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What's in Algae Media?

Microalgae Media Implications for Biomanufacturing
(Part 1)

Biomanufacturing continues to grow…as do the cells used in their bioprocesses. 

In its earliest days, biomanufacturing was primarily relegated to biopharmaceutical production, allowing companies to make insulin, blockbuster antibody drugs, and other biologic therapeutics. 

As biomanufacturing technologies and knowledge develop further, there’s little doubt that bioproduction will become an increasingly important manufacturing tactic across nearly all sectors. The increasing consumer demand for sustainable bio-based ingredients and goods supports this prediction. 

Companies from a wide range of industries are already realizing the potential of bioproducts thanks to their improved functional properties and reduced reliance on petroleum-based ingredients. With increasing accessibility and decreasing implementation costs, harnessing life to make valuable commercial products is becoming easier. 

However, it’s still critically important for these enterprises to research and develop their bioprocesses to bring down the cost of goods (COGs). In doing so, companies can offer products to the marketplace at an approachable price while maintaining margins needed for business growth.

What's in Algae Media? Implications for Biomanufacturing & Bioproduction Part 1

Cell Culture Media & Its Outsized Impact

Cell culture media selection represents an important bioprocess development step for cell-based biomanufacturing and a major cost consideration. Thanks to the rapid growth of the biomanufacturing industry, the global cell culture media market alone was estimated to be worth over 4 billion USD in 2022. With an expected compound annual growth rate of 12.23% from 2023 to 2030, the influence of the media market isn’t slowing down any time soon. 

Media ingredients, storage, and preparation (particularly in regulated environments) represent a dominant factor in bioproduction costs. Thus, understanding your cellular system of choice and the media needed to make your target product is crucial for successful commercialization. Though heterotrophic cell media varies significantly across cell kingdoms and species, they all generally need sugars (for energy), amino acids (for protein synthesis), vitamins, inorganic salts, and other vital nutrients (like lipids, nucleotides, co-factors, transcription factors, etc.) for efficient, rapid growth. In short, heterotrophic systems need complex media simply because these organisms cannot synthesize their own chemical energy and materials without harvesting them from other organisms. Naturally, this implies that autotrophic cell culture media, like those used for microalgal, tobacco, or carrot cells, require less complex media simply because they can use photosynthesis to generate much of what they need. 

Since many life science enterprises and biomanufacturers are less familiar with microalgae cultivation, this two-part blog series aims to share an inside look at microalgae culture medium ingredients (Part 1) and their approximate costs (Part 2). So, let’s start by reviewing the three common microalgae media (F/2, HSM, and MLA) and discussing the biological roles of algal cell culture media components

Why Work With Microalgae?

Algae Media Ingredients

At a basic level, all microalgae media must supply the microorganisms with a nitrogen source (nitrates, ammonium, etc.) and a phosphorous source (phosphates, etc.) as well as essential inorganic metal ions (Fe, Cu, Zn, Mn, Mo, etc.) and key vitamins (B12, Biotin, & Thiamine). 

Naturally, the nitrogen source is needed to synthesize amino acids and nucleic acids, whereas phosphorous is critical for membrane lipid and nucleic acid synthesis. The metal ions primarily operate as co-factors in both respiration and photosynthesis. Iron is arguably the most important mineral element for algae, as it plays a central role in photosynthesis, acting as a core co-factor in the electron transport system and light harvesting. 

Otherwise, the key vitamins are critical to life are found in nearly all culture media, especially eukaryotes that cannot produce their own necessary vitamins. In particular, most microalgal species cannot synthesize B12, despite it being an essential co-enzyme. Instead, they naturally rely on symbiotic relationships with bacterial species to acquire these nutrients. In exchange, these symbiotic bacteria receive sugars and oxygen. In biomanufacturing and bioproduction conditions, where axenic culture is greatly preferred, these vitamins are added directly to the algae media in lieu of symbiotic bacteria. 

