Multitron

Control or biopretreated wheat straw slurries with a consistency of 5% (w dm/v, in 50 ml Falcon^® tubes) were first subjected to a mild alkali treatment with 0.1% sodium hydroxide at 50°C and 120 rpm for 1 hr (Multitron, Infors AG, Switzerland). Afterward, the pH was adjusted to 4.8 by the addition of citrate phosphate buffer (pH 4, 100 mM) and supplemented with 12 FPU/g dm substrate of the commercial cellulase cocktail GC220 from Trichoderma reesei (Genencor Danisco, NY, USA) and 60 U/g dm substrate of β‐glucosidase SP188 from Aspergillus niger (Novozyme, Demark). Tetracycline (0.15 mg/ml) and cycloheximide (0.04 mg/ml) were added to prevent any microbial contamination. Then, the slurries consistencies dropped to 2.5% (w dm/v). The reaction was carried out at 50°C and 120 rpm for 72 hrs. The released reducing sugars and glucose were quantified at end point after centrifugation (5000 rpm, 5 min) using the dinitrosalicylic acid method and the Glucose RTU kit (Biomérieux, Marcy‐l’étoile, France), respectively.
The hydrolysis of the untreated and the noninoculated wheat straw (control) by the commercial enzymatic cocktail led to 25 and 23% (w/w) net cellulose and holocellulose conversion yields, respectively.

Picochlorum sp. BPE23, isolated from a coastal bay on Bonaire, was pre-cultivated in shake flasks in an orbital shaker incubator (Multitron, Infors HT) under continuous light at an intensity of 100 μmol m⁻² s⁻¹ photosynthetically active radiation (PAR) [18 ]. The temperature was set at 40 °C, and the relative humidity of the air in the headspace was set to 60% and enriched with 2% CO₂. Cells were cultured in artificial seawater enriched with nutrients and trace elements. Elements were provided at the following concentrations (in g L⁻¹): NaCl, 24.50; MgCl₂·6H₂O, 9.80; Na₂SO₄, 3.20; NaNO₃, 2.12; K₂SO₄, 0.85; CaCL₂.2H₂O, 0.80; KH₂PO₄, 0.23; Na₂EDTA·2H₂O, 0.11; NaFeEDTA, 3.96·10⁻²; MnCl₂·2H₂O, 1.71·10⁻³; ZnSO₄·7H₂O, 6.60·10⁻⁴; Na₂MoO₄·2H₂O, 2.42·10⁻⁴; Co(NO₃)₂·6H₂O, 7.00·10⁻⁵; NiSO₄·6H₂O, 2.63·10⁻⁵; CuSO₄·5H₂O, 2.40·10⁻⁵; K₂CrO₄, 1.94·10⁻⁵; Na₃VO₄, 1.84·10⁻⁵; and H₂SeO₃, 1.29·10⁻⁵. HEPES (4.77 g L⁻¹) was added to Erlenmeyer cultures as a pH buffer. The medium pH was adjusted to 7.0, after which it was filter sterilized before use. During photobioreactor cultivation, Antifoam B (J.T.Baker, Avantor, USA) was added at a concentration of 0.5 mL L⁻¹ from a 1% w/w% stock. Also, 0.168 g L⁻¹ Sodium bicarbonate (NaHCO₃) was added at inoculation to provide sufficient CO₂ at the start of the cultivation. The photobioreactor was inoculated at a starting cell density of OD₇₅₀ 0.2.

Cryopreserved cells of E. coli FUS4 (pF81_kan) and E. coli 3RP (pF81_kan) were streaked on minimal medium agar plates with glycerol as carbon source prepared according to Weiner et al. (2014) [29 (link)] and incubated at 37°C for at least 66 h. One single colony was then used for inoculation of a 100 mL shake flask with 10 mL minimal medium with 7 g/L of glycerol as carbon source prepared according to the same protocol as the agar plates [29 (link)]. After cultivation at 37°C and 150 rpm for approximately 24 h in an orbital shaker (Multitron, Infors HT, Switzerland), the optical density at 600 nm (OD₆₀₀) was measured (Genesys 10UV, Thermo Fisher Scientific, USA). A defined volume of cell suspension was transferred to two 500 mL shake flasks each one with 100 mL minimal medium to yield a starting OD₆₀₀ of 0.01. These cultures were further cultivated at 37°C and 250 rpm for at least 24 h. When the cells reached exponential growth phase with an OD₆₀₀ above 0.5, the cultures were centrifuged at 3260×g for 10 min at 4°C. The supernatant was discarded and the cell pellets were suspended in fresh minimal medium. Bioreactor cultivations were inoculated with washed cells to a starting OD₆₀₀ of 0.1.

Pre‐cultures of P. taiwanensis VLB120∆C have been used to inoculate the main culture with an initial biomass concentration of 0.09 g_cdw L⁻¹ in 25 mL M9 medium (0.5% glucose) using screw‐cap baffled 250 mL Erlenmeyer flasks and subsequently respective butanol amount was added. The flasks were incubated in a horizontal shaker (30°C and 200 r.p.m.; Multitron; Infors HT, Bottmingen, Switzerland) and growth was monitored by analysing the final biomass density achieved. Only an end‐point measurement was performed to avoid butanol losses through the head‐space by sampling.

Pre‐cultures of P. taiwanensis VLB120 and P. taiwanensis VLB120∆C (Park et al., 2007) and P. taiwanensis VLB120∆Cegfp (Halan et al., 2011) were grown overnight in 25 mL M9 medium (0.5% glucose) using baffled 250 mL Erlenmeyer flasks in a horizontal shaker (30°C and 200 r.p.m.; Multitron; Infors HT, Bottmingen, Switzerland).

Provision of cell biomass for the inoculation for the cultivation in a stirred-tank bioreactor was realized by a two-step preliminary cultivation in shake flasks. First, a single colony of cells grown on minimal medium agar plates ( $>$ 66 h at 37°C) was picked for inoculation of a single 100-ml shake flask with 10 ml minimal medium and cultivated at 37 °C and 150 rpm in an orbital shaker (Multitron, Infors HT, Switzerland) for 24 h. Afterward, the cells were transferred for further cultivation in two 500-ml shake flasks with 100 ml minimal media each and a starting optical density at 600 nm (OD₆₀₀) of 0.01. After incubation at 37°C and 250 rpm for at least 24 h, the cells were centrifuged (4,500 rpm, 10 min) and resuspended in fresh minimal medium. These cell suspensions were used for inoculation of cultivations in the stirred-tank bioreactor with a starting OD₆₀₀ of 0.1.