Acetic anhydride (4 mL) was added a

Acetic anhydride (4 mL) was added and the solution was shaken
again. Then 1-methylimidazole (600 lL) was added. After mixing,
the sample was allowed to stand for 10 min, and excess acetic
anhydride was decomposed by adding distilled water (10 mL).
Then dichloromethane (3 mL) was added, mixed gently, and
centrifuged (2000g, 10 s) for phase separation. After removing
the upper phase, the lower phase was washed with distilled
water (3  20.0 mL). The collected lower phase was dehydrated
by adding anhydrous sodium sulfate and stored at 20 C until
analysis by GC–MS (SHIMADZU GCMS-QP2010 Plus).
GC–MS Analytical Conditions: Restek Rxi-5 ms, 30 m 
0.25 mm ID  0.25 lm df column. Carrier gas: He. Injection
method: Split. Injection port: 250 C, interface: 250 C. Injection
volume: 1.0 lL. The temperature program: from 170 C (held for
12 min) to 220 C (held for 8 min) at 3 C/min. Ion source temperature:
200 C, ACQ mode: SIM. The mass spectrometer was operated
in the EI mode with ionization energy of 70 ev. Mass spectra
were acquired with full scans based on the temperature program
from 50 to 500 m/z in 0.45 s. Calibration curves of all analytes
routinely yielded correlation coefficients 0.999 or better.
2.6. Detection of total lignin and monolignin composition
Total lignin level of biomass samples was detected by two-step
acid hydrolysis method according to analytical procedure of the
National Renewable Energy Laboratory with minor modification
by Wu et al. (2013). The acid-insoluble lignin was calculated gravimetrically
after correction for ash, and the acid-soluble lignin was
measured using UV spectroscopy. All experiments were carried out
in biological triplicates.
Monolignin determination was described by Li et al. (2013) and
Wu et al. (2013) with minor modification. Standard chemicals:
p-Hydroxybenzaldehyde (H), vanillin (G) and syringaldehyde (S)
were purchased from Sinopharm Chemical Reagent Co., Ltd. After
removal of soluble sugars from biomass sample, the bagasse was
extracted with benzene-ethanol (2:1, v/v) in a Soxhlet for 4 h,
and the remaining pellet was collected as cell wall residue
(CWR). The supernatants from 1% NaOH and 1% H2SO4 pretreatments
as described below, were neutralized with HCl and NaOH
respectively, and then mixed with 10 mL 4 M NaOH as wall
pretreatment supernatant (WPS). About 0.05 g CWR was added
with 5 mL 2 M NaOH and 0.5 mL nitrobenzene, and a stir bar was
put into a 25 mL Teflon gasket in a stainless steel bomb for
monolignin detection of CWR. 10 mL WPS was added with 1 mL
nitrobenzene, and a stir bar was put into a 25 mL Teflon gasket
in a stainless steel bomb for WPS detection.
The bomb was tightly sealed and heated at 170 C (oil bath) for
3.5 h and stirred at 20 rpm. The bomb was then cooled with cold
water. The chromatographic internal standard (ethyl vanillin)
was added to the oxidation mixture. The alkaline oxidation mixture
was washed 3 times with 30 mL CH2Cl2/ethyl acetate mixture
(1/1, v/v) to remove nitrobenzene and its reduction by-products.
The alkaline solution was acidified to pH 3.0–4.0 with 6 M HCl,
and then extracted with CH2Cl2/ethyl acetate (3  30 mL) to obtain
the lignin oxidation products which were in the organic phase. The
organic extracts were evaporated to dryness under reduced pressure
(20 mm Hg) at 40 C. The oxidation products were dissolved
in 10 mL chromatographic-grade pure methanol.
HPLC analysis: The solution was filtered with membrane filter
(0.22 lm). 20 lL solution was injected into HPLC (Waters 1525
HPLC) column Kromat Universil C18 (4.6 mm  250 mm, 5 lm)
operating at 28 C with CH3OH:H2O:HAc (25:74:1, v/v/v) carrier
liquid (flow rate: 1.1 mL/min). Calibration curves of all analytes
routinely yielded correlation coefficients 0.999 or better, and the
detection of the compounds was carried out with a UV-detector
at 280 nm.