Pentaacetyl chitopentaose

Chitin from shrimp shells (practical grade coarse flakes), chitosan CHIT50.1 (Product number: 900,344) and CHIT50.2 (Product number: 448,869), N-acetyl-D-glucosamine (GlcNAc) and biotin were from Sigma Aldrich (St. Louis, MO, USA). Chitosan from shrimp shells CHIT100 and CHIT600 were from Across Organics Chemicals (ThermoFisher Scientific, Waltham, Massachusetts, USA). Colloidal chitin (CC) was obtained as referred previously [30 (link)]. Basically, 10 g chitin in 10 M HCl was maintained 16 h at 4 ºC, filtered through glass thick fibres into ethanol and then chitin floccules were precipitated at 4 ºC, collected (5000xg during 10 min) and maintained in 0.1 M sodium acetate pH 4.0-4.5. Chitosan was dissolved in 0.1 M sodium acetate pH 4.0-4.5, in a final concentration of 10 g l^− 1. Diacetyl-chitobiose ((GlcNAc)₂), triacetyl-chitotriose ((GlcNAc)₃), tetraacetyl-chitotetraose ((GlcNAc)₄), pentaacetyl-chitopentaose ((GlcNAc)₅) and hexaacetyl-chitohexaose ((GlcNAc)₆) were from Megazyme (Bray, Irland). Nitrogen base w/o amino acids (YNB) was from Difco (BD, Sparks, MD, USA). Yeast extract, peptone, tryptone and agar were from Laboratorios Conda S.A. (Madrid, Spain).

We used the wildtype strain Vibrio natriegens ATCC 14048 and Alteromonas macleodii sp. 4B03 (non-clumping variant) isolated from marine particles [8 (link)]. Strains were cultured in Marine Broth (MB, Difco 2216) and grown overnight at 25 °C. In total, 1 ml of cell culture was centrifuged (13,000 rpm for 2 min) in a 1.5 ml microfuge tube. After discarding the supernatant, the cells were washed with 1 ml of MBL minimal medium medium without carbon source. Cells were centrifuged again and the cell pellet was resuspended in 1 ml of MBL (marine minimal medium) [30 , 34 (link)] adjusted to an 0.002 OD600. Cells from these cultures were used for experiments in MBL minimal medium containing 0.1% (weight/volume) Pentaacetyl-Chitopentaose (Megazyme, Ireland). The carbon source was added to the MBL minimum medium and filter sterilized using 0.22 μm Surfactant-Free Cellulose Acetate filters (Corning, USA). A total of 500 µl of the prepared cultures (250 µl + 250 µl for co-cultures) were added to 9.5 ml of MBL + 0.1 % chitopentaose (v/w) in serum flasks. This resulted in a starting OD of 0.0001. The flasks included a stirrer and were sealed with a rubber seal. Serum flasks were stored on a bench top magnetic stirrer (500 rpm) and connected to the microfluidics setup via Hamilton NDL NO HUB needles (ga21/135 mm/pst 2).