OTUs showing a statistically significant difference by the Kruskal-Wallis sum rank test (*p* < 0.05). Different superscripts in the same row indicate that groups are significantly different by the Wilcoxon test for multiple pairwise comparisons. Please see Supplementary Table S4 for standard errors of the means and Supplementary Table S1 for a complete list of OTUs and their respective abundances. Abbreviations: *F.*: *Falsiporphyromonas*; *La.*: *Lactococcus*; *Le.*: *Leuconostoc*; *Pe.*: *Peptoniphilus*; *Ps.*: *Peptostreptococcus*; *Pl.*: *Plesiomonas*; *R.*: *Ralstonia*; *Sp.*: *Sphingomonas*; *St.*: *Streptococcus*.

#### **4. Discussion**

As a result of challenges such as cost and availability, the inclusion of fishmeal as a primary ingredient in aquaculture has become difficult to sustain [6]. Fishmeal use in aquaculture and livestock diets is also cause for ethical and social sustainability concerns, as increased demand for fishmeal would not only risk reducing the supply of fish available as food for humans but also promote overexploitation of species that are not for human consumption [57]. While other animal protein sources, such as poultry meal and feather meal, have served as suitable alternatives in aquaculture diets, there remains a critical need to find more economical replacement ingredients. While lower cost and availability make plant-based protein ingredients attractive alternatives, the presence of anti-nutritional factors such as non-starch polysaccharides, protease inhibitors, lectins, saponins, phytic acid, phytoestrogens, and allergens limits the extent to which they can be included in fish diets [58].

Thus, even if soybean meal provides a well-balanced amino acid profile, a favorable protein content, and lower amounts of anti-nutrients relative to other plant-based protein sources [59,60], its inclusion in diets of carnivorous species still needs to be restricted. Bioprocessing, i.e., modification of plant-based primary ingredients by microbial metabolism, has provided a solution to this problem. Indeed, fermentation of soybean meal into 'bioprocessed' soybean meal results in a product with an enhanced nutritional profile, as it is highly digestible and has a high protein content with increased lysine and methionine concentrations, while its anti-nutritional factor levels are greatly reduced [61].

One possible effect of the high inclusion of bioprocessed soybean meal in aquaculture diets is its potential impact on the composition of intestinal microbial communities. Considering their contributions to the health and nutrition of their host, alterations in the composition of symbiotic gut microbial communities could have unintended consequences on aquaculture production. In this context, the study presented in this report aimed at investigating the potential effects of bioprocessed soybean meal on the intestinal bacterial

composition of aquaculture-raised hybrid striped bass. In addition to gaining further insight on an alternative feed ingredient with great potential for aquaculture, this report is also the first to provide insight on the gut microbiome of the hybrid striped bass using a culture-independent method.

The first observation was the absence of major differences in intestinal bacterial composition between fish fed a diet with the bioprocessed soybean meal (BP) and fish fed a diet without bioprocessed soybean meal (CON). Based on its limited sequence identity to its closest valid relative (*Peptostreptococcus russellii*, 91%), the main OTU (SD\_McMs-00001) in these samples was predicted to correspond to a currently uncharacterized or uncultured bacterial species. Because of its taxonomic affiliation, SD\_McMs-00001 would be predicted to utilize proteins as a main substrate. Indeed, strains of *P. russellii* were originally isolated from swine manure, and they were reported to produce elevated amounts of ammonia when grown in culture with various peptide-based ingredients [62]. This activity was interpreted as *P. russellii* playing an active role in the digestion and fermentation of proteinaceous material [63,64], which is consistent with the high protein content in carnivorous fish diets and the dietary treatments used in this study. Bacteria affiliated to the genus *Peptostreptococcus* have been identified as one of the main bacterial groups in rainbow trout [31] and proposed as an indicator taxon of fast-growing fish for this host [65]. This bacterial group was also reported to be well represented in aquaculture raised Arctic char (*Salvelinus alpinus*) [66]. Interestingly, many species of the genus *Peptostreptococcus* can increase the production of indoleacrylic acid and decrease the susceptibility of epithelial injury in mice [60]. Furthermore, research in humans has revealed that increased production of indoleacrylic acid could provide relief to inflammatory bowel disease [67]. Together, these previously published studies suggest that diets resulting in increased *P. russellii* may improve fish health by benefiting the host's intestinal mucus layer.

