Substrates
Debarked pine (Pinus taeda) (from USA pulp mill), poplar, (from USA pulp mill) and Alamo switchgrass (from University of Georgia, Athens, GA) were milled to a 2 mm particle size with a Wiley Mill and extractives were removed by Soxhlet extraction with dichloromethane for 48 h. The extracted biomass samples were air-dried and frozen at − 20 °C until usage.
Dilute-acid pre-treatment, equipment and process
Dilute-acid pre-treatment was conducted based on work by Saha et al. (2005). The treatment parameters were as follows: water to biomass ratio of 10:1 (w/w dry biomass) and 1.5% H2SO4 (w/w dry biomass). Samples were heated for approximately 45 min while maintaining a mean temperature of 180 °C. Upon completion, the heater for the Parr reactor was turned off and the pressure was slowly vented to return the vessel to atmospheric pressure and the pretreated biomass was removed from the glass liner and cooled to room temperature before freezing at − 20 °C until use.
Bacterial strains, media, and cultivation
Strains
The D5A ethanol-producing strain of Saccharomyces cerevisiae (ATCC® 200062™) was obtained from ATCC (https://www.atcc.org/). Rhodococcus opacus (DSMZ 1069, hereby referred to as DSM 1069) and R. opacus PD630 (DSMZ 44193, hereby referred to as PD630) were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ, http://www.dsmz.de).
Media
All media components were obtained from Fisher Scientific (Hampton, NH, USA) or Sigma Aldrich (St. Louis, MO, USA) and used as received. The D5A yeast strain was initially cultivated in YP media amended with 5% w/v final glucose as described by Dowe and McMillan (NREL/TP-510-42630) (Dowe and McMillan 2008). During simultaneous saccharification and fermentation (SSF) the yeast was cultured in 7.5% w/w cellulose, 1% w/v yeast extract, 2% w/v peptone, 0.05 M citrate buffer (pH 4.8), 15 units cellulase enzyme from Trichoderma reesei (Sigma C2730), per gram cellulose and 50 units beta-glucosidase from almond (Sigma G4511) in 100 mL media.
Shake flask fermentations were carried out with two types of media for each of the Rhodococci strains: a full media and a minimal media, which were composed of different carbon and nitrogen sources. The full media was prepared according the formulations as recommended by DSMZ: Medium 65, GYM Streptomyces Medium for DSM 1069 and Medium 535, Trypticase Soy Broth medium for PD630. The minimal media for DSM 1069 was composed of the following chemicals: 0.40 g KH2PO4, 1.60 g K2HPO4, 0.20 g MgSO4·7H2O, 0.03 g FeCl3, 0.50 mg MnSO4·H2O, 1.00 mg CuSO4·5H2O, 1.00 mg ZnSO4·7H2O, 0.50 mg CaCl2, 0.10 mg KCl, and 0.50 mg H3BO3/L distilled water (Kosa and Ragauskas 2012). For PD630, the minimal media contained: 9.00 g Na2HPO4·12H2O, 1.50 g KH2PO4, 0.20 g, MgSO4·7H2O, 1.20 mg FeNH4 citrate, 20.00 mg CaCl2, 2.00 mL Hoagland solution (Sigma, H2395), and 0.50 g NaHCO3/L distilled water (Schlegel et al. 1961). The nitrogen source for minimal media for DSM 1069 was NH4NO3 and the source for PD630 was NH4Cl. During the cell adaptation phase of cell culture, nitrogen was supplemented at 0.1 and 0.05% w/v during lipid accumulation phase.
Yeast cultivation
Simultaneous saccharification and fermentation of D5A yeast using pine, poplar, and switchgrass biomasses as the carbohydrate source were carried out anaerobically at 35 °C, 130 rpm for 160 h as described by Dowe and McMillan (NREL/TP-510-42630) (Dowe and McMillan 2008). Small aliquots (1 mL) were sterilely removed from each flask at 24, 96, and 160 h, in duplicate, and saved for ethanol detection with an EnzyChrom Ethanol Assay Kit (ECET-100). During the sampling times, the fermentation broth was streaked on solid YP media to test for viability and contamination.
