**4. Materials and Methods**

#### *4.1. Sampling*

*Ascophyllum nodosum* samples were obtained in the intertidal zone at Rinville in Galway Bay, Ireland at 53◦14 40 North, 8◦58 2 West in late January, 2016. Approximately 2 kg of seaweed was sampled and packaged in sterile air-tight plastic bags and stored on dry ice at the sampling location. *Ascophyllum* samples were subsequently stored briefly at 4 ◦C in the laboratory before further analyses.

#### *4.2. Experimental Design*

Three sets of approximately 450 g of the seaweed were suspended in separate 950 mL sterile artificial seawater (3.33% *w*/*v* synthetic seawater salts Instant Ocean, Aquarium Systems, in distilled water), with each 2 L flask incubated at a different temperature (18 ◦C, 25 ◦C, and 30 ◦C) on a shaking platform at 125 rpm for a six-week period. The incubation flasks were single replicates (*n* = 1) per temperature treatment. Three separate ichip devices were subsequently inoculated four weeks after the initial incubation into each of the flasks under sterile conditions in a laminar flow hood (BioAir Safeflow 1.2—EuroClone, Pero, Italy), as previously described [32], and removed at the end of the decay period. To inoculate each ichip device, 1 mL suspension from the incubation flask containing the seaweed decaying at one of the three temperatures (18 ◦C, 25 ◦C, and 30 ◦C), with an estimated microbial density of 1.0 × <sup>10</sup><sup>12</sup> cells mL−<sup>1</sup> was diluted appropriately in sterile artificial seawater (3.33% *<sup>w</sup>*/*<sup>v</sup>* synthetic seawater salts Instant Ocean, Aquarium Systems, in distilled water) to attain an average of one cell per through-put hole in the iChip central plate (i.e., one cell per μL of inoculum) and suspended in molten 0.5% agar (Sigma Aldrich, Munich, Germany) solution. The cell-agar suspension was poured over the ichip central plate to allow cells that were immobilized within the suspension to be trapped in the small throughput holes on the device as the agar solidified. The device was then assembled and placed in the flask containing the decaying seaweed for another two weeks. Approximately 10 g of the fresh intact macroalga (T0) was collected before incubation into the artificial seawater and 10 g of the decaying seaweed was collected from each incubating flask at two-week intervals (weeks 2, 4, and 6) during the decay period. All *Ascophyllum* samples were stored at −20 ◦C for further analyses.

#### *4.3. 16S rRNA Gene Amplicon Library Preparation and MiSeq Sequencing*

Metagenomic DNA was extracted from approximately 0.5 g of the intact seaweed (T0) and 0.5 g each of decaying *Ascophyllum nodosum* samples collected at three phases of the decay period (week 2, week 4 and week 6) at 18 ◦C (2\_18, 4\_18, 6\_18), 25 ◦C (2\_25, 4\_25, 6\_25), and at 30 ◦C (2\_30, 4\_30, 6\_30) as previously described [58]. PCR amplicon libraries were generated using forward (5 *TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG*CCTACGGGNGGCWGCAG-3 ) and reverse (*GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG*GACTACHVGGGTATCTAATCC-3 ) primers complementary to the V3-V4 16S rRNA gene region [59] with ligated Illumina adapter overhang sequences in italic text. This primer pair was identified as the most promising pair required for a good representation of bacterial diversity and has been successfully applied in a number of studies on a wide range of environments [59–62]. PCR amplification was performed under the following conditions: 98 ◦C for 30 s, followed by 30 cycles of denaturation (98 ◦C for 10 s), primer annealing (57 ◦C for 30 s), primer extension (72 ◦C for 30 s), and 72 ◦C for 5 min. PCR amplicons were purified using Agencourt AMPure XP beads (Beckman Coulter) according to the manufacturer's instructions and a subsequent reduced-cycle (8 cycles) reaction was performed to further attach unique dual eight-base Nextera XT multiplexing indices and sequencing adapters under similar cycling conditions. Index PCR products were purified using Agencourt AMPure XP beads (Beckman Coulter; Fisher Scientific, Dublin, Ireland) according to the manufacturer's instructions. All PCR reactions from each sample were performed

in triplicates to minimize bias, replicate amplicons were pooled together and sequenced using the Illumina MiSeq platform by Macrogen (Seoul, Korea).

