Russell Tran 12 May 2021
Asparagopsis taxiformis is a common red macroalgae (seaweed) native to Australia, southeast Asia, and southern California, among other regions, especially the warm-water tropics. In fact, it is sufficiently common that it is an invasive species in other parts of the world, such as off the Iberian Peninsula/Spain.
Someone should achieve Agrobacterium-mediated genetic transformation of Asparagopsis taxiformis as part of building a toolkit to enable engineering the species and other red macroalgae. A broadly reliable protocol to genetically transform macroalgae does not yet exist, and the use of modern tools like CRISPR has not been achieved either (Mikami 2014, Ramessur et al. 2018).
A. taxiformis does not have a history of commercial cultivation. At most, it has been wild harvested in Hawaii as a delicacy. However, the species has been gaining attention as a potential climate change breakthrough in its use as a feed supplement to prevent enteric methanogenesis in cattle and other ruminant livestock. Namely, 1% of feed by mass of Asparagopsis has shown to reduce in vivo methane emissions from cows by 70-90%. Cow methane production is one of the largest contributors to anthropogenic climate change.1 The mechanism for inhibiting gut bacteria methanogenesis is due to A. taxiformis’s relatively high secondary metabolite concentration of bromoform and bromoform-like compounds (aka halogenated methane analogues - HMAs), which block some of the pathway enzymes. (Evolutionarily, some algae produce bromoform as a latent defense against microbes). Being able to genetically modify A. taxiformis would facilitate study of the species and accelerate its domestication.
Preexisting literature reviews can be found here, here, and here. But generally, I have been able to gauge the 7-year chronology and summarize the research as follows:
Canadian farmer hypothesizes that his cows grazing along the beach might be healthier because of seaweed in their diet2
Australian microbiologists take up the hypothesis and test the effect of various alga on cow gut microbiota in vitro. Unintentionally discover that addition of Asparagopsis taxiformis prevents virtually all (99%) methane emissions from their samples (Machado et al. 2016)
Research of the seaweed continues in Australia across University of Sunshine Coast, CSIRO, James Cook University. Eg, small livestock trials, study on suspension of seaweed in vegetable oil to prevent decay of bromoform vs freeze drying (Magnusson et al. 2020)
Australian aquaculture company (Australis Aquaculture/Greener Grazing) tries to cultivate the seaweed in its farms in Vietnam. Starts a seed bank of A. taxiformis geographic varieties, and other characterizations3
A startup (Blue Ocean Barns) evangelizes research in California, securing funding for experiments on cattle at UC Davis and cultivation research at UC San Diego
UC Davis (147 day trial) and Penn State research reliably show in vivo methane reduction of 80-98% using only 1-2% A. taxiformis in feed by mass. No negative impact on cattle health, milk production, or milk flavor, and no bromoform detected in milk or tissues (Roque et al. 2020)
Other studies on the East Coast and in New Zealand confirm methane reduction in sheep4
Another UCSD researcher examines native California seaweeds in hopes of finding other species similar to A. taxiformis in bromoform production5
Different startup (Symbrosia) attempts to cultivate the seaweed in Hawaii6
At the moment, the success of this seaweed in cows is hindered by economic feasibility and the need to achieve a planetary scale-up of currently nonexistent aquaculture infrastructure for A. taxiformis. Vijin et al. (2020) calculate that 3-3.4 million metric tons of seaweed (dry mass) per year is necessary for the 1% inclusion in feed level to support the 93 million cattle in the United States, which is half the current global seaweed output. They do not even bother to calculate the requisite seaweed mass for the total 1.4 billion cows on Earth. There exists further speculation that the bromoform-based methanogenesis inhibition may fail in the long term if the gut microbiota simply adapt (Hristov at a conference, 2019).