Simultaneous saccharification and fermentation (SSF) treatment
SSF was conducted based on NREL protocol TP-510-42630 (Dowe and McMillan 2008). The yeast implemented was Saccharomyces cerevisiae D5A (S. cerevisiae Meyen ex E.C. Hansen ATCC® 200062TM). The enzyme loading was 15 units/g cellulose, at 7.0 g cellulose/g biomass, 105 units of cellulase enzyme from Trichoderma reesei (Sigma Aldrich, C2730-50ML) was loaded into each 100 mL flask, and 50 units of β-glucosidase from almonds was also added. The flasks were incubated at 35 °C, 130 rpm. The ethanol production data (% ethanol, g/L, % cellulose conversion) for 0, 24, 96, and 160 h were also determined as a function of time (in Additional file 1). Post-SSF, the solid residue was separated from the fermentation broth via vacuum filtration using a Büchner funnel fitted with Whatman Grade 1 filter paper (Sigma Aldrich, WHA1001150) and a side-arm flask. The solid portion of the pretreatment residue was washed with 500 mL of water three times to remove residual yeast fermentation broth and was freeze-dried with a VirTis Freezemobile 25 L freeze dryer for 48 h.
Rhodococcus cultivation
Rhodococcus strains were incubated at 28 °C, 150 rpm with 1.5% (w/v) freeze-dried DAP-SSF residue (2.25 g/150 mL) loading in minimal media. The strains underwent initial adaptation on the residues at 1.5% solids in 10 mL of 0.1% w/v nitrogen minimal media for 24 h. After the initial adaptation phase, all cells and residues were transferred to 150 mL of 0.05% w/v nitrogen minimal media and 1.5% solids loading, that was sterilized by autoclaving for 20 min at 121 °C. The flasks were incubated for 96 h, with sampling at 0, 12, 24, 48, 72, and 96 h. At each sampling time point, the insoluble solid fraction of the fermentation broth was separated from the liquid media and saved for analysis as described above.
Serial dilution and plating/colony count
Due to the solid-state nature of the biomass/feedstock, optical density (OD) could not be utilized to monitor cell growth as a function of time. Instead, serial dilution and plating was used to track cell viability at 0, 12, 24, 48, 72, and 96 h. For each strain/biomass, 1 mL was removed sterilely from the culture flask and diluted in 9 mL of sterile physiological salt solution, and repeated to dilutions of 10−2, 10−3, 10−4, and 10−6. A sample of each dilution, 100 μL, was plated on full media agar for the respective Rhodococci strains. The plates were incubated at 28 °C overnight and colonies were counted.
Sugar release and quantification of monosaccharides using high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD)
Quantification of monosaccharides in the respective fermentation broths were performed using high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The soluble/liquid portion, removed directly from the fermentation broth at 0 and 96 h, and freeze-dried substrate from Rhodococcus fermentation of DAP-SSF pretreated residues after acid hydrolysis were analyzed. For the latter preparation, the freeze-dried residues of the initial starting material (0 h) and at the end of the fermentation (96 h) was treated with a two-step H2SO4 hydrolysis following NREL/TP-510-42618 (Sluiter et al. 2008). In the first step, the DAP-SSF residues were hydrolyzed with 72% (w/w) H2SO4 at 30 °C for 1 h. In the second step, the hydrolyzed samples were diluted to 3% H2SO4 (w/w) of final concentration, followed by autoclaving at 121 °C for 1 h. The hydrolysate was filtered from solid residue and the recovered liquid fraction was analyzed with a Dionex ICS-3000 ion chromatography system (Thermo Fisher Scientific, Sunnyvale, CA) to determine the sugar profile of the fermentation residues. Specifically, a Dionex ICS-3000 ion chromatography with CarboPac PA-1 column was utilized. The temperature of the column was set to 30 °C and the eluents A and B were 100% DI water and 200 mM NaOH, respectively. The flow rate was set to 0.35 mL/min. Duplicate samples of the fermentation broths were analyzed and each analyte was quantified using a standard curve based on calibration curves of an external standard of glucose, xylose, mannose, galactose, arabinose.
Gel permeation chromatography (GPC)
The DAP-SSF residues, before and after exposure to Rhodococcus, and after vacuum filtration as previously described above, were dried under vacuum at 40 °C overnight and then were acetylated with acetic anhydride/pyridine (1/1, v/v) at ambient temperature for 24 h in a sealed flask under an inert atmosphere. The concentration of the lignin in the solution was approximately 20 mg/mL. After 24 h, the solutions were diluted with ~ 20 mL of ethanol and stirred for an additional 30 min, after which the solvents were removed with a rotary evaporator, followed by drying in a vacuum oven at 40 °C. Prior to GPC analysis the acetylated pre- and post-Rhodococcus fermentation residue samples were dissolved in tetrahydrofuran (1.0 mg/mL), filtered through a 0.45 µm filter, and placed in a 2 mL auto-sampler vial. The molecular weight distributions of the acetylated lignin samples were then analyzed on an Agilent GPC SECurity 1200 system equipped with four Waters Styragel columns (HR1, HR2, HR4, HR6), an Agilent refractive index (RI) detector, and an Agilent UV detector (270 nm), using tetrahydrofuran (THF) as the mobile phase (1.0 mL/min), with an injection volume of 20 μL. A standard polystyrene sample was used for calibration. The number-average molecular weight (Mn) and weight-average molecular weight (Mw) were determined by GPC.
Fourier transform infrared spectroscopy (FTIR)
A PerkinElmer Spectrum 100 FTIR spectrometer with a universal attenuated total reflectance (ATR) sampling accessory (Perkin-Elmer Inc., Wellesley, MA, United States) was used to monitor the structural changes in the residues. The residue samples were pressed uniformly against the crystal surface via a spring-anvil, and the spectra were obtained by 32 scans accumulation from 4000 to 500 cm−1 at 4 cm−1 resolution. The ATR correction and the baseline correction were carried out using Perkin-Elmer Spectrum software (Perkin-Elmer Inc., Norwalk, CT, United States). The background correction of the obtained spectra was performed using the Spectrum program from Perkin-Elmer.
Lipid characterization
For methanolysis/transesterification, approximately 15–20 mg of freeze dried cells/residual biomass, for each substrate at several time points, was dissolved in 1.00 mL CHCl3, 0.85 mL methanol and 0.15 mL concentrated H2SO4. The samples were incubated at 100 °C for 140 min in a sandbath to obtain fatty acid methyl esters (FAME) from all lipids present. Subsequently, 0.50 mL of distilled water was added and the samples were shaken vigorously (vortexed) for 1 min, and after phase separation, the FAME-containing organic phase was removed. Approximately 0.6 mL containing the FAME was recovered. Each sample was diluted to 2 mL with chloroform (1:3.33 dilution) before analysis.
GC–MS was used to identify FAME produced by Rhodococcus DSM 1069 and PD630, which was performed on an Agilent 7890A GC/5975C MSD system on a HP-5MS column, with 1 μL sample injection. The sample program was run as follows: 0.5 min equilibration time, 50 °C for 2 min, 15 °C/min to 200 °C then held for 5 min, 10 °C/min to 250 °C then held for 2 min, and 25 °C/min to 300 °C then held for 4 min. The Supelco® 37 Component FAME Mix (Sigma-Aldrich CRM47885) was used as an external standard.
AMDIS software by NIST was used to determine the integrated areas of peaks identified as “methyl esters” by the NIST Chemical Library. The integrated areas of each identified peak with 90% or greater compound m/z spectrum similarity of the external standard at five dilutions (undiluted, 1:2, 1:10, 1:20, and 1:40, wt%) were used to generate standard curves for each component. The integrated areas of peaks for identified fatty acid methyl esters at each sampling time point for the pine, poplar, and switchgrass samples were used to approximate the total lipid content (mg/L) at each time point.