Scythe (v0.994 BETA) [63] and Sickle [64] programs were used to quality trim raw reads and remove adapter sequences. This service was provided by Macrogen Inc (Seoul, Korea) as part of a next generation sequencing package. Trimmed paired end reads were merged (using the join\_paired\_reads.py script with the fastq-join method [65] ) in QIIME version 1.9.1 (QIIME.org) [34] and processed using standard QIIME version 1.9.1 protocols (http://qiime.org/tutorials/illumina\_ overview\_tutorial.html). Briefly, a further quality step was applied by excluding reads with a Phred score less than 20 using the split\_libraries\_fastq.py QIIME script. The USEARCH algorithm [66] was used to remove chimeras and assign sequences to OTUs based on the SILVA database (version 123) (Max Plank Institute, Bremen, Germany) [35] at a threshold of 97% identity. Singletons were identified and filtered from the OTU table and the OTU table was CSS (cumulative sum scaling) normalized [67]. The taxonomy identified from the dataset was then represented through bar plots. Species diversity and richness within samples were calculated using alpha and beta diversity analyses (Chao1, Good's coverage, Shannon indices and principle coordinates analysis) using QIIME (version 1.9.1) (http://qiime.org/tutorials/illumina\_overview\_tutorial.html) scripts (alpha\_diversity.py and beta\_diversity\_through\_plots.py) [34].

#### *4.4. Bacterial Isolation from Intact and Decaying A. nodosum Using Maceration Method*

Surface-attached bacteria were isolated from the intact (T0) and decaying seaweed samples collected at weeks 2, 4 and 6 of the decay period, each at three different temperatures (18 ◦C, 25 ◦C, and 30 ◦C) using the maceration method adapted from [68]. Briefly, approximately 0.5 g of the algal sample was cut into small pieces of about 1 cm<sup>2</sup> and suspended in 1 mL of sterile artificial seawater (3.33% *w*/*v* synthetic seawater salts Instant Ocean, Aquarium Systems, in distilled water) [69]. Serial dilutions of the suspension were plated on SYP-SW agar plates which consisted of soluble starch (Sigma Aldrich, Munich, Germany) 10 g L−1; yeast extract (Sigma Aldrich, Germany) 4 g L−1; peptone (Merck, Germany) 2 g L<sup>−</sup>1; Instant Ocean (Aquarium Systems) 33.3 g L−1; agar (Sigma Aldrich, Germany) 15 g L−<sup>1</sup> and incubated at 28 ◦C for 72 h. The culture isolation procedure was conducted aseptically in a laminar flow hood (BioAir Safeflow 1.2—EuroClone, Pero, Italy). Individual colonies were selected and further streaked to isolate pure cultures which were grown at 28 ◦C overnight in SYP-SW medium and maintained in glycerol (20% *w*/*v*) stocks at −80 ◦C.

#### *4.5. Bacterial Isolation from Decaying A. nodosum Using ichip Device*

Three separate ichip devices were inoculated into each of the incubating flasks (18 ◦C, 25 ◦C, and 30 ◦C) at week 4 of the decay period and were removed at the end of the decay period (week 6). Macroalgal-associated bacteria from decaying *A. nodosum* were recovered from small throughput holes on the central plate of each ichip device and plated directly onto 96-well plates containing SYP-SW agar and incubated at 28 ◦C for 72 h. Individual colonies were selected and further streaked to isolate pure cultures which were grown at 28 ◦C overnight in SYP-SW medium and maintained in glycerol (20% *w*/*v*) stocks at −80 ◦C.

#### *4.6. Taxonomic Identification of A. nodosum Cultivable Surface Microbiota Populations*

Bacterial isolates recovered from both intact and decaying *A. nodosum* samples using both the traditional maceration method and the ichip method were taxonomically identified using 16S rRNA gene sequencing. Genomic DNA was extracted from the bacterial isolates grown overnight at 28 ◦C in SYP-SW medium using a modified Tris-EDTA boiling DNA extraction method [70]. Bacterial 16S rRNA PCR amplification was performed with the universal forward (8F; 5 -AGAGTTTGATCCTGGCTCAG-3 or 27F; 5 -AGAGTTTGATCMTGGCTCAG-3 ) and reverse (1492R; 5 -GGTTACCTTGTTACGACTT-3 ) primers [71,72] under the following conditions: initial denaturation (95 ◦C for 30 s), followed by 35 cycles of denaturation (95 ◦C for 1 min), primer annealing (55 ◦C for 1 min), primer extension

(72 ◦C for 1 min) and a final primer extension step (72 ◦C for 5 min). PCR products were analyzed by gel electrophoresis on a 1% agarose gel and purified using a QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions.

Sanger sequencing was performed on the amplified PCR products by GATC Biotech, (Konstanz, Germany) and Macrogen (Amsterdam, The Netherlands). Low quality 5' and 3' sequence ends were trimmed using FinchTV (http://www.geospiza.com/finchtv) depending on the data set. The BLAST program (NCBI) (https://blast.ncbi.nlm.nih.gov/Blast.cgi) was used to compare trimmed sequences against the GenBank database and closest relatives to the bacterial isolates were identified. 16S rRNA gene sequences were checked for chimeras using USEARCH algorithm [66] and the data sets were de-replicated using the Fastgroup database [73] and Avalanche NextGen Workbench version 2.30 (http://www.visualbioinformatics.com/html/)(bioinformatics.org) with a 99% cut-off value. Sequence alignment and phylogenetic tree construction were performed with MEGA (version 7) (Penn State University, PA, USA) [74]. The evolutionary history was inferred using the Neighbor-joining method [75].

#### *4.7. Enzyme Screens*

Bacterial isolates obtained from both intact *A. nodosum* and the decaying seaweed, using both the maceration method and the ichip device, were screened for the production of macroalgal cell wall degrading enzymes including pectinase, hydroxyethyl cellulase, and enzymes involved in lichenin degradation. Bacterial isolates were grown at 28 ◦C for 72 h on LB gellan gum (Sigma Aldrich, Munich, Germany) plates supplemented with the appropriate substrate, at a concentration of 0.2% (*w*/*v*) for pectin (Sigma Aldrich, Germany), 0.5% (*w*/*v*) for hydroxyethyl cellulose (Sigma Aldrich, Germany) and 0.05% (*w*/*v*) for lichenin (Megazyme). Enzymatic activities on lichenin and HE-cellulose were indicated by a surrounding zone of clearance upon flooding with Congo red solution (0.1% *w*/*v* Congo red in 20% *v*/*v* ethanol) for 30 min and wash with 1M NaCl for 5 min [76,77] while pectin supplemented plates were flooded with Lugol's iodine solution [78].

#### *4.8. Enzyme-Assisted Extraction*

Entire specimens of *Fucus vesiculosus* (2–3 kg fresh weight) were collected at low tide on 6 July 2016 at Finavarra, Co., Clare, Ireland (53◦08 59 North–9◦08 09 West). In the laboratory, on the day of collection, algal biomass was cleaned of epiphytes and rinsed in distilled water to remove excess salt. Samples were patted dry with tissue paper and stored at −20 ◦C. Then, the biomass was freeze-dried in a Labconco Freezone® freeze-dryer system (Labconco Corporation, Kansas City, MO 64132-2696, USA). Dried biomass was ground using a coffee-grinder and sieved to produce two types of powder, Ps < 0.5 mm and 0.5 < Ps < 2.5 mm), prior to subsequent enzymatic-assisted extraction (EAE) of total phenolic compounds. Three commercially available enzymes; cellulase (from *Aspergillus* sp., Sigma Aldrich, ≥1000 U/g), xylanase (from *Trichoderma* sp., Megazyme, 2.86 U/mL) and protease (from *Bacillus licheniniformis*, Sigma Aldrich, ≥2.4 U/g) were used for the hydrolysis of the seaweed. The potential of three bacterial strains (IC18\_D7, IC18\_D5 and ANT0\_A6 with ≥ 98% sequence similarity to *Vibrio anguillarum* X0906, *Vibrio oceanisediminis* S37 and *Winogradskyella* sp. MGE\_SAT\_697, respectively) isolated from *A. nodosum* and shown to be producers of algal cell wall degrading enzymes in this study was also tested. Bacterial isolates were grown overnight at 28 ◦C in SYP-SW medium and equal volumes of supernatants from the overnight cultures obtained by centrifugation at 4300× *g* for 10 min were used. Three sets of approximately 4 g dry weight of the crushed algae was incubated at 50 ◦C for 24 h on a shaking platform (185 rpm) with sodium acetate buffer (100 mL; 0.1 M; pH 5.2), each with 100 μL of one of the three commercial enzymes. Another set of the algal biomass was also incubated at 28 ◦C with a mixture of culture supernatants obtained from the bacterial isolates (enzymatic bacterial supernatants, EBS) to a final volume of 100 μL. All experiments were performed in triplicate and control experiments without the addition of either commercial enzymes or culture supernatants (EBS) were also conducted under the same conditions. The hydrolysate mixture from

each experimental set was centrifuged at 4300× *g* for 10 min at 4 ◦C to eliminate the algal debris from the extract. The different extracts produced were freeze dried, weighed, and stored at −80 ◦C until further analysis for total phenolics.