Because of these scaling issues, I have wondered for quite some time why one would not simply use synthetic bromoform, which is already on the market as an industrial product. I am not fully satisfied with the answers I have found so far, an important caveat to resolve before I begin the proposed research project. Tomkins et al. (2009) showed a 93% reduction in cattle methane using synthetic bromochloromethane at a dose rate of 0.30 g/100 kg LW, but with the disclaimer “The experiments reported here were completed in 2004 before the Australian Government prohibited the manufacture and use of BCM”.7 There is also some ambiguity about the carcinogenicity of bromoform. The International Agency for Research on Cancer (IARC) says bromoform is not a human carcinogen, whereas the EPA classifies it as a probable human carcinogen (Agency for Toxic Substances and Disease Registry). As alluded in the above bullets, there have thus far been no measurable health issues from a 147 day trial (Roque et al. 2021). Per advice I received from an Asparagopsis cultivation startup about synthetic bromoform, “[1] pure bromoform does not have the full nutritional profile of the algae and [2] instead causes gas build up in the stomach of the animal, making them think they are full and thus they stop eating. Else, [3] it would be extremely difficult/impossible to get regulatory approval to feed synthetic bromoform to livestock” (numbering mine). [1] is a commercial reason, and [3] might demonstrate the useful role of A. taxiformis as a perception “wrapper” or “Trojan horse” of sorts for bromoform, but [2] is not self-explanatory and I currently do not have an answer for it and how the distinction is caused by A. taxiformis.
Suppose A. taxiformis as consumed by the cow is in fact more viable than isolated/synthetic bromoform.8 Then we are back to the economic problem, for which I imagine two possible bioengineering solutions. The first is that we would want to engineer taxiformis into a truly valuable livestock feed supplement by adding or enhancing other features of the seaweed. Feed supplements today make use of brown seaweed (kelp and rockweed, Laminariales and A. nodosum) in powdered form because of its nutrient content, etc. (Makkar et al. 2016 cited by Vijin 2020). There is evidence that the methanogenic inhibition by A. taxiformis in cows may improve energy utilization from feed because the enteric methanogenic pathway is of no metabolic use to cows (hydrogen metabolism to propionate production) (Kinley et al. 2020 cited by Vijin 2020). This is one way in which the seaweed may be economically useful to farmers on its own. We would want to further this by making A. taxiformis into a superior feed supplement by engineering its nutrient content to outperform kelp and rockweed. The second solution is that if cultivation of A. taxiformis still fails to be economically viable, then continue with the prior strategy but move the bromoform and nutrient production to a different biological chassis, such as spirulina or Pichia, and sell that as the supplement.
I assumed genetic engineering of macroalgae was well-established because the federal government and oil companies have invested heavily in microalgae biofuel research, and algae biofuels were a notorious poster child of biotechnology in the 2000s.9 But macroalgae engineering is more analogous to plant engineering, and tracks behind it in progress (Ramessur et al. 2018). Per the review by Mikami (2014), if you want to genetically modify seaweed, there are 4 steps that have to be solved:
For macroalgae species other than A. taxiformis: (1) has already been solved. (2) has worked via particle bombardment, microinjection, and Agrobacterium for various species, but not yet electroporation or glass beads. Mikami’s experiments showed that glass bead methods did not work.
One might be encouraged to try Agrobacterium transformation because it is cost effective compared to the alternatives and thus more likely to become a tool if it works.10
The red macroalgae species that have been transformed by Agrobacterium thus far are Pyropia yezoensis (Cheney et al. 2001),11 Gracilaria changii (Gan et al. 2005 cited by Ramessur 2018), Kappaphycus alvarezii (Handayani et al. 2014 cited by Ramessur 2018; Triana et al. 2016 cited by Ramessur 2018), and Chondrus crispus (Ramessur et al. 2018). As a novice, I would have my methodology heavily rely on the methodology provided in Ramessur et al. 2018. The research would consist largely of reproducing the methodology to see if it works in A. taxiformis. The paper is a wellspring of useful idiosyncrasies that are useful to successful Agrobacterium-seaweed transformation, such as the importance of a transformation environment which is agreeable to both species. For instance, they used the LBA4404 strain of Agrobacterium because it is comparatively salt-tolerant. For the marine unicellular algae Parachlorella kessleri, Rathod et al. (2013) had to use the freshwater medium TAP because both P. kessleri and Agrobacterium could tolerate the salinity level, so this consideration is common. Ramessur et al. were also able to report transformation of Chondrus crispus with needle wounding without the external application of acetosyringone, implying that Chondrus crispus releases its own phenolics when wounded. It would be interesting to observe whether A. taxiformis releases its own phenolics as well.
