Cellobiose

The oxygen reactivity of PMOs was measured by a time resolved quantification of H₂O₂ formation in 96-well plates (total volume of 200 μL) using a Perkin Elmer EnSpire Multimode plate reader. All reactions were performed in 100 mM sodium phosphate buffer, pH 6.0 at 22°C. Based on preliminary studies ascorbate and CDH were used in concentrations of 30 μM and 0.3 μM (0.025 mg mL^-1), respectively to prevent a limitation in the PMO reduction step. As electron donor for CDH 500 μM cellobiose was used. When ascorbate was used as reductant, it was added to a final concentration of 30 μM and enzyme assays were started by mixing 20 μL of the respective PMO with 180 μL of the ready-made assay solution containing 30 μM ascorbate, 50 μM Amplex Red and 7.14 U mL^-1 peroxidase in 96-well plate wells. In reference experiments without PMO the background signal (H₂O₂ production by CDH) was measured and subtracted from the assays. When CDH was used as reductant, the PMO assays were started by mixing 20 μL of sample solution and 20 μL CDH solution with 160 μL of the reaction mix containing cellobiose, Amplex Red and peroxidase. Initial fluorescence scans of resorufin showed highest signal intensities and lowest interference with matrix compounds when using an excitation wavelength of 569 nm and an emission wavelength of 585 nm for the selected conditions. The stoichiometry of H₂O₂ conversion to resorufin formation is 1:1. By using a high concentration of Amplex Red (50 μM) the linearity of the detection reaction was ensured and the molar ratio of Amplex Red:H₂O₂ was always greater than 50:1
[22 (link)]. The H₂O₂ concentration in the assays was far below 1 μM, which leads to a linear concentration/activity response of horseradish peroxidase, which has a K_M for H₂O₂ of 1.55 μM. The high final activity of horseradish peroxidase (7.14 U mL^-1) assures a fast conversion of the formed H₂O₂ and prevents the final reaction to be rate limiting. Additionally, it prevents the accumulation of H₂O₂, which could have detrimental effects on enzyme stability in the assay. The stability of resorufin fluorescence under these conditions was tested by addition of varying concentrations of hydrogen peroxide (0.1 – 5 μM) to the assay. A stable signal that remained constant throughout the measured period of 45 minutes was observed and maximum signal intensity was reached already during the mixing period before starting the assay. A linear relation between fluorescence and H₂O₂ concentrations in the range of 0.1 – 2 μM H₂O₂ was observed and the slope (28450 counts μmol^-1) was used for the calculation of an enzyme factor to convert the fluorimeters readout (counts min^-1), into enzyme activity. PMO activity was defined as one μmol H₂O₂ generated per minute under the defined assay conditions.

Fresh faecal samples were obtained from six consenting healthy adult human donors (1 faecal sample per donor: minimum 0.5 g) and were placed in anaerobic conditions within 1 h of passing to preserve the viability of anaerobic bacteria. All sample processing and culturing took place under anaerobic conditions in a Whitley DG250 workstation at 37 °C. Culture media, PBS and all other materials that were used for culturing were placed in the anaerobic cabinet 24 h before use to reduce to anaerobic conditions. The faecal samples were divided in two. One part was homogenized in reduced PBS (0.1 g stool per ml PBS) and was serially diluted and plated directly onto YCFA^{7 (link)} agar supplemented with 0.002 g ml⁻¹ each of glucose, maltose and cellobiose in large (13.5 cm diameter) Petri dishes. This sample was also subjected to metagenomic sequencing to profile the entire community. The other part was treated with an equal volume of 70% (v/v) ethanol for 4 h at room temperature under ambient aerobic conditions to kill vegetative cells. Then, the solid material was washed three times with PBS and it was eventually resuspended in PBS. Plating was performed as described earlier.
For the ethanol-treated samples, the medium was supplemented with 0.1% sodium taurocholate to stimulate spore germination. Colonies were picked 72 h after plating from Petri dishes of both ethanol-treated and non-ethanol-treated conditions harbouring non-confluent growth, (that is, plates on which the colonies were distinct and not touching). The colonies that were picked were re-streaked to confirm purity. No statistical methods were used to predetermine sample size. The experiments were not randomized. The investigators were not blinded to allocation during experiments and outcome assessment.

Saccharomyces cerevisiae BY4741 was used and precultured anaerobically in YPD media with 2.0% glucose (w/v) (Wako, Osaka, Japan) at 30 °C for 72 h without shaking. YPD media was used for one litter of medium: 10 g of yeast extract (Bacto, MD, USA), 20 g of Pepton (Bacto), 20 g of glucose, and adjusted to pH 6.
The preculture and culture medium for C. cellulovorans 743B (ATCC 35296) and C. beijerinckii NCIMB8052 (ATCC 51743) was partially modified and used [5]. For one litter of medium, it was prepared with 4 g of yeast extract, 1 mg of Resazurin salt, 1 g of l-cysteine HCl, 5 g of NaHCO₃, 0.45 g of K₂HPO₄, 0.45 g of KH₂PO₄, 0.3675 g of NH₄Cl, 0.9 g of NaCl, 0.1575 g of MgCl₂∙6H₂O, 0.12 g of CaCl₂∙2H₂O, 0.85 mg of MnCl₂∙4H₂O, 0.942 mg of CoCl₂∙6H₂O, 5.2 mg of Na₂EDTA, 1.5 mg of FeCl₂∙4H₂O, 0.07 mg of ZnCl₂, 0.1 mg of H₂BO₃, 0.017 mg of CuCl₂∙2H₂O, 0.024 mg of NiCl₂∙6H₂O, 0.036 mg of Na₂MoO₄∙2H₂O, 6.6 mg of FeSO₄∙7H₂O, 0.1 g of p-aminobenzoic acid), and was adjusted to pH 7 for C. cellulovorans and to pH 5 for C. beijerinckii, respectively. C. cellulovorans and C. beijerinckii were anaerobically precultured in the above medium with 0.5% (w/v) cellobiose (Sigma, MO, USA) and with 2.0% (w/v) glucose, respectively, at 37 °C for 23 h without shaking.

The
recent and publicly available toolkit AutoSolvate^{37 (link)} was run in an Ubuntu 22.04 LTS environment, encompassing
Python 3.7, Openbabel 2.4.0, AmberTools 22, MDTraj 1.9.4, and NGLView
3.0.3. The process involved Antechamber with the AM1-BCC model to
assign point charges, LEaP for Generalized Amber Force Field parameters,
and the B3LYP hybrid exchange-correlation functional for density functional
theory (DFT) calculations. Both water and chloroform at 298 K were
tested as solvents.
Three XYZ files (ESI-01) were studied for the structures of DTZ: dithizone_planar.xyz corresponds
to the highly conjugated symmetric form, with the nonaromatic hydrogen
atoms on the outer nitrogen atoms (Figure 1a); dithizone_thione.xyz differentiates the
azo group at one side of the thiocarbazone chain and two secondary
amino groups at the other side; and dithizone_anion.xyz corresponds
to the deprotonated thiol tautomer of dithizone with ammonium as counterion.
Finally, oxycellobiose.xyz is a proxy for CNFs, simply containing
a cellobiose molecule with regioselective oxidation on carbon 6 in
one of its two glucose units. Bond angles, bond distances, and molecular
surfaces of these three forms were calculated and displayed by Jmol
14.
With the same toolkits (AutoSolvate, AmberTools),
a simulation
of molecular dynamics was run on the basis of molecular mechanical
(MM) energy minimization.^{38 (link)} Then, a solvation
shell considering the most neighboring solvent molecules was modeled
and 10 XYZ files, also included in ESI-01, were extracted. Each file corresponds to every 10 frames, spaced
by a timelapse of 4 ps, of the dynamic simulation.

According to the results of transcriptomic and proteomic analyses, the carbohydrate substance (mannose, galactose, cellobiose, and D-ribose) was added to the mono-culture of L. paraplantarum RX-8 at the concentrations of 2, 20, and 200 mM. Meanwhile, the amino acid substance (arginine, cysteine, glutamate, and glutamine) was added to the mono-culture of L. paraplantarum RX-8 at the concentrations of 0.5, 1.25, and 2.5 g/L. After being cultured for 24 h at 37°C, the supernatants of each sample were collected for the plantaricin production assay. The mono-culture of L. paraplantarum RX-8 was used as a control.

Lignin was modeled as guaiacyl glycerol-β-guaiacyl ether, which is a β-O-4 dimer representing the building blocks of natural lignin^{51 (link)}. Cellulose was modeled with cellobiose, which is a glucose dimer containing a β-O-4 glycosidic bond^{52 (link)}. Four units of whewellite were retrieved from the whewellite monoclinic structure for use as the model for calculations^{53 (link)}. The structures of lignin, cellulose, whewellite, lignin-cellulose composite, and lignin-cellulose-whewellite composite were optimized by employing the PM6 Hamiltonian with Grimme’s D3 dispersion correction implemented in the Gaussian 16 package⁵⁴ . The M062X hybrid exchange-correlation functional with the 6-31 + G(d,p) basis set and Grimme’s D3 dispersion correction were used for the energy calculations, since they have been found to be effective in describing intermolecular interactions^{51 (link)}. The binding energy of the composite (E_bind) was obtained via Eq. 8. ${\mathrm{E}}_{\mathrm{bind}}=\mathrm{E}-{\mathrm{E}}_1-{\mathrm{E}}_2-{\mathrm{E}}_3$ in which E is the energy of the composite, and E₁, E₂, and E₃ represent the energy of each constituent. Moreover, the electrostatic potential and electron density difference analyses were conducted with Multiwfn 3.8 (dev) code^{55 (link)} and visualized via Visual Molecular Dynamics software^{56 (link)}.
In addition, the redox potential for tetracycline hydroxylation (Eq. 9) was calculated based on the standard-state Gibbs free energy of gas-phase molecule (G_gas, via CBS-4M), the energy of the gas-phase molecule (E_gas, via M052X/6-31 G(d)), and the energy of the molecule dissolved in water (E_dis, via M052X/6-31 G(d) with the solvation model of water based on density). Namely, the aqueous Gibbs free energy (G_aq) of each species in Eq. 9 was derived via Eq. 10, according to which the aqueous Gibbs free energy change of the reaction (ΔG_aq) was acquired^{57 (link)}. Furthermore, the redox potential was calculated with the Nernst equation and referenced to the standard hydrogen electrode (Eq. 11)^{57 (link)}. $\mathrm{T}\mathrm{etracycline}+{\mathrm{H}}_2\mathrm{O}=\mathrm{Hydroxylated\; t}\mathrm{etracycline}+2{\mathrm{H}}^{+}+2{\mathrm{e}}^{-}$ ${\mathrm{G}}_{\mathrm{aq}}={\mathrm{G}}_{\mathrm{gas}}+\left({\mathrm{E}}_{\mathrm{gas}}-{\mathrm{E}}_{\mathrm{dis}}\right)$ $\mathrm{E}=\frac{{△\mathrm{G}}_{\mathrm{aq}}}{\mathrm{nF}}-4.28$