Utilizing proteomics to identify and optimize microalgae strains for high-quality dietary protein: a review.

Algae-derived protein has immense potential to provide high-quality protein foods for the expanding human population. To meet its potential, a broad range of scientific tools are required to identify optimal algal strains from the hundreds of thousands available and identify ideal growing conditions for strains that produce high-quality protein with functional benefits. A research pipeline that includes proteomics can provide a deeper interpretation of microalgal composition and biochemistry in the pursuit of these goals. To date, proteomic investigations have largely focused on pathways that involve lipid production in selected microalgae species. Herein, we report the current state of microalgal proteome measurement and discuss promising approaches for the development of protein-containing food products derived from algae.

Heterosigma akashiwo in Patagonian Fjords: Genetics, Growth, Pigment Signature and Role of PUFA and ROS in Ichthyotoxicity.

Heterosigma akashiwo is the only raphidophyte described for Chilean waters. A recent 2021 fish-killing bloom event of this raphidophyte ignited scientific research, but the ichthyotoxic mechanism and environmental conditions that promote its growth are still unclear. This is the first study confirming the occurrence of H. akashiwo in Chilean waters on the basis of the region D1/D2 of the 28S ribosomal gene. The pigment signature of the CREAN_HA03 strain revealed chlorophyll-a, fucoxanthin, and violaxanthin as the most abundant pigments, but profiles were variable depending on culture and field conditions. A factorial temperature−salinity growth experiment showed a maximal growth rate of 0.48 d−1 at 17 °C and 35 in salinity, but reached a maximal cell abundance of ~50,000 cells mL−1 at 12 °C and 25 in salinity. The fatty acid profile included high levels of saturated (16:0) and polyunsaturated (18:4 ω3; 20:5 ω3) fatty acids, but superoxide production in this strain was low (~0.3 pmol O2− cell−1 h−1). The RTgill-W1 bioassay showed that the H. akashiwo strain was cytotoxic only at high cell concentrations (>47,000 cells mL−1) and after cell rupture. In conclusion, salmon mortality during H. akashiwo bloom events in Patagonian fjords is likely explained by the high production of long-chain PUFAs at high cell densities, but only in the presence of high ROS production.

Docosahexaenoic Acid Is Naturally Concentrated at the sn-2 Position in Triacylglycerols of the Australian Thraustochytrid Aurantiochytrium sp. Strain TC 20.

The Labyrinthulomycetes or Labyrinthulea are a class of protists that produce a network of filaments that enable the cells to glide along and absorb nutrients. One of the main two Labyrinthulea groups is the thraustochytrids, which are becoming an increasingly recognised and commercially used alternate source of long-chain (LC, ≥C20) omega-3 containing oils. This study demonstrates, to our knowledge for the first time, the regiospecificity of the triacylglycerol (TAG) fraction derived from Australian thraustochytrid Aurantiochytrium sp. strain TC 20 obtained using 13C nuclear magnetic resonance spectroscopy (13C NMR) analysis. The DHA present in the TC 20 TAG fraction was determined to be concentrated in the sn-2 position, with TAG (16:0/22:6/16:0) identified as the main species present. The sn-2 preference is similar to that found in salmon and tuna oil, and differs to seal oil containing largely sn-1,3 LC-PUFA. A higher concentration of sn-2 DHA occurred in the thraustochytrid TC 20 oil compared to that of tuna oil.

Disentangling the environmental processes responsible for the world's largest farmed fish-killing harmful algal bloom: Chile, 2016.

