae. It also provides a method for the determination of the culture growth rate and neutral lipid content.

There is significant interest in the study of microalgae for engineering applications such as the production of biofuels, high value products, and for the ...

Dendra2 Photoswitching through the Mammary Imaging Window

In the last decade, intravital microscopy of breast tumors in mice and rats at single-cell resolution1-4 has resulted in important insights into mechanisms of metastatic behavior such as migration, invasion and intravasation of tumor cells5, 6, angiogenesis3 and immune cells response7-9. We have recently reported a technique to image orthotopic mammary carcinomas over multiple intravital imaging sessions in living mice10. For this, we have developed a Mammary Imaging Window (MIW) and optimized imaging parameters for Dendra211 photoswitching and imaging in vivo. Here, we describe the protocol for the manufacturing of MIW, insertion of the MIW on top of a tumor and imaging of the Dendra2- labeled tumor cells using a custom built imaging box. This protocol can be used to image the metastatic behavior of tumor cells in distinct microenvironments in tumors and allows for long term imaging of blood vessels, tumor cells and host cells.

Intravital photoswitching and tracking of Dendra2-labeled tumor cells through the Mammary Imaging Window is a technique which allows us to image the metastatic behavior of tumor cells in chosen tumor microenvironments over a timescale of days.

In the last decade, intravital microscopy of breast tumors in mice and rats at single-cell resolution1-4  has resulted in important insights into ...

Microalgal Cultivation for Biomethane Obtention from Swine Waste Biogas

Biogas Purification through the use of a Microalgae-Bacterial System in Semi-Industrial High Rate Algal Ponds

In recent years, a number of technologies have emerged to purify biogas into biomethane. This purification entails a reduction in the concentration of polluting gases such as carbon dioxide and hydrogen sulfide to increase the content of methane. In this study, we used a microalgal cultivation technology to treat and purify biogas produced from organic waste from the swine industry to obtain ready-to-use biomethane. For cultivation and purification, two 22.2 m3 open-pond photobioreactors coupled with an absorption-desorption column system were set up in San Juan de los Lagos, Mexico. Several recirculation liquid/biogas ratios (L/G) were tested to obtain the highest removal efficiencies; other parameters, such as pH, dissolved oxygen (DO), temperature, and biomass growth, were measured. The most efficient L/Gs were 1.6 and 2.5, resulting in a treated biogas effluent with a composition of 6.8%vol and 6.6%vol in CO2, respectively, and removal efficiencies for H2S up to 98.9%, as well as maintaining O2 contamination values of less than 2%vol. We found that pH greatly determines CO2 removal, more so than L/G, during cultivation because of its participation in the photosynthetic process of microalgae and its ability to vary pH when solubilized due to its acidic nature. DO, and temperature oscillated as expected from the light-dark natural cycles of photosynthesis and the time of day, respectively. Biomass growth varied with CO2 and nutrient feeding as well as reactor harvesting; however, the trend remained primed for growth.

Author Spotlight: Scaling Microalgal Biotechnology for Enhanced Biomethane Production 

Air pollution impacts the quality of life of all organisms. Here, we describe the use of microalgae biotechnology for the treatment of biogas (simultaneous removal of carbon dioxide and hydrogen sulfide) and the production of biomethane through semi-industrial open high-rate algal ponds and subsequent analysis of treatment efficiency, pH, dissolved oxygen, and microalgae growth.

In recent years, a number of technologies have emerged to purify biogas into biomethane. This purification entails a reduction in the concentration of ...

