The existing literature has established the potential of torula yeast as a valuable protein source for the aquafeed industry 15,18. The present study addressed essential knowledge gaps regarding the effect of replacing ingredients currently used in salmonid feeds with increasing inclusions of torula yeast on both growth performance and gut microbiome of freshwater stage Atlantic salmon (Salmo salar). Two separate base diets were investigated. A marine protein base diet where fish meal (FM) was replaced with increasing levels of torula yeast to provide a simplified replacement and a marine protein and plant protein combination where mixed plant meals (MIX) were replaced with increasing levels of torula yeast to provide a commercial relevant dietary replacement.
Growth performance for fish fed the FM diets was comparable between all inclusion levels for both the Specific Growth Rate SGR (%) and Condition Factor (K) up to 20% inclusion. This matches well with existing research where torula yeast has successfully replaced up to 40% of protein from fish meal in the diets of freshwater (FW) Atlantic salmon without negatively impacting growth performance 18. Existing research suggests that the addition of torula yeast to a standard fish meal diet can even enhance the growth of FW Atlantic salmon compared to fish meal alone 13. The present study provides further evidence that even in Atlantic salmon less than 2g body weight, torula yeast is a suitable partial replacement for marine protein. The lack of difference in K across the different inclusion levels suggest torula yeast also provides comparable energy levels to marine protein 53. Growth performance for fish fed MIX diets was best for fish fed the moderate inclusion MIX10, even better than both the control and the highest inclusion level, and K was comparable across all diets. Few studies have explored the replacement of plant proteins with torula yeast despite the prevalence of these materials in commercial salmonid. The reduced growth performance of fish fed the MIX20 compared with MIX10 was also found when torula yeast replaced gluten and starch in a soybean meal-based diet also at an inclusion of 20% 13 however MIX20 was comparable with the MIX00 control, suggesting MIX10 may be optimal in this juvenile study. In comparison, when 25% inclusion level of torula yeast replaced a mixture of plant and marine protein sources in Atlantic Salmon through the freshwater to seawater transfer, growth performance and feed intake were improved compared to the control 14. This difference in results could be due to the strain and growing conditions suggesting this process can be optimised to improve the application for salmonids 18. In an experiment with Tilapia (Oreochromis mossambicus) fed a mixed diet with both animal, and plant protein combined, increasing levels of torula yeast were fed to fry, and showed similar trend to this study, with moderate levels, in this case 30% torula yeast supporting better growth than lower or higher levels 54. This study suggests that there is potential for growth benefits with moderate inclusion levels of torula yeast as a replacement for plant protein. The decline in growth performance at higher inclusions levels could be driven by potential over-feeding of yeast presenting a detrimental impact 22 however given that the higher inclusion is comparable with the control, a detrimental impact is unlikely in this study. This is supported by good growth performance of the 20% inclusion for the FM in this study. However, this study along with (Øvrum Hansen) et al., 2019, indicates that higher inclusion levels of torula yeast are more applicable in combination with a marine protein diet than they are with a mixed source protein diet, but that the strain and growth conditions could be improved to optimise utilisation in mixed diets 18.
