Feedstocks and chemicals
Switchgrass (Panicum virgatum L.) air-dried whole herbage (late maturity, low quality, harvested after overwintering in February 2013) was generously provided by K. J. Shinners, University of Wisconsin-Madison. The material was ground through Wiley mill (1 mm) but otherwise was not subjected to additional pretreatment, and was stored at room temperature in the dark. Analysis (in triplicate) using the detergent fiber method of Goering and Van Soest (1970; without α-amylase treatment) revealed a composition [g (kg DM)−1] of: neutral detergent fiber, 878 ± 9; acid detergent fiber, 537 ± 1; and acid-detergent lignin, 86 ± 5. The N content, determined using a Leco TruMac (St. Joseph, MI, USA) combustion analyzer, was 5.2 ± 0.1 g (kg DM)−1. DM content of the ground switchgrass was 924 g (kg DM)−1.
The following redox mediators were used in their oxidized form: methyl viologen (MV, Acros, 98% dye content); neutral red (NR, Sigma, 95% dye content); safranin O (Aldrich, 96% dye content), resazurin (Sigma, ~85% dye content); tannic acid (TA, Aldrich).
Inocula were obtained from two lactating Holstein cows each fitted with a ruminal cannula (Bar-Diamond, Parma, ID, USA). The cows were fed a total mixed ration that contained corn silage, alfalfa haylage, ground corn grain, soybean meal and a vitamin and mineral mix. Ruminal contents (solids and liquids) from each cow were collected manually, then processed and the separate diluted ruminal fluid from each cow combined as described previously (Mouriño et al. 2001).
All fermentations were conducted in triplicate within treatment, under a CO2 gas phase in volume-calibrated glass serum vials (Wheaton) of ~60 mL volume fitted with butyl rubber closures and aluminum crimp seals. Experiments were conducted using freshly collected and diluted ruminal inocula. Vials contained Goering–Van Soest medium (1970) reduced with cysteine and Na2S, along with the switchgrass [19 mg DM (mL liquid volume)−1]. Total liquid volume in the vials was typically 10 mL, except for the NR concentration experiment (22 mL). Unless otherwise indicated, resazurin was added at low concentrations (0.008 mM) as a redox indicator to confirm (via decolorization upon reduction) establishment of reducing conditions in the culture media. Each redox mediator was dissolved in N2-gassed, deionized water to achieve a ~20 mM stock solution and added to fermentation vials to achieve the indicated concentration in the medium. Each experiment included control vials that lacked redox mediators, as well as blank vials that contained media and inoculum but lacked switchgrass or redox mediators. All experimental setup and incubations were conducted under non-aseptic conditions, with no sterilization of vessels, apparatus, biomass feedstocks, or culture media. Incubations were performed 39°C in a static upright position for 72 h.
Analysis of residual substrate and fermentation products
Analysis of gas phase H2 and methane was conducted by removal of fixed volumes (0.20–0.40 mL) of headspace using a pressure-lock syringe, and direct injection into a Shimadzu 8A gas chromatograph fitted with a 1.88 m × 3.18 mm (i.d.) stainless steel column packed with Carbosieve S-II (Sigma-Aldrich, St. Louis, MO, USA). The following chromatographic conditions were used: carrier gas, He; injector T, 120°C; oven T, 70°C; detector T, 120°C; detector type, thermal conductivity; detector current, 120 mA. External standard curves were used for quantification of H2 and CH4.
Culture pH was measured immediately after removal of the rubber stopper (to minimize alkalinization of medium that results from CO2 outgassing), using a Mettler-Toledo FiveEasy Plus pH meter calibrated with pH 4.01 and 7.00 buffers. Volatile fatty acids, nonvolatile acids (lactate, succinate) and ethanol in the culture liquid phase were determined by HPLC, as described previously (Weimer et al. 1991). For all gaseous and nongaseous products, net product formation was calculated after subtraction of products contained in substrate-free blank vials inoculated and incubated with the blank vials. Total enthalpy of combustion of products was calculated from enthalpies of combustion of individual end products (Weast 1969) at their measured net molar concentrations.
Total substrate consumption was calculated as initial dry weight of substrate minus neutral detergent fiber (NDF) residue (equivalent to plant cell wall residue). Residual NDF was determined gravimetrically by a modified Goering and Van Soest method (Weimer et al. 1990).
Statistical tests were performed using PROC MIXED in SAS, v.9.4 (SAS, Cary, NC, USA), using the model Yi = μ + Si + εi, where Yi = dependent variable; μ = overall mean; Si = effect of redox dye or its concentration; and εi = residual error. For analysis of data from the experiment conducted at different resazurin concentrations, the model Yi = μ + Si + Ri +SRij + εI was used, where Ri = resazurin concentration. For fermentations conducted at different neutral red concentrations, PROC REG was used for linear and quadratic regression analysis. Data are reported as least-square means. Means separation tests were conducted using the Tukey procedure. Significance was declared at P < 0.05, and trends identified at 0.05 < P < 0.10.