Qualitative Characterization of the Aqueous Fraction from Hydrothermal Liquefaction of Algae Using 2D Gas Chromatography with Time-of-flight Mass Spectrometry

Two-dimensional gas chromatography coupled with time-of-flight mass spectrometry is a powerful tool for identifying and quantifying chemical components in complex mixtures. It is often used to analyze gasoline, jet fuel, diesel, bio-diesel and the organic fraction of bio-crude/bio-oil. In most of those analyses, the first dimension of separation is non-polar, followed by a polar separation. The aqueous fractions of bio-crude and other aqueous samples from biofuels production have been examined with similar column combinations. However, sample preparation techniques such as derivatization, solvent extraction, and solid-phase extraction were necessary prior to analysis. In this study, aqueous fractions obtained from the hydrothermal liquefaction of algae were characterized by two-dimensional gas chromatography coupled with time-of-flight mass spectrometry without prior sample preparation techniques using a polar separation in the first dimension followed by a non-polar separation in the second. Two-dimensional plots from this analysis were compared with those obtained from the more traditional column configuration. Results from qualitative characterization of the aqueous fractions of algal bio-crude are discussed in detail. The advantages of using a polar separation followed by a non-polar separation for characterization of organics in aqueous samples by two-dimensional gas chromatography coupled with time-of-flight mass spectrometry are highlighted.

A two-dimensional gas chromatography-time-of-flight mass spectrometry method is described for characterization of the aqueous fraction of bio-crude produced from hydrothermal liquefaction of algae. This protocol can also be employed to analyze the aqueous fraction of liquid products from fast pyrolysis, catalytic fast pyrolysis, catalytic deoxygenation and hydro-treating.

Two-dimensional gas chromatography coupled with time-of-flight mass spectrometry is a powerful tool for identifying and quantifying chemical components in ...

Bioengineering

The Logic, Experimental Steps, and Potential of Heterologous Natural Product Biosynthesis Featuring the Complex Antibiotic Erythromycin A Produced Through E. coli

The heterologous production of complex natural products is an approach designed to address current limitations and future possibilities. It is particularly useful for those compounds which possess therapeutic value but cannot be sufficiently produced or would benefit from an improved form of production. The experimental procedures involved can be subdivided into three components: 1) genetic transfer; 2) heterologous reconstitution; and 3) product analysis. Each experimental component is under continual optimization to meet the challenges and anticipate the opportunities associated with this emerging approach.
Heterologous biosynthesis begins with the identification of a genetic sequence responsible for a valuable natural product. Transferring this sequence to a heterologous host is complicated by the biosynthetic pathway complexity responsible for product formation. The antibiotic erythromycin A is a good example. Twenty genes (totaling &gt;50 kb) are required for eventual biosynthesis. In addition, three of these genes encode megasynthases, multi-domain enzymes each ~300 kDa in size. This genetic material must be designed and transferred to E. coli for reconstituted biosynthesis. The use of PCR isolation, operon construction, multi-cystronic plasmids, and electro-transformation will be described in transferring the erythromycin A genetic cluster to E. coli.
Once transferred, the E. coli cell must support eventual biosynthesis. This process is also challenging given the substantial differences between E. coli and most original hosts responsible for complex natural product formation. The cell must provide necessary substrates to support biosynthesis and coordinately express the transferred genetic cluster to produce active enzymes. In the case of erythromycin A, the E. coli cell had to be engineered to provide the two precursors (propionyl-CoA and (2S)-methylmalonyl-CoA) required for biosynthesis. In addition, gene sequence modifications, plasmid copy number, chaperonin co-expression, post-translational enzymatic modification, and process temperature were also required to allow final erythromycin A formation.
Finally, successful production must be assessed. For the erythromycin A case, we will present two methods. The first is liquid chromatography-mass spectrometry (LC-MS) to confirm and quantify production. The bioactivity of erythromycin A will also be confirmed through use of a bioassay in which the antibiotic activity is tested against Bacillus subtilis. The assessment assays establish erythromycin A biosynthesis from E. coli and set the stage for future engineering efforts to improve or diversify production and for the production of new complex natural compounds using this approach.