The gut microbiome of fish fed FM diets and MIX diet is altered by the replacement of proteins with torula yeast, but the impact trends differ for the different dietary bases. The lack of difference in alpha diversity measures between any of the FM diets suggests that all three diets provide a comparable substrate to support a community with similar defining characteristics, however the actual community composition established is different, which is shown by the beta diversity, and this is most apparent at the genus level. The MIX diets support gut microbiome communities with different alpha diversity characteristics, especially for the highest inclusion level MIX20 and with different community compositions between the three inclusion levels which are also most clear at the genus level. Compared to the existing literature on the Atlantic salmon (Salmo salar) gut microbiome during the freshwater stage, the alpha diversity measures for both the FM and MIX fish in this study fall within normal levels for captive individuals 55. The alpha diversity results of FM diets concur with comparable experiments with other salmonid species. In Rainbow trout (Oncorhynchus mykiss) kept in freshwater up to 20% replacement of fish meal with the yeasts Saccharomyces cerevisiae and Wickerhamomyces anomalus did not significantly alter the gut microbial community diversity, but higher replacement levels of 40% and 60% reduced bacterial diversity, and even led to increasing presence of the pathogenic Candida albicans at 60% inclusion levels, even reducing the presence of LABs 40. This suggests that inclusion of greater than 20% of yeasts may be problematic for salmonid health which could in turn affect production even when in combination with marine protein only 22. To the knowledge of these authors this is the first experiment to assess the gut microbiome of Atlantic salmon when plant proteins in a mixed protein source diet are replaced with a yeast protein source, yet this is a highly relevant concern for commercial salmonid aquaculture. This study reveals that the impact to the alpha diversity is more pronounced than in marine protein diets for observed diversity, Shannon diversity and Chao1 diversity which were all higher in the 0% control and moderate 10% inclusion of torula yeast than in the higher inclusion of 20% in the MIX diets. Higher levels of alpha diversity measures do not necessarily mean a healthier or more resilient community assemblage, so we must take this outcome in context with the other findings about the gut microbiome community discussed here 56. In early stage Rainbow trout (Oncorhynchus mykiss) alpha diversity measures decreased when animal protein was replaced with a mix of plant proteins, but plant proteins levels were at 50% and 97% replacement 35 which were much higher than in the present study. This indicates drastic changes in diet can be detected in the alpha diversity measures, suggesting that replacing plant proteins with torula yeast in our MIX diets drastically influenced the community, more than replacing fish meal in a marine protein diet for our FM diets. In another monogastric animal, weaning pigs, a 40% replacement of conventional proteins (a combination of plant and marine proteins) with torula yeast (Cyberlindnera jadinii), both the alpha diversity and the beta diversity were significantly altered by the replacement, with lower alpha diversities with the 40% diet 41, suggesting a similar trend as found in the present study for the MIX diets.
Existing research on the gut microbiome of freshwater Atlantic salmon suggests that even when alpha diversity is not altered, difference may be present in the beta diversity, for example, Chao1 richness and Shannon diversity were not altered by acute cold stress or a chronic environmental stress but the beta diversity was altered 57. In early stage salmonids when the dietary protein composition is altered, existing studies observed a significant shift in the beta diversity and composition of the gut microbiome, in Atlantic salmon (Salmo salar) 58, in Rainbow trout (Oncorhynchus mykiss) 35,59, and in Arctic charr (Salvelinus alpinus) 60. Similarly, in the present study, beta diversity was distinct between each inclusion level of torula yeast irrespective of the dietary base (FM or MIX) and which protein component was being replaced. Since gut microbiota changes between freshwater and seawater transfer 31, future studies should follow Atlantic salmon fed torula yeast as a dietary protein through-out development, from first feeding to harvest to clarify the impact across different environmental conditions. In other cultured fish species, the presence of yeast species either as a supplement or as a protein replacement for conventional proteins also significantly altered the community compositions, in early-stage zebrafish (Danio rerio) 33, in the gilthead sea bream (Sparus aurata) 61 and the grass carp (Ctenopharyngodon idellus) 62.
