Animals and fungal isolation
Twenty white Tianzhu yaks aged 5–6 years with a body weight of 250 ± 20 kg, grazed on wild grass at Wushaoling (37,812–4790°N, 102,851–6950°E; research farm building at 3154 m above sea level) in the Tibetan Autonomous County of Tianzhu (Gansu Province, China) were randomly chosen for fungal isolation. The pasture is alpine meadow with Polygonum viviparum and Kobresia capillifolia as the main species. The use of animals and the protocol of rumen liquid collection were approved by local farms in the Tibetan Autonomous County and Animal Ethics Committees of Lanzhou University (Gashu, China). Fresh rumen fluids were collected from the rumen of each grazing yak through a stomach tube and were individually inoculated into anaerobic tubes filled with a mixture of 9.0 ml basal liquid medium and 100 mg air-dried chopped wheat straw which was autoclaved at 121 °C for 20 min, supplemented with 1600 IU/ml penicillin and 2000 IU/ml streptomycin. The basal liquid culture medium was modified from that described by Bauchop (1979) and consisted of following components (per liter): yeast extract, 1.0 g; tryptone, 1.0 g; NaHCO3, 7.0 g; resazurin 1 mg; l-cysteine hydrochloride, 1.7 g; the rumen fluid of yaks prior to feeding cleared by centrifugation at 10,000×g for 20 min at 4 °C, 170 ml; Salt solution I, 165 ml; Salt solution II, 165 ml; and distilled water added to adjust the volume to 1000 ml. Salt solution I contained 6 g/L NaCl, 3 g/L (NH4)2SO4, 3 g/L KH2PO4, 0.4 g/L CaCl2·2H2O, and 0.6 g/L MgSO4·2H2O. Salt solution II contained 4 g/L K2HPO4.
The anaerobic cultures were incubated at 39 °C and transferred every 4 days without shaking. After several rounds of subculturing, the cultures were serially diluted with the basal liquid medium under anaerobic condition and subsequently inoculated into Hungate roll-tubes that contained molten agar medium supplemented with 0.1% (w/v) glucose. The inoculated agar tubes were promptly rolled in ice water, followed by incubation at 39 °C. The single fungal colonies that formed after 2–3 days’ incubation were picked and inoculated into fresh anaerobic 0.1% (w/v) glucose liquid medium without wheat straw. The procedure was repeated several times between anaerobic liquid glucose medium without wheat straw and agar roll-tubes medium until the fungal colonies on the roll-tubes appeared uniform under the microscope. The cultures were subsequently individually examined for the production of CH4 by gas chromatography as described previously (Cheng et al. 2009) to ensure the presence of methanogen. Bacterial contamination in each culture was examined by PCR with the 968f/1401r primer pair (Su et al. 2008). All the cultures were maintained at 39 °C in anaerobic medium with 1% (w/v) wheat straw and transferred every 4 days.
Fungus and methanogen identification
The co-cultures incubated for 2–3 days in Hungate tubes in 0.1% glucose liquid medium without straw were observed under light microscope. The anaerobic fungi were identified according to the morphological features (Ho and Barr 1995). The co-cultures incubated in 0.1% glucose liquid medium without straw for 4 days were collected by centrifugation at 10,000×g at 4 °C for the extraction of the total genomic DNA. The internal transcribed spacer 1 (ITS1) of the anaerobic fungi was amplified with the primer Neo18S For and Neo5.8S Rev as previously described (Edwards et al. 2008). The related ITS1 sequences were deposited in GenBank under accession numbers: KP123392-KP123394. PCR-DGGE (denaturing gradient gel electrophoresis) analysis was used to evaluate the diversity of methanogens in each co-culture, using the 519f/915rGC primers as previously described (Cheng et al. 2009). To identify the methanogens in the co-cultures, the 16S rRNA genes of methanogens were PCR amplified using the Met86F and Met1340R primers (Wright and Pimm 2003) followed by direct sequencing (Beijing Genomics Institute, Beijing, China). The related sequences were deposited in GenBank under accession numbers: KP123413-KP123415. One of the identified (Piromyces + M. ruminantium) co-cultures, Yak-G18 co-culture, was selected for further characterization. The (Piromyces + M. ruminantium) Yak-G18 co-culture is available from the authors upon request.
Scanning electronic microscope
The co-cultures were grown at 39 °C for 72 h in the anaerobic medium containing 100 mg chopped wheat straw and were subsequently collected by centrifugation at 1000×g for 5 min. After being washed four times with phosphate-buffered saline (PBS), pH 7.2, the sediments were processed for electron microscopy as described previously (Wei et al. 2016b).
