Seaweeds as an alternative crop for the production of protein

The total global demand for protein from both humans and livestock will rise substantially into the future due to the combined increase in population and per capita consumption of animal protein. Currently, net protein is primarily produced by agricultural crops. However, the future production of agricultural crops is limited by a finite supply of arable land, fresh water and synthetic fertilisers. Alternative crops such as seaweeds have the potential to help meet the protein demand without applying additional stress on traditional agricultural resources. This thesis investigates the potential of seaweeds as an alternative crop for the production of protein.

Chapter 1 provides a general introduction to the thesis. The chapter begins by introducing the current supply and future demand of protein globally, with a specific focus on the demands of mono-gastric livestock (poultry, swine and fish). This is followed by a summary of potential alternative protein sources that are currently being explored. Finally, seaweeds are introduced in the context of their potential as a biomass crop for the production of protein.

Many seaweed species have considerable plasticity in nitrogen content, yet the relationship between nitrogen content, protein concentration, protein quality and growth rate are poorly understood. Therefore, in Chapter 2, the plasticity in protein content in the green seaweed Ulva ohnoi was investigated. This was done by assessing the quantitative and qualitative changes in protein in Ulva ohnoi and relating these to changes in internal nitrogen content and growth rate. To do this water nitrogen concentrations and water renewal rates were varied simultaneously to manipulating the supply of nitrogen to outdoor cultures of U. ohnoi. Both internal nitrogen content and growth rate varied substantially, and the quantitative and qualitative changes in total amino acids were described in the context of three internal nitrogen states; nitrogenlimited, metabolic, and luxury. The nitrogen-limited state was defined by increases in all amino acids with increasing nitrogen content and growth rates up until 1.2 % internal nitrogen. The metabolic nitrogen state was defined by increases in all amino acids with increasing internal nitrogen content up to 2.6 % with no increases in growth rate. Luxury state was defined by internal nitrogen contents above 2.6 % which occurred only when nitrogen availability was high but growth rates were reduced. In this luxury circumstance, excess nitrogen was accumulated as free amino acids, in two phases. The first phase is distinguished by a small increase in the majority of amino acids up to ≈ 3.3 % internal nitrogen, and the second by a large increase in glutamic acid/glutamine and arginine up to 4.2 % internal nitrogen. This chapter demonstrates that the relationship between internal nitrogen content and amino acid quality is dynamic but predictable, and could be used for holding seaweeds in a desired nitrogen state during culture.

In Chapter 3, I assessed the relative importance of direct and indirect effects of salinity on protein in seaweed. Indirect effects, through altering growth rates, and direct effects, through altering the synthesis of specific amino acids and osmolytes, were examined in the context of the concentration and quality of protein in Ulva ohnoi. To do this, U. ohnoi was cultured under a range of salinities without nutrient limitation. Both the concentration and quality of protein varied across the salinity treatments. Protein concentration was strongly related to the growth rate of the seaweed and was highest in the slowest growing seaweed. In contrast, the quality of protein (individual amino acids as a proportion of total amino acid content) was strongly related to salinity for all amino acids, although this varied substantially amongst individual amino acids. Increases in salinity were positively correlated with the proportion of proline (46 % increase), tyrosine (36 % increase) and histidine (26 % increase), whereas there was a negative correlation with alanine (29 % decrease). The proportion of methionine, with strong links to the synthesis of the osmolyte dimethylsulphonioproprionate (DMSP), did not correlate linearly with salinity and instead was moderately higher at the optimal salinities for growth. This chapter demonstrates that salinity simultaneously affects the concentration and quality of protein in seaweed through both indirect and direct mechanisms, with growth rates playing the overarching role in determining the concentration of protein.