Though microalgae medium formulations have a lot of similarities, they also must be tuned to the organism at hand. Microalgae species thrive in a wide range of environments, which provide different critical nutrients at different abundances. Thus, different microalgae species evolved unique metabolisms to thrive in their niche with what’s available. 

First and foremost, algae media can generally be divided into two camps: saltwater and freshwater. Since some microalgae thrive in marine environments and others thrive in freshwater or terrestrial habitats, species evolved osmotic balance tuned with their niche. 

Though many formulations exist, let’s discuss some common microalgae media used for autotrophic culture and their unique attributes. For simplicity, core active nutrients are bolded.

F/2 (Saltwater)

F/2 Algae Media Ingredients Table
Source: Adapted from CSIRO Media Formulation
Marine environments are some of the most critical reservoirs for microalgae species. Generally speaking, marine species require a salinity level similar to ocean water, approximately 34.7 psu (practical salinity unit). Thus, algae cultivation and biotechnology need reliable saltwater media. These media are often made using seawater to reach high salinity and to bring water costs down. That said, significant filtration is usually required to ensure that seawater doesn’t contaminate cultures with bacteria, viruses, other algae, and toxic solutes. 

One of the most common marine microalgae media is F/2. F/2 is a modified version of F media, first described in 1962. As the name implies, F/2 is simply F media prepared at half strength to reduce media inputs without negatively impacting cultivation. F/2 has become popular for cultivating coastal microalgae species, especially diatoms.

F/2 is primarily comprised of sodium nitrate, iron citrate, sodium dihydrogen phosphate dihydrate, and EDTA disodium salt, as well as a trace metal (including copper sulfate, zinc sulfate, cobalt chloride, manganese(II)  chloride, & sodium molybdate and vitamin micronutrient (B12Biotin, & Thiamine) solutions. The majority of these inputs are essential nutrients. That said, EDTA acts as a chelating agent that helps to balance available ion concentrations and buffer the system. Some research shows that EDTA helps increase the reproducibility of algae media.

Interestingly, F/2 also includes a sizable amount of sodium metasilicate. This ingredient provides a source of silica, which many diatoms use to construct their unique cell wall, known as frustule.

Microalgae Frustule Shapes (credit: Wikimedia Commons)
Microalgae Frustule Shapes (credit: Wikimedia Commons)

Sueoka's High-Salt Media (Freshwater)

For freshwater species, Sueoka’s High-Salt Media (HSM) is one of the most common choices. First described in 1960, HSM’s name makes the media sound like it might be intended for marine species. Though referred to as “high-salt media,” HSM is closer to tap water in salinity than seawater. HSM has risen to prominence because it is a common choice for cultivating Chlamydomonas reinhardtii, the most well-characterized eukaryotic microalgae and a common model organism. 

HSM is primarily comprised of ammonium chloride, magnesium sulfate calcium chloride, and phosphates as well as a Hutner’s trace elements solution (which includes EDTA disodium salt, manganese (II) chloride, copper sulfate, zinc sulfate, cobalt chloride, ammonium heptamolybdate, iron sulfate, and boric acid). 

While HSM shares similarities with F/2, a few differences are worth noting. First and foremost, it’s probably intuitive that most of the nutrient counter ions in F/2 carry either sodium or chloride ions, indicative of saltwater conditions.

Sticking with ion composition, HSM also includes calcium and magnesium ions, whereas saltwater media (like F/2) do not. These critical ions are already abundant in ocean water but not freshwater. By using ocean water or sea salt in saltwater media formulations, users obviate the need for ions like Ca2+ and Mg2+. In particular, magnesium is often essential in chlorophyll and autotrophic growth. Thus, these ions must be added to freshwater media.

Source: Chlamydomonas Resource Center

Another key difference is the nitrogen source; instead of nitrates, HSM contains ammonium. Most phytoplankton can use either nitrate or ammonium as their nitrogen source. Still, various studies have displayed preferences between algae species, resulting in higher growth and photosynthesis rates when grown on one or the other. Most likely, these preferences relate to the nitrogen source most readily available in a given species’ environment and the adaptation of algae to better utilize the available nutrients. 