Lastly, a genome of A. taxiformis has not yet been published to GenBank. At present, there are only sequences for its plastid and mitochondria.12_ _A low-hanging fruit to get started would be to sequence its genome.
Failure to cultivate the specimen will significantly kneecap progress.
Because A. taxiformis does not have a prior history of cultivation, best practices for cultivation are not common knowledge. As recent as 2019, the life cycle of the species had not yet been completed in captivity (stuck in the sporophyte phase).13 Currently, at least 1 company claims to have completed the life cycle, though that information may be proprietary.14 Replication can still be achieved in the sporophyte phase through fragmentation of the specimen, but it is unknown whether other phases might be more amenable to genetic transformation.
Abbott, D. W., Aasen, I. M., Beauchemin, K. A., Grondahl, F., Gruninger, R., Hayes, M., … & Xing, X. (2020). Seaweed and Seaweed Bioactives for Mitigation of Enteric Methane: Challenges and Opportunities. Animals, 10(12), 2432.
Álvarez-Viñas, M., Flórez-Fernández, N., Torres, M. D., & Domínguez, H. (2019). Successful approaches for a red seaweed biorefinery. Marine drugs, 17(11), 620.
Alimuddin, A., Widyastuti, U., Suryati, E., & Parenrengi, A. (2014). Binary Vector Construction and Agrobacterium tumefaciens-mediated Transformation of Lysozyme Gene in Seaweed Kappaphycus alvarezii. BIOTROPIA-The Southeast Asian Journal of Tropical Biology, 21(2), 82-92.
Cheney, D., Metz, B., & Stiller, J. (2001). Agrobacterium-mediated genetic transformation in the macroscopic marine red alga Porphyra yezoensis. J. Phycol, 37(11).
Gan, S. Y., Othman, R. Y., & Phang, S. M. (2005, July). AGROBACTERIUM-MEDIATE D TRANSFORMATION OF GRACILARIA CHANGII (GRACILARIALES, RHODOPHYTA), A TROPICAL RED ALGA. In Phycologia (Vol. 44, No. 4, pp. 35-35). NEW BUSINESS OFFICE, PO BOX 1897, LAWRENCE, KS 66044-8897 USA: INT PHYCOLOGICAL SOC.
Holdt, S. L., & Kraan, S. (2011). Bioactive compounds in seaweed: functional food applications and legislation. Journal of applied phycology, 23(3), 543-597.
Kinley, R. D., Martinez-Fernandez, G., Matthews, M. K., de Nys, R., Magnusson, M., & Tomkins, N. W. (2020). Mitigating the carbon footprint and improving productivity of ruminant livestock agriculture using a red seaweed. Journal of Cleaner Production, 259, 120836.
Machado, L., Magnusson, M., Paul, N. A., Kinley, R., de Nys, R., & Tomkins, N. (2016). Dose-response effects of Asparagopsis taxiformis and Oedogonium sp. on in vitro fermentation and methane production. Journal of Applied Phycology, 28(2), 1443-1452.
Machado, L., Magnusson, M., Paul, N. A., Kinley, R., de Nys, R., & Tomkins, N. (2016). Identification of bioactives from the red seaweed Asparagopsis taxiformis that promote antimethanogenic activity in vitro. Journal of Applied Phycology, 28(5), 3117-3126.
Magnusson, M., Vucko, M. J., Neoh, T. L., & de Nys, R. (2020). Using oil immersion to deliver a naturally-derived, stable bromoform product from the red seaweed Asparagopsis taxiformis. Algal Research, 51, 102065.