The dictyochophyte microalga Pseudochattonella verruculosa was responsible for the largest farmed fish mortality ever recorded in the world, with losses for the Chilean salmon industry amounting to US$ 800 M in austral summer 2016. Super-scale climatic anomalies resulted in strong vertical water column stratification that stimulated development of a dynamic P. verruculosa thin layer (up to 38 µg chl a L-1) for several weeks in Reloncaví Sound. Hydrodynamic modeling (MIKE 3D) indicated that the Sound had extremely low flushing rates (between 121 and 200 days) in summer 2016. Reported algal cell densities of 7000-20,000 cells mL-1 generated respiratory distress in fish that was unlikely due to low dissolved oxygen (permanently >4 mg L-1). Histological examination of salmon showed that gills were the most affected organ with significant tissue damage and circulatory disorders. It is possible that some of this damage was due to a diatom bloom that preceded the Pseudochattonella event, thereby rendering the fish more susceptible to Pseudochattonella. No correlation between magnitude of fish mortality and algal cell abundance nor fish age was evident. Algal cultures revealed rapid growth rates and high cell densities (up to 600,000 cells mL-1), as well as highly complex life cycle stages that can be easily overlooked in monitoring programs. In cell-based bioassays, Chilean P. verruculosa was only toxic to the RTgill-W1 cell line following exposures to high cell densities of lysed cells (>100,000 cells mL-1). Fatty acid profiles of a cultured strain showed elevated concentrations of potentially ichthyotoxic, long-chain polyunsaturated fatty acids (PUFAs) (69.7% ± 1.8%)- stearidonic (SDA, 18:4ω3-28.9%), and docosahexaenoic acid (DHA, 22:6ω3-22.3%), suggesting that lipid peroxidation may help to explain the mortalities, though superoxide production by Pseudochattonella was low (< 0.21 ± 0.19 pmol O2- cell-1 h-1). It therefore remains unknown what the mechanisms of salmon mortality were during the Pseudochattonella bloom. Multiple mitigation strategies were used by salmon farmers during the event, with only delayed seeding of juvenile fish into the cages and towing of cages to sanctuary sites being effective. Airlift pumping, used effectively against other fish-killing HABs in the US and Canada was not effective, perhaps because it brought subsurface layers of Pseudochattonella to the surface, or and it also may have lysed the fragile cells, rendering them more lethal. The present study highlights knowledge gaps and inefficiency of contingency plans by the fish farming industry to overcome future fish-killing algal blooms under future climate change scenarios. The use of new technologies based on molecular methods for species detection, good farm practices by fish farms, and possible mitigation strategies are discussed.

Taxonomy, ecology and biotechnological applications of thraustochytrids: A review.

Thraustochytrids were first discovered in 1934, and since the 1960's they have been increasingly studied for their beneficial and deleterious effects. This review aims to provide an enhanced understanding of these protists with a particular emphasis on their taxonomy, ecology and biotechnology applications. Over the years, thraustochytrid taxonomy has improved with the development of modern molecular techniques and new biochemical markers, resulting in the isolation and description of new strains. In the present work, the taxonomic history of thraustochytrids is reviewed, while providing an up-to-date classification of these organisms. It also describes the various biomarkers that may be taken into consideration to support taxonomic characterization of the thraustochytrids, together with a review of traditional and modern techniques for their isolation and molecular identification. The originality of this review lies in linking taxonomy and ecology of the thraustochytrids and their biotechnological applications as producers of docosahexaenoic acid (DHA), carotenoids, exopolysaccharides and other compounds of interest. The paper provides a summary of these aspects while also highlighting some of the most important recent studies in this field, which include the diversity of polyunsaturated fatty acid metabolism in thraustochytrids, some novel strategies for biomass production and recovery of compounds of interest. Furthermore, a detailed overview is provided of the direct and current applications of thraustochytrid-derived compounds in the food, fuel, cosmetic, pharmaceutical, and aquaculture industries and of some of the commercial products available. This review is intended to be a source of information and references on the thraustochytrids for both experts and those who are new to this field.

Screening of new British thraustochytrids isolates for docosahexaenoic acid (DHA) production.

Thraustochytrids isolated from hot tropical and sub-tropical waters have been well studied for DHA and biodiesel production in the last decades. However, little research has been performed on the oils of cold water thraustochytrids, in particular from the North Sea region. In this study, thraustochytrid strains from British waters showed high relative levels of omega-3 long-chain (≥C20) polyunsaturated fatty acids (LC-PUFA), including docosahexaenoic acid (DHA, 22:6ω3). The relative levels of DHA (as % of total fatty acids, TFA) in the different British strains are hitherto amongst the highest recorded from any thraustochytrid screening study, with strain TL18 reaching up to 67% DHA in modified Glucose-Yeast Extract-Peptone (GYP) medium. At this screening stage, low final biomass and fatty acid yield were observed in modified GYP and MarChiquita-Brain Heart Broth (MCBHB), while PUFA profiles (as % of PUFA) remained unaltered regardless of the culture medium used. Hence, optimizing the medium and culture conditions to improve growth and lipid content, without impacting the relative percentage of DHA, has the potential to increase the final DHA concentration. With this in mind, three strains were identified as promising organisms for the production of DHA. In the context of possible future industrial exploitation involving a winterization step, we investigated the recycling of the residual oil for biodiesel use. To do this, a mathematical model was used to assess the intrinsic properties of the by-product oil. The results showed the feasibility of producing primary DHA-rich oil, assuming optimized conditions, while using the by-product oil for biodiesel use.

Comparison of Thraustochytrids Aurantiochytrium sp., Schizochytrium sp., Thraustochytrium sp., and Ulkenia sp. for production of biodiesel, long-chain omega-3 oils, and exopolysaccharide.

Heterotrophic growth of thraustochytrids has potential in coproducing biodiesel for transportation, as well as producing a feedstock for omega-3 long-chain (≥C20) polyunsaturated fatty acids (LC-PUFA), especially docosahexaenoic acid (DHA) for use in nutraceuticals. In this study, we compared eight new endemic Australian thraustochytrid strains from the genera Aurantiochytrium, Schizochytrium, Thraustochytrium, and Ulkenia for the synthesis of exopolysaccharide (EPS), in addition to biodiesel and LC-PUFA. Aurantiochytrium sp. strains readily utilized glucose for biomass production, and increasing glucose from 2 to 4 % w/v of the culture medium resulted in increased biomass yield by an average factor of 1.7. Ulkenia sp. strain TC 010 and Thraustochytrium sp. strain TC 033 did not utilize glucose, while Schizochytrium sp. strain TC 002 utilized less than half the glucose available by day 14, and Thraustochytrium sp. strain TC 004 utilized glucose at 4 % w/v but not 2 % w/v of the culture suggesting a threshold requirement between these values. Across all strains, increasing glucose from 2 to 4 % w/v of the culture medium resulted in increased total fatty acid methyl ester content by an average factor of 1.9. Despite an increasing literature demonstrating the capacity of thraustochytrids for DHA synthesis, the production of EPS from these organisms is not well documented. A broad range of EPS yields was observed. The maximum yield of EPS was observed for Schizochytrium sp. strain TC 002 (299 mg/L). High biomass-producing strains that also have high lipid and high EPS yield may be better candidates for commercial production of biofuels and other coproducts.

High cell density cultivation of a novel Aurantiochytrium sp. strain TC 20 in a fed-batch system using glycerol to produce feedstock for biodiesel and omega-3 oils.

A recently isolated Australian Aurantiochytrium sp. strain TC 20 was investigated using small-scale (2 L) bioreactors for the potential of co-producing biodiesel and high-value omega-3 long-chain polyunsaturated fatty acids. Higher initial glucose concentration (100 g/L compared to 40 g/L) did not result in markedly different biomass (48 g/L) or fatty acid (12-14 g/L) yields by 69 h. This comparison suggests factors other than carbon source were limiting biomass production. The effect of both glucose and glycerol as carbon sources for Aurantiochytrium sp. strain TC 20 was evaluated in a fed-batch process. Both glucose and glycerol resulted in similar biomass yields (57 and 56 g/L, respectively) by 69 h. The agro-industrial waste from biodiesel production-glycerol-is a suitable carbon source for Aurantiochytrium sp. strain TC 20. Approximately half the fatty acids from Aurantiochytrium sp. strain TC 20 are suitable for development of sustainable, low emission sources of transportation fuels and bioproducts. To further improve biomass and oil production, fortification of the feed with additional nutrients (nitrogen sources, trace metals and vitamins) improved the biomass yield from 56 g/L (34 % total fatty acids) to 71 g/L (52 % total fatty acids, cell dry weight) at 69 h; these yields are to our knowledge around 70 % of the biomass yields achieved, however, in less than half of the time by other researchers using glycerol and markedly greater than achieved using other industrial wastes. The fast growth and suitable fatty acid profile of this newly isolated Aurantiochytrium sp. strain TC 20 highlights the potential of co-producing the drop-in biodiesel and high value omega-3 oils.