The Logic, Experimental Steps, and Potential of Heterologous Natural Product Biosynthesis Featuring the Complex Antibiotic Erythromycin A Produced Through E. coli

The heterologous biosynthesis of erythromycin A through E. coli includes the following experimental steps: 1) genetic transfer; 2) heterologous reconstitution; and 3) product analysis. Each step will be explained in the context of the motivation, potential, and challenges in producing therapeutic natural products using E. coli as a surrogate host.

The heterologous production of complex natural products is an approach designed to address current limitations and future possibilities.  It is ...

Imaging Through the Pupal Case of Drosophila melanogaster

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled analysis of cell and developmental biology from a variety of methodological angles. Live imaging is an emerging method for observing dynamic cell processes, such as cell division or cell motility. Having isolated mutations in uncharacterized putative cell cycle proteins it became essential to observe mitosis in situ using live imaging. Most live imaging studies in Drosophila have focused on the embryonic stages that are accessible to manipulation and observation because of their small size and optical clarity. However, in these stages the cell cycle is unusual in that it lacks one or both of the gap phases. By contrast, cells of the pupal wing of Drosophila have a typical cell cycle and undergo a period of rapid mitosis spanning about 20 hr of pupal development. It is easy to identify and isolate pupae of the appropriate stage to catch mitosis in situ. Mounting intact pupae provided the best combination of tractability and durability during imaging, allowing experiments to run for several hours with minimal impact on cell and animal viability. The method allows observation of features as small as, or smaller than, fly chromosomes. Adjustment of microscope settings and the details of mounting, allowed extension of the preparation to visualize membrane dynamics of adjacent cells and fluorescently labeled proteins such as tubulin. This method works for all tested fluorescent proteins and can capture submicron scale features over a variety of time scales. While limited to the outer 20 &#181;m of the pupa with a conventional confocal microscope, this approach to observing protein and cellular dynamics in pupal tissues in vivo may be generally useful in the study of cell and developmental biology in these tissues.

Imaging Through the Pupal Case of Drosophila melanogaster

This paper demonstrates the use of a fast scanning confocal microscope to image cell behavior directly through the puparium. By leaving the pupal case intact, this method allows observation and measurement of dynamic cell processes at a stage of Drosophila development that is difficult to study directly.

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled ...

Larval RNA Interference in the Red Flour Beetle, Tribolium castaneum

The red flour beetle, Tribolium&#160;castaneum, offers a repertoire of experimental tools for genetic and developmental studies, including a fully annotated genome sequence, transposon-based transgenesis, and effective RNA interference (RNAi). Among these advantages, RNAi-based gene knockdown techniques are at the core of Tribolium research. T. castaneum show a robust systemic RNAi response, making it possible to perform RNAi at any life stage by simply injecting double-stranded RNA (dsRNA) into the beetle&#8217;s body cavity.
In this report, we provide an overview of our larval RNAi technique in T. castaneum. The protocol includes (i) isolation of the proper stage of T. castaneum larvae for injection, (ii) preparation for the injection setting, and (iii) dsRNA injection. Larval RNAi is a simple, but powerful technique that provides us with quick access to loss-of-function phenotypes, including multiple gene knockdown phenotypes as well as a series of hypomorphic phenotypes. Since virtually all T. castaneum tissues are susceptible to extracellular dsRNA, the larval RNAi technique allows researchers to study a wide variety of tissues in diverse contexts, including the genetic basis of organismal responses to the outside environment. In addition, the simplicity of this technique stimulates more student involvement in research, making T. castaneum an ideal genetic system for use in a classroom setting.

Larval RNA Interference in the Red Flour Beetle, Tribolium castaneum

RNA interference (RNAi)-based gene knockdown techniques are at the core of Tribolium research. Here, we provide an overview of our larval RNAi technique in Tribolium castaneum. Larval RNAi is a simple, but powerful technique that provides quick access to loss-of-function phenotypes, allowing researchers to study gene functions in diverse contexts.

Valorization of the Red Seaweed Gracilaria gracilis Through a Biorefinery Approach

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