Ramessur, A. D., Bothwell, J. H., Maggs, C. A., Gan, S. Y., & Phang, S. M. (2018). Agrobacterium-mediated gene delivery and transient expression in the red macroalga Chondrus crispus. Botanica Marina, 61(5), 499-510.
Makkar, H. P., Tran, G., Heuzé, V., Giger-Reverdin, S., Lessire, M., Lebas, F., & Ankers, P. (2016). Seaweeds for livestock diets: A review. Animal Feed Science and Technology, 212, 1-17.
Mikami, K. (2014). A technical breakthrough close at hand: feasible approaches toward establishing a gene-targeting genetic transformation system in seaweeds. Frontiers in plant science, 5, 498.
Ramessur, A. D., Bothwell, J. H., Maggs, C. A., Gan, S. Y., & Phang, S. M. (2018). Agrobacterium-mediated gene delivery and transient expression in the red macroalga Chondrus crispus. Botanica Marina, 61(5), 499-510.
Rathod, J. P., Prakash, G., Pandit, R., & Lali, A. M. (2013). Agrobacterium-mediated transformation of promising oil-bearing marine algae Parachlorella kessleri. Photosynthesis research, 118(1), 141-146.
Roque, B. M., Venegas, M., Kinley, R. D., de Nys, R., Duarte, T. L., Yang, X., & Kebreab, E. (2021). Red seaweed (Asparagopsis taxiformis) supplementation reduces enteric methane by over 80 percent in beef steers. Plos one, 16(3), e0247820.
Silwer, H. (2018). Macroalgae as feed supplement for reduction of methane emission in livestock.
Tomkins, N. W., Colegate, S. M., & Hunter, R. A. (2009). A bromochloromethane formulation reduces enteric methanogenesis in cattle fed grain-based diets. Animal Production Science, 49(12), 1053-1058.
Vijn, S., Compart, D. P., Dutta, N., Foukis, A., Hess, M., Hristov, A. N., … & Kurt, T. D. (2020). Key Considerations for the Use of Seaweed to Reduce Enteric Methane Emissions From Cattle. Frontiers in Veterinary Science, 7, 1135.
Widyastuti, U., Suryati, E., & Parenrengi, A. (2016). The method of Agrobacterium tumefaciens-mediated MmCu/Zn-SOD gene transformation in the red seaweed Kappaphycus alvarezii. Pakistan Journal of Biotechnology, 13(4), 221-230.
Collective emissions of cattle and industry amount to 5.0 gigatons of CO2 equivalent annually. For comparison, China’s emissions are 10.2 and the United States 5.3: https://www.gatesnotes.com/energy/my-plan-for-fighting-climate-change ↩
https://e360.yale.edu/features/how-eating-seaweed-can-help-cows-to-belch-less-methane ↩
https://www.theverge.com/22297685/seaweed-methane-greenhouse-gas-climate-change-livestock ↩
https://civileats.com/2019/06/03/can-we-grow-enough-seaweed-to-help-cows-fight-climate-change/ ↩
Other synthetic compounds were also tried on ruminants for methanogenesis inhibition before A. taxiformis appeared on the radar: chloroform and chloral hydrate, which resulted in varying degrees of liver and blood cell damage, toxicity, and death (Patra cited by Silwer, 2011). ↩
For instance, no work has been done so far to decouple the efficacy of the other bromoform-like compounds from A. taxiformis (tease out any synergies that exist) such that A. taxiformis as consumed by the cow is holistically more performant than just synthetic bromoform. Or A. taxiformis is like the coating on a pill for better delivery. ↩
The Wikipedia article for Algae fuel is extensive, if that says anything https://en.wikipedia.org/wiki/Algae_fuel ↩
Conversation with Keoni Gandall ↩
Cheney also patented seaweed + Agrobacterium in 2000; the patent is now expired https://patents.google.com/patent/WO2000062601A1/en ↩