Unexpectedly, the main intestinal OTUs in fish fed different post-fermentation modified versions of the bioprocessed soybean meal, i.e., diets BP-F1, BP-E, and BP-W, were very different from BP. The most abundant OTU in four of the six combined samples for BP-E and BP-W samples, SD\_McMs-00007, was predicted to be a strain of *Lactococcus lactis* based on their high nucleotide sequence identity. *L. lactis* is known for its broad use in the food industry [68] because of its basic ability to utilize proteins and ferment carbohydrates into lactate. Strains of this species have been isolated from a number of distinct sources, including drain water and human vaginal samples [69,70], indicating that *L. lactis* is suited to many different types of environments. While it is not considered a typical resident of the gastrointestinal tract, *L. lactis* is capable of surviving in the gut environment [71,72], where it may interact with the mucus layer [73]. This species has been used as a probiotic in red sea bream (*Pagrus major*), resulting in a higher final weight, percent weight gain, and specific growth rate [74], as well as in olive flounder (*Paralichythys olivaceus*), where it was found to increase levels of growth-promoting metabolites, such as short-chain fatty acids, citrulline, taurine and vitamins [75].

The other two main OTUs in samples from diets BP-F1, BP-E, and BP-W may have represented potential pathogens. SD\_McMs-00002 was 99% identical to *Plesiomonas shigelloides*, a bacterial species predominantly found in freshwater fish [40,76,77]. Notably, *P. shigelloides* was reported as a pathogen in cultured tilapia, with infected fish suffering tissue damage in the liver, spleen, kidney, heart, and intestine [78]. Since *P. shigelloides* has been reported as pathogenic in another report [76], future investigations will be required to gain further insight into the biological roles of SD\_McMs-00002 and other related strains to determine whether this species represents a pathogen or a commensal in the hybrid striped bass. Similarly, SD\_McMs-00003 was very closely related to *Ralstonia pickettii* (99%), also a bacterial species of the phylum Proteobacteria. While it has been proposed as a normal resident of the fish gastrointestinal tract [79], and it has been identified in soil and water samples [80], *R. pickettii* was also reported as a low virulence pathogen in certain cases of invasive infections in humans [81].

Based on the results presented in this report, the inclusion of bioprocessed soybean meal did not dramatically alter the intestinal bacterial composition of hybrid striped bass in the context of an aquaculture-based diet that consisted of a combination of fishmeal and alternative protein sources. However, inclusion of bioprocessed soybean meal that had been further processed after fermentation resulted in different and less consistent intestinal bacterial composition patterns. It will be of interest to further investigate these different effects of post-fermentation treated bioprocessed soybean meal on gut bacterial communities of aquaculture-raised fish in order to determine if the benefits of these alternative feed ingredients on performance are worth potential risks to gut microbiome function.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/microorganisms9051032/s1.

**Author Contributions:** Conceptualization M.B.; methodology M.B. and B.S.-P.; investigation, B.W., E.C.F. and P.P.; formal analysis, E.C.F., B.W. and B.S.-P.; data curation, M.B. and B.S.-P.; writing original draft preparation, E.C.F.; writing—review and editing, M.B. and B.S.-P.; funding acquisition, M.B. All authors have read and agreed to the published version of the manuscript.

**Funding:** Funding for this research was provided by the South Dakota State University Agricultural Experiment Station, the South Dakota Soybean Research and Promotion Council, and the United Soybean Board.

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Animal Care and Use Committee (IACUC) of South Dakota State University (Approval Number 16-089A, issued on 11/22/2016).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Raw sequence data are available from the NCBI Sequence Read Archive under Bioproject PRJNA718291.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