Experimental design and sampling
For each fermentation experiment, a total of 18 anaerobic bottles were used with each bottle starting with 45 ml basal medium supplemented with 1600 IU/ml penicillin and 2000 IU/ml streptomycin, 5 ml inoculum of co-cultures, and 500 mg of substrate as indicated. The substrates comprise wheat straw (ws), corn stalk (cs), rice straw (rs), Chinese wildrye (cw) and medicago sativa (ms). The fermentation was carried out at 39 °C for 7 days without shaking. During the 7-day incubation, three bottles were taken out every day and analyzed extensively. First, the total gas and methane in the headspace was determined. Then, the cultures were centrifuged at 5000×g for 10 min at 4 °C, the resulting supernatants were subject to the analysis of fibrolytic enzyme activities followed by a second centrifugation at 10,000×g for 5 min at 4 °C to yield the supernatants subjected to end-product profiling while the resulting pellets were collected for the analysis of IVDMD, NDF and ADF. The medium devoid of inoculum was used as the control. The fermentation experiment was repeated three times.
Fiber digestibility determination
According to AOAC (1999, ID 930.5), the sample in each bottle was centrifuged and dried at 105 °C for 24 h to determine the in vitro dry matter digestibility (IVDMD). Acid detergent fiber (ADF) and neural detergent fiber (NDF) were determined as described by Van Soest et al. (1991). Alpha amylase was not used, but sodium sulfite was added to each sample for NDF determination. IVDMD or ADFD or NDFD (%) = [(initial DM or ADF or NDF of feed taken for incubation − DM or ADF or NDF of residue at end of incubation)/(initial DM or ADF or NDF of feed taken for incubation)] × 100. The DM, ADF and NDF of dried residue from the uninoculated control were taken as initial DM, ADF and NDF, respectively.
Total gas production and CH4 measurement
The total gas production was measured using the pressure transducer technique described by Theodorou et al. (1994). CH4 was measured by gas chromatography according to Cheng et al. (2009).
Determination of enzyme activities
The supernatants were analyzed for the activities of xylanase, filter paperase (FPase), ferulic acid esterase (FAE), acetyl esterase (AE), and p-coumaric acid esterase (CAE). The supernatants were diluted 30-fold for the xylanase activity assay, and the xylanase activities were determined at pH 6.8 and 39 °C with substrate solution of 10 g/l birchwood xylan (Sigma, USA). The FPase activities were assayed using Whatman No. 1 filter paper, and detected by a spectrophotometer at 540 nm using the dinitrosalicylic acid method at pH 6.8 and 39 °C as previously described (Lowe et al. 1987). For determining the FAE activities, the supernatants were incubated with methyl ferulate in phosphate buffer solution (pH 6.8) and the absorbances were read with a microplate reader 680XR (Bio-rad, USA) at 340 nm. The FAE activities were subsequently calculated using standard ferulic acid and methyl ferulate curves according to the equation previously published (Yue et al. 2009). The AE activities were assayed in a 96-well reaction microplate by measuring the amount of p-nitrophenol released from 1.0 mM p-nitrophenyl acetate (Sigma, USA) at pH 6.8 and 39 °C with a micro plate reader 680XR at 415 nm. For assaying the CAE activities, 100 mg de-starched wheat bran were suspended in 1 ml 100 mM MOPS buffer at pH 6.8, followed by incubation with 1 ml supernatant at 39 °C for 20 min. The reaction was terminated by boiling the sample for 3 min, and the p-coumaric acid produced was measured by high-performance liquid chromatography (HPLC) (Waters, USA) comprising a Wufeng analytical instrument (Wufeng Co., Ltd, China) and a symmetry reversed-phase C18 column (250 × 4.6 mm, 5 µm, pH 2–8, Waters, USA), according to Wang et al. (2013). The CAE activities were subsequently calculated using the standard curve.
Determination of ferulic acid and p-coumaric acid releases
The levels of ferulic acid and p-coumaric acid in the fermentation supernatants were determined by the same method described above for assaying the FAE and CAE activities.
Analysis of fermentation end-products
One milliliter of supernatant was mixed with 0.3 ml of 250 g/l orthophosphoric acid solution in a polypropylene microtube. The mixture was cooled at 4 °C for 2 h, followed by centrifugation at 15,000×g for 10 min at 4 °C. The resulting supernatant was analyzed for acetate, propionate and butyrate levels with HPLC (Agilent 1200, USA) equipped with a ShodexRspak KC-811S-DVB gel col column (Waters, USA) and the SPD-M10AVP detector.
The lactic acid in the supernatant was detected by HPLC (Agilent 1200, USA) equipped with a lactic acid chiral separation column MCI GEL-CRS10W (Mitsubishi Chemical Holdings, Tokyo, Japan) and the SPD-M10AVP detector.
For assaying the succinate level in the supernatant, 5 μl of each sample was separated by ultra-performance liquid chromatography (UPLC) (Waters, USA), followed by measurement with Triple Quadrupole Mass Spectrometer (AB SICEX, USA).
Statistical analyses
Statistical analyses of the data were performed with SPSS 18.0 software (Microsoft) employing one-way ANOVA followed by Tukey test. All the data were expressed as mean ± SEM and p < 0.05 was regarded as statistically significant.