The gut microbiome composition of early stage Atlantic salmon in this study for both FM and MIX diet bases was similar at the phylum level regardless of the torula yeast inclusion level. Similarly in other studies that have used next generation sequencing in salmonids during the freshwater growth stages, the dominance of Firmicutes followed by Proteobacteria have been noted with varying levels of Actinobacteria depending on the study 31,40,63,64. Suggesting that at lower taxonomic resolution a core phyla composition can be expected regardless of diet and influenced by a range of other factors. The drivers of differing community compositions can be seen in this study at the genus level. The dominance of Firmicutes at the phyla level for both FM and MIX diets at all inclusion levels is explained by the high relative abundance of Staphylococcus at the genus level. This dominance of Staphyloccocus species is consistent with other Atlantic salmon (Salmo salar) and many other fish gut microbiome characterisations 65,66 and it has been associated with nutritional processes in the salmonid, Arctic charr (Salvelinus alpinus) 67. However, this study revealed different trends for FM and MIX diets with increasing inclusion of torula yeast further suggesting that torula yeast interacts differently with marine protein diets and mixed source protein diets, this could be to do with potentially differing levels of dietary fibre in FM based diets and the MIX diets in this study. Torula yeast has a high (20%) Total Dietary Fibre (TDF) at 20% TDF, which is higher than standard fish meal which is largely devoid of TDF but may see values as high as 5% 68, which may explain why we see a slight trend toward increased diversity indices with increasing torula yeast inclusion, although the effect is minimal. Conversely in the MIX diets, a similarly high TDF% from the combined plant meals 68 is being replaced by the torula yeast. This suggests that TDF is not the only driver of the decreased diversity indices seen for the high yeast MIX20 diet. The torula yeast was 12% insoluble fibre and 8% soluble fibre (20% TDF) (R.Ekmay 2021, personal communication 26 May). Future investigations should assess the balance of soluble and insoluble fibre components of formulated feeds with torula yeast and their impact on fish gut microbiota since dietary fibre is known to influence microbiome in other animals 69. It will be important to elucidate the significance of such differing trends for health and development of cultured salmonids. The Lactic Acid Bacteria (LABs) from the genus Weissella and Leuconostoc 37 did not show a strong trend in the FM diets, but slightly increased with increasing inclusion of torula yeast. Conversely, for the MIX diets, the LABs Lactobacillus, Leuconostoc, and Weissella 37 were present, and for the former two they declined with increasing inclusion of torula yeast, whereas the latter increased slightly in the presence of torula yeast. In the fin-fish literature, LABs are widely regarded to be linked to positive benefits such as disease resistance, improved performance and are associated with innate immune activities 37,70, however the exact impact is dependent on the species of bacteria and the specific host. In the FM diets of this study Clostridium_sensu_stricto_1, and Preptostreptococcus were both higher in diets containing torula yeast, and highest in the modest 10% inclusion. These genera (Clostridium and Preptostreptococcus) have been associated with faster growth in Rainbow trout (Oncorhynchus mykiss) faecal bacteria samples, although in our study Clostridium_sensu_stricto_7 did not show a strong trend. However, the genus Paeniclostridium was associated with slower growing individuals 71 and this was also highest in FM10 of the fish from this study presenting a complicated picture. Since the growth was not significantly different in the FM diets of this study, it might suggest that Atlantic salmon (Salmo salar) may have different bacterial indicators of growth than other salmonids. In the MIX diets the genus Corynebacterium was lowest in the moderate 10% inclusion of torula yeast, and highest in the 20% inclusion level, and in Rainbow trout (Oncorhynchus mykiss) this genus was associated with slow growing individuals 71 which does correspond with the growth results for the present study of Atlantic salmon (Salmo salar), suggesting it may be a potentially useful indicator for this salmonid species. It would be highly valuable for future research to identify bacteria in the digestive tract of Atlantic salmon that are associated with fast and slow growing individuals.
It appears from this study that when torula yeast can effectively partially replace FM based diet up to an inclusion of 20% without apparent negative impact to the gut microbiome community assemblage, and some potential benefits in terms of raising the levels of some LABs. Conversely, when torula yeast is added to a mix of protein sources it is more effective for growth performance and promoting desirable gut bacteria at moderate levels of 10% inclusion than at the higher inclusion level of 20%. The finding that moderate levels of torula yeast in a mixed protein diet are preferable to higher inclusions is reflected in the existing literature for Atlantic salmon (Salmo salar) 21. It should be noted that this study sampled the gut digesta and mucosa together, which may have masked further differences in the gut microbiome community composition, since the digesta appears most impacted by diet. Future studies should focus on the digesta where possible 32 although this has proved challenging with very small Atlantic salmon (Salmo salar), it would be valuable to investigate the gut microbiome down to the species taxonomic resolution. Additionally this early developmental stage in the salmonid life cycle is a time of a highly dynamic gut microbiome that is malleable and has the potential to change when the dietary input changes and may not be consistent or have consistent function over time 30,35.