3.1 Research framework
Fig 5. Research framework
3.2 Raw material and analysis
The aboveground samples of M. micrantha were collected in January 2009 at Lu-Ku township, Nan-Tou county, central Taiwan. M. micrantha samples were collected from a low-elevation site in Lu-Ku, Nantou county, central Taiwan. Samples were air dried, milled to collect portions 40 and 60 mesh screen, and homogenized to a single lot for further experiments. The average composition of materials determined according to standard methodology: moisture content (CNS 452), ash content (CNS 3084), alcohol-toluene solubility (CNS 4713), holocellulose (CNS 3085), pentosan (CNS 7749), Klason lignin (CNS 14907)..
3.3 Pretreatments of M. mikania samples
Milled M. mikania powders were extracted with a 2:1 (v/v) toluene-ethanol mixture using a soxhlet extractor. Solvent extracted Mikania powders (250 g air-dried powder in 2.5 L solution) were further extracted, using an Erlenmeyer flask, with 12%
(w/v) NaOH solution at 150 rpm, 60 oC for 16 h. The extracts were centrifuged for 15
min at 12000 rpm, 4oCin a high-speed refrigerated centrifuge (HITACHI, HIMAC CR21GⅡ). After the treatments, the residue was centrifuged after washing one time
with distilled water, air-dried to ensure quantitative non-extracted xylan by pentosan analysis. The supernatants above were mixed and neutralized with HCl to a final pH 7.
3.4 Extraction of M. micrantha xylan (MX) from various treatments
Three different treatments have been used for MX extraction:
a. Ethanol- precipitation (EMX)
The pH of the supernatant was adjusted to 6 by adding drops of 1N HCl. The above mixture was allowed to settle for one day at room temperature with 1.5 times of 95% (w/w) ethanol. Then the precipitate was collected after centrifugation at 12,000 rpm, 4oC for 15 min. Repeat washed the precipitate with 70 and 95% ethanol for twice. The collected precipitate was vacuum-dried at room temperature. The dried solids were defined as EMX and stored in 4oC.
b. Nanofiltration (NMX)
A nanofiltration process was employed to concentrate and to remove the mineral salts in the raw MX solutions. The pH of the supernatant was adjusted to 7 by adding drops of 1N HCl, and then concentrated MX and removed NaCl by nanofiltration. Each experiment filtration run was setup with a pressure gauge before the filtration unit and a back pressure regulator after the filtration unit to fine tune the trans-membrane pressure. Membrane filtration runs were conducted in a batch recirculation mode. Influent reservoir, containing raw MX suspension in a beaker, was constantly mixed with a magnetic stirrer. One filtration run with 10 kD MWCO
ultrafiltration membrane and four runs with thin-film composite nanofiltration membrane were conducted. Four TFC membrane runs were conducted using same membrane without change. Permeate flux was monitored using a volumetric cylinder. A filtration run was terminated after steady-state fluxes was reached. The concentrated MX liquid was vacuum-dried at room temperature. Final obtained solid was defined as NMX.
c. Ethanol- precipitation and ultrafiltration (EUMX)
This method was combining the above-mentioned ways. The pH of the supernatant was adjusted to 6 by adding drops of 1N HCl. The mixture was allowed to settle out for a day at room temperature with 1.5 times of 95% ethanol. Instead of precipitation and centrifugation steps, the mixture was subjected to staged-ultrafiltration processes to concentrate and to remove NaCl. The set-up and operation principles were the same as described in the previous section. Successive filtration runs with 5, 10, and 30 kD MWCO ultrafiltration membranes were conducted. Membranes with larger pore size were used in later stages to accelerate separation processes. Four consecutive 5 kD membrane runs were conducted using the same membrane without change. The final concentrated MX liquor was vacuum-dried at room temperature. The obtained solid was defined as EUMX.
Extracted NMX and EUMX were used membrane filtration to remove out NaCl
and low molecular weight saccharides. Na+ was detected on an Ion Chromatography;
total sugar recovery was measured by total sugars assay. The pentosan contents of EMX,
NMX and EUMX were determined according to the method provided by CNS 7749.
3.5 Membrane filtration
Fig. 6 was the schematics of a cross-flow filtration module and the filtration cell. A gear pump: Model 75211-10, 50-5000 rpm, 0.07 HP, Cole-Parmer Instrument Company.