During my investigations into the protein physiology and nutrition of seaweeds, it became evident that there were many inconsistencies and potential inaccuracies with the way protein concentrations are reported. Therefore, in Chapter 4, I assessed these issues on a broad scale by systematically analysing the literature to assess the way that people measure and report protein in seaweeds with the aim to provide an evidence-based conversion factor for nitrogen to protein that is specific to seaweeds. Almost 95 % of studies on seaweeds determined protein either by direct extraction procedures (42 % of all studies) or by applying an indirect nitrogen-to-protein conversion factor of 6.25 (52 % of all studies), with the latter the most widely used method in the last 6 years. Metaanalysis of the true protein content, defined as the sum of the proteomic amino acids, demonstrated that direct extraction procedures under-estimated protein content by 33 %, while the most commonly used indirect nitrogen-to-protein conversion factor of 6.25 overestimated protein content by 43 %. I then questioned whether a single nitrogen-toprotein conversion factor could be used for seaweeds and evaluated how robust this would be by analysing the variation in N-to-protein conversion factors for 103 species across 44 studies that span three taxonomic groups, multiple geographic regions and a range of nitrogen contents. This resulted in an overall median nitrogen-to-protein conversion factor of 4.97 and a mean nitrogen-to-protein conversion factor of 4.76. Based on these results I proposed that the value of 5 be adopted as the universal seaweed nitrogen-to-protein (SNP) conversion factor. This chapter highlighted that most of the quantitative data on the protein contents of seaweeds have been under- or overestimated and was in need of review in regards to the potential applications of seaweed protein.

Therefore, in Chapter 5, seaweeds were quantitatively assessed as a protein source in livestock feeds using the dataset established in Chapter 4 as a platform to compare the quality and concentration of protein to traditional protein sources (soybean meal and fishmeal) and then benchmarking the seaweeds against the amino acid requirements of mono-gastric livestock (chicken, swine and fish). The quality of seaweed protein (% of essential amino acids in total amino acids) is similar to, if not better than, traditional protein sources. However, seaweeds without exception have substantially lower concentrations of essential amino acids, including methionine and lysine, than traditional protein sources (on a whole biomass basis, % dw). Correspondingly, seaweeds in their whole form contain insufficient protein, and specifically insufficient essential amino acids, to meet the requirements of most mono-gastric livestock. This chapter highlights that the protein from seaweeds must be concentrated or extracted, and these techniques are the most important goals for developing seaweeds as alternative source of protein for mono-gastric livestock.

Therefore, in Chapter 6, I examined multiple techniques to isolate and concentrate protein in a seaweed, returning to the model organism the green seaweed Ulva ohnoi. The aim of this chapter was to compare the protein isolation and concentration efficiency of a mechanical-based method (as applied to leaves) to the solvent based method (as applied to seed crops). Protein isolate yields ranged from 12.28 ± 1.32 % to 21.57 ± 0.57 % and were higher using the methods established for leaves compared to those for seeds. Protein isolates from all treatment combinations were ~ 250 – 400 % higher in the concentration of protein and essential amino acids compared to the original whole biomass, reaching a maximum concentration of 56.04 ± 2.35 % and 27.56 ± 1.16 % for protein and total essential amino acids, respectively. In contrast, protein and essential amino acid concentrations were only ~ 30 – 50 % higher in protein concentrates compared to the original whole seaweed, reaching a maximum of 19.65 ± 0.21 % and 9.52 ± 0.11 % for protein and total essential amino acids, respectively. This chapter demonstrated that the methodologies used for the isolation of protein in leaves are more suited to seaweeds than those that are based on seed crops, which have traditionally been applied to seaweeds. This chapter also demonstrated that protein isolation methods are more suited to seaweeds with low concentrations of protein, such as Ulva ohnoi, compared to protein concentration methods.

In summary, the research presented throughout this thesis establishes that seaweeds, irrespective of cultivation conditions and species, are not viable as a protein source for mono-gastric livestock in a whole form and will need to be processed post-harvest to concentrate their protein. Therefore, it is proposed that the most important strategy for developing seaweeds as a protein crop is the development of protein isolates and concentrates from seaweeds produced under intensive cultivation.