Biodiscovery of new Australian thraustochytrids for production of biodiesel and long-chain omega-3 oils.

Heterotrophic growth of thraustochytrids has potential in co-producing a feedstock for biodiesel and long-chain (LC, ≥C(20)) omega-3 oils. Biodiscovery of thraustochytrids from Tasmania (temperate) and Queensland (tropical), Australia, covered a biogeographic range of habitats including fresh, brackish, and marine waters. A total of 36 thraustochytrid strains were isolated and separated into eight chemotaxonomic groups (A-H) based on fatty acid (FA) and sterol composition which clustered closely with four different genera obtained by 18S rDNA molecular identification. Differences in the relative proportions (%FA) of long-chain C(20), C(22), omega-3, and omega-6 polyunsaturated fatty acids (PUFA), including docosahexaenoic acid (DHA), docosapentaenoic acid, arachidonic acid, eicosapentaenoic acid (EPA), and saturated FA, as well as the presence of odd-chain PUFA (OC-PUFA) were the major factors influencing the separation of these groups. OC-PUFA were detected in temperate strains of groups A, B, and C (Schizochytrium and Thraustochytrium). Group D (Ulkenia) had high omega-3 LC-PUFA (53% total fatty acids (TFA)) and EPA up to 11.2% TFA. Strains from groups E and F (Aurantiochytrium) contained DHA levels of 50-61% TFA after 7 days of growth in basal medium at 20 °C. Groups G and H (Aurantiochytrium) strains had high levels of 15:0 (20-30% TFA) and the sum of saturated FA was in the range of 32-51%. ß,ß-Carotene, canthaxanthin, and astaxanthin were identified in selected strains. Phylogenetic and chemotaxonomic groupings demonstrated similar patterns for the majority of strains. Our results demonstrate the potential of these new Australian thraustochytrids for the production of biodiesel in addition to omega-3 LC-PUFA-rich oils.

Transcriptomic and phenotypic responses of Listeria monocytogenes strains possessing different growth efficiencies under acidic conditions.

In an experiment delineating aciduric strains, food and clinical Listeria monocytogenes isolates tended to produce the most biomass whereas ovine and avian strains produced comparatively less biomass when exposed to high levels of sodium diacetate (SD) and potassium sorbate. Compared to reference strains that exhibited greater acid sensitivity, representative food isolates with comparatively good growth capacities in the presence of 21 mM SD at pH 5.0 accumulated reduced levels of acetate anion and K(+) ion. The aciduric nature of SD-resistant strains was also reflected by comparatively high tolerance to pH 2.4 (HCl) acid challenges, a property boosted by the presence of SD. Exposure to elevated levels of SD (21 mM SD at pH 5.0) was found to have broad effects on gene expression, as differentiated from effects caused by mildly acidic conditions (pH 5.0). SD-resistant strain FW04/0025 was more responsive to elevated SD, increasing the expression of 222 genes (>2-fold change [P < 0.05]), compared to the more sensitive EGD reference strain, which exhibited increases in expression of 112 genes. Key differences between the strains in relation to SD-enhanced transcripts were notably associated with the cell envelope, oxidative stress management, and intermediary metabolism. SD thus appears to differentially influence growth efficiency and survival of strains, under conditions relevant to acidic foods, that could be due to altered cell wall and metabolic phenotypes.