Generally speaking, ammonium is the preferred nitrogen source for Chlamydomonas species. To their benefit, ammonium provides some general biochemical advantages. Chiefly, ammonium can be used more directly in amino acid synthesis and metabolism. In turn, this conserves energy consumption by circumventing nitrogen reduction steps. 

In addition, you might note that HSM does not include a vitamin solution. B12 requirements act as a primary driver for the use of vitamin solutions in algae media. While most microalgae need B12 as a co-enzyme for B12-dependent Met synthase enzyme (METH) for growth, Chlamydomonas reinhardtii has a B12-independent isoform of METH known as METE. Thus, Chlamydomonas reinhardtii is capable of B12-independent growth. Thus, this main HSM formulation is ideal for species like C. reinhardtii that do not require this vitamin. That said, process developers can also add vitamins to the media if their target species is auxotrophic. 

Lastly, it’s worth noting that some algae cultivators will sometimes opt to supplement HSM with acetate. Acetate provides the algae with another carbon source beyond CO2, which enables algae to grow in mixotrophic conditions.

MLA (Freshwater & Cyanobacteria)

MLA Algae Media Ingredients Table
Source: Bolch et al. 1996, J. Phycology

Continuing our discussion of freshwater media, MLA has become another popular choice for cultivating freshwater species. First described in 1996, MLA has become a common algae growth medium for freshwater aquaculture species, especially cyanobacteria (aka, blue-green algae), like those from genus Arthrospira, Anabaena, Dolichospermum, and Aphanizomenon

MLA is primarily comprised of sodium nitrate, dipotassium hydrogen phosphate, magnesium sulfate, boric acid, selenious acid, sodium bicarbonate, and calcium chloride as well as micronutrient (including EDTA disodium salt, iron chloride, copper sulfate, zinc chloride, cobalt chloride, manganese(II) chloride, and sodium molybdate) and vitamin (B12, Biotin, & Thiamine) solutions. 

Interestingly, MLA shares similarities with both HSM and F/2. Like HSM and other freshwater media, MLA includes magnesium, calcium, and a more diverse collection of nutrient counter ions. Like F/2, MLA uses nitrates as the nitrogen source and contains vitamins. Though many cyanobacteria can synthesize B12 de novo and don’t require it in their media, MLA is also used to grow eukaryotic microalgae, like Chlorella vulgaris and Tetraselmis, which require those essential nutrients. 

In addition, to the core components discussed throughout, MLA includes a few unique ingredients that help specific species thrive. 

The most intriguing of which is selenite. Though toxic to organisms at high levels, selenium is a trace element needed to generate selenoproteins, which bear selenocysteine residues. Though completely absent in higher plants and fungi, selenoproteins are particularly enriched in aquatic species (like algae) playing an essential role in redox homeostasis. It’s thought that in cyanobacteria (aka, blue-green algae, the first photosynthetic organisms), selenoproteins help protect cells from reactive oxygen species. As a result of their functional activities, including selenium in algae media helps stimulate some green and blue-green algae growth

Another difference is that MLA, by default, includes sodium bicarbonate. Microalgae species can also use bicarbonate as a carbon source for photosynthesis. Thus, the inclusion of some bicarbonate acts as a supplementary source to drive photosynthetic processes in bioproduction in addition to carbon dioxide. 

Lastly, like HSM, MLA also includes boric acid, which serves as a buffer, helping to regulate culture pH. In addition, boric acid also acts as a micronutrient for some species, with a suspected role in cell wall formation and integrity. Like magnesium, boron is found naturally in ocean water; thus, its inclusion is only necessary for freshwater media.

Concluding Remarks

As microalgae biomanufacturing becomes more commonplace, bioprocess developers and engineers will need to fully understand their microalgae culture medium and its components. While this blog doesn’t provide a comprehensive view of all microalgae media, we hope it serves as a helpful introduction to this unique cell culture area. 

In part 2, we dive deeper into the COGs considerations of these media ingredients, comparing them to the cell culture media of other more traditional biomanufacturing cell systems.

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