*4.9. Determination of Total Phenolic Content (TPC)*

The total phenolic content (TPC) of the *F. vesiculosus* crude extracts was determined using a slightly modified version of the Folin–Ciocalteu assay [79] as described by [80]. A known amount of crude extract was re-suspended in methanol to a concentration of 1 mg.mL−1. 100 μL of each crude extract was placed in a 1.5 mL Eppendorf tube along with 100 μL of methanol, 100 μL of Folin–Ciocalteu reagent (2N) and 700 μL of 20% sodium carbonate, to a final volume of 1 mL. Samples were vortexed and immediately afterwards placed in darkness to incubate for 20 min at room temperature. Samples were then centrifuged at 4300× *g* for 3 min before measuring the absorbance of the supernatant at 735 nm using a Cary UV50 Spectrophotometer and CaryWIN software (Varian Inc., Palo Alto, CA 94304, USA). A sample treated according to the same protocol, but where 100 <sup>μ</sup>L methanol instead of 100 <sup>μ</sup>L of crude extract (1 mg·mL<sup>−</sup>1) was added, was used as a blank. Phloroglucinol was used as the external standard and a calibration curve was performed by serial dilution of a 2 mg mL−<sup>1</sup> stock solution (10, 20, 50, 80, 120, 160 μg mL<sup>−</sup>1). Total phenolic content (TPC) was expressed as milligram of phloroglucinol equivalents (PE) per gram of dry weight extract (mg PE g−<sup>1</sup> DWE) or per gram of dry weight biomass (mg PE g−<sup>1</sup> DWB) [36]. TPC quantification was performed in triplicate for each crude extract. The yield of extraction was calculated after exhaustive solid-liquid extraction (i.e., three successive extraction) of the total phenolics of 50 mg of *F. vesiculosus* freeze-dried ground biomass (Ps < 0.5 mm) using 80% methanol.

#### *4.10. Accession Numbers*

The metagenomic sequencing data (raw reads) was deposited in the European Nucleotide Archive (ENA) under the accession numbers ERR2608102 -ERR2608111. The 16S rRNA gene sequences for the bacterial isolates were deposited in GenBank under the accession numbers KY224981–KY225289, KY327837, KY327838, MG693225–MG693716, MG760723–MG760725, and MK480287–MK480325. Bacterial isolates IC18\_D7 and ANT0\_A6 with enzymatic activities and applicability in EAE approach were deposited in DSMZ culture collection bank under the accession numbers DSM 107285 and DSM 107318, respectively.

#### *4.11. Statistical Analysis*

Prior to performing statistical analyses on data obtained by enzyme-assisted extraction (EAE) of total phenolics from *F. vesiculosus*, tests of normality were carried out with the Kolmogorov–Smirnov test for normal distributions and Levene's test for homogeneity of variance. A one-way ANOVA and post hoc Tukey's pairwise test was performed to assess significant differences (*p* < 0.05) between commercial enzymes and control (50 ◦C); and a *t*-test was applied to assess significance differences (*p* < 0.05) between EBS and control (28 ◦C). All data treatments and statistical analyses were performed using IBM SPSS Statistics V22.0 (IBM Corporation, Armonk, NY, USA).

#### **5. Conclusions**

In conclusion, we have demonstrated, using both metagenomic and culture based approaches, that changes occur in the composition and abundance of *A. nodosum*-associated epibiotic communities which are both time and temperature dependent and that the microflora of *A. nodosum* is composed of diverse and complex bacterial communities which produce a wide range of hydrolytic enzymes, some of which may be useful in future EAE based strategies in the agricultural, food, cosmeceutical, and pharmaceutical sectors.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1660-3397/17/4/ 200/s1; Table S1: Table showing enzymatic activities, Figure S1: *Ascophyllum* cultivable surface microbiota at genus classification level, Figures S2–S6; Phylogenetic trees of intact and decaying *A. nodosum* associated microbial communities.

**Author Contributions:** A.D.W.D., M.W.I. and D.S. conceived and designed the study; F.G., H.M. and M.W.I. performed the experiments and together with L.M.M. and S.A.J. analyzed the data. M.W.I., D.C., S.A.J., and A.D.W.D. wrote the manuscript.

**Funding:** This research was funded by the European Commission, PharmaSea project (www.pharma-sea.eu) (contract no. 312184), Marine Biotechnology ERA/NET, NEPTUNA project (contract no. PBA/MB/15/02), SMI-BIO project (15/F/698), Science Foundation Ireland (SSPC-2, 12/RC/2275), Department of Agriculture, Food and the Marine (DAFM) SMI-BIO project (15/F/698), DAFM, (FIRM 1/F009/MabS) and by the Beaufort Marine Research Award, part of the Sea Change Strategy and the Strategy for Science Technology and Innovation (2006–2012), with the support of The Marine Institute under the Marine Research Sub-Programme of the National Development Plan 2007–2013.

**Conflicts of Interest:** The authors declare that they have no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.