The way to filtrate effluent is cross-flow filtration and filtration area is 0.0084 m2, room temperature, pH 6-7, operation pressure is 45 psi. Table 6 was the specification of membranes which has been used in this experiment.
Fig 6. The schematics of a cross-flow filtration module and the filtration cell.
Table 6. The specification of membranes Material Operation pH
3.6 Xylan Hydrolysis
Hydrolysis of xylan was conducted by mixing 1 mL of 50.0 units/mL of xylanase from Paenibacillus sp. isolate BL11 with birchwood xylan, EMX, NMX and EUMX which pentosan contents content were 0.9% in 0.1 M tris-buffer, pH 7, and allowing the incubation to proceed at 60oC and shake (150 rpm) for 24 h.
In order to optimize enzyme concentration and reaction time on production of XOs, the reaction was performed using the method described above with different enzyme dosage (10, 50, 100 U/mL) and reaction time (in the range of 0-96 h); the substrate concentration was using 1% birchwood xylan (pentosan content: 0.9%) to substitute for three M. micrantha xylan (EMX, NMX, EUMX). At 0, 1, 2, 3, 6, 12, 24, 48, 72 and 96 h, a 1 mL sample was taken at regular interval and heated to 100oC for 5 min to inactivate the enzyme, centrifuged (10 min, 3500 rpm) analyzed by the HPLC method.
3.7 HPLC separation of XOs
XOs were chromatographed on a (Transgnomic) HPLC system equipped with a
UV detector (RI-810) and column oven (Col Box). Before injection, samples were filtered through a 0.45 μm filter. Aliquots of filtered sample (20 μl) were injected onto the HPLC system. XOs were eluted using 0.0085 N H2SO4 as the mobile phase. The column (300 mm × 7.8 mm) (ICSep ICE-ION-300) was used at 70oC and a flow rate of 0.4 mL/min. A complete analysis of XOs was carried out in 30 min.
3.8 Total sugars assay
Phenol, in a 5% (w/v) solution is added to a glass test tube containing a clear sample solution. Concentrated sulfuric acid is added in a rapid stream directly to the surface of the liquid in the test tube. The mixture is thoroughly combined using a vortex mixture and then permitted to stand a sufficient time to allow for color development.
The solution absorbance is read at 480 nm using a spectrophotometer, depending on the type of sugar present. Mixing and standing time should be kept the same for all samples to assure reproducible results (Dubois et al., 1956).
3.9 DNSA (dinitrosalicylic acid) assay
The simple, robust and highly reproducible method according to König et al. was
used to estimate the activity of xylanase, β-glucanase, and cellulose. The method is based on measurement of reducing sugars released from xylan, β-glucan, or carboxymethylcellulose (CMC) by the enzymes digestion. Xylanase activity was
measured by the increase in reducing sugars, as given by the dinitrosalicylic acid method using xylose as a standard. One unit of xylanase activity was defined as the amount of enzyme producing 1 mol of reducing equivalents per min under the assay conditions. The effects of temperature and pH on the stability of the recombinant enzymes activity were determined.
Reducing sugar that resulted from the enzymatic reaction was determined by measuring
the absorbance at 540 nm using xylose as the standard after the reaction mixture cooled down. One unit (IU) is defined as the amount of enzyme that releases 1 μmol of reducing sugar per min. Enzyme was tested for its ability to hydrolyze the variety of substrates, including 1.5% birchwood xylan (Sigma X0502). For the pH and temperature, the enzyme activity was assayed in reaction Buffers: 0.1 M Tris buffer adjust to pH 7, 20 mM CaCl2 (E. Merck TA144482), 0.04% (v/v) Tween 20 (Bio Basic D0560) under temperatures 60oC at 20 min.
3.10 Ion Chromatography
Na+ was detected on a Ion Chromatography (DKK-TOA, IA-300) (IC) analyzer.
Before injection, samples were filtered through a 0.45 μm filter. Aliquots of filtered sample (20 μL) were injected onto the IC system. Na+ was eluted using 6 mM methanesulfonic acid as the mobile phase. The column (250 mm × 4.6 mm) (PCI-322)
was used at 40oC and a flow rate of 0.8 mL/min. A complete analysis of Na+ was carried out in 18 min.