1. LITERATURE OVERVIEW
4.4 METNIN™ fractions in NaOH solution
4.4.3 Enzymatic treatment of 10-70 kDa fraction of HL
The same result was obtained with 10-70 kDa fractions as shown in figure 23. where 10-70 kDa fractions were tested with 1ml L371 laccase and the samples were taken at Start, S+E, 1 h,3 h, 5 h and 22 h which is used to compare the Mn, Mw and PDI.
Figure 23. Repolymerization of 10-70 kDa treated with L371 and analyzed in SEC at 280nm.
As shown in table 7 the measurement of Mn, Mw and PDI shows increase in molecular weight during the reaction referring to repolymerization of 10-70 kDa fractions.
0 0.005 0.01 0.015 0.02 0.025 0.03 0.035
10 100 1000 10000 100000
Area
Molecular Weight (Da) 10-70 (pH 10)
22 h Start
36
Table 7. Mn, Mw and PDI of 10-70 kDa treated with L371 and analyzed in SEC at 280 nm.
Time Mn (Da) Mw (Da) PDI
S 241 443 1.84
S+E 230 480 1.9
1H 235 500 1.9
3H 240 690 2
5H 365 800 2.2
22H 440 850 2.34
Similarly, L371 was tested with >3 kDa fractions. Results can be found in Appendix Figure 14 and the Mn, Mw and PDI can be found in Appendix Table 7.
37
5 SUMMARY
Lignocellulosic biomass is the main source of renewable chemicals. Biorefineries allow the fractionation of polysaccharides and hydrolysis lignin (HL), which can already be utilized in high value products. Biorefineries are presently focused on the valorization of sugars from polysaccharides, while neglecting the potential of HL. It is suggested that in the near future the value-added utilization of HL will be a game changer for the biorefinery idea. One pos-sible way for the valorization can be the treatment of HL by enzymes.
However, there are many barriers to using enzymes in lignin solutions. Many of them require acidic or near neutral pH, however lignin solubility in these solutions is very low. While there are some enzymes working in alkaline conditions, these are only used with low lignin concentration. For these reasons the application of enzymes on an industrial scale has re-mained elusive.
The goal of the present work was to investigate possibilities to use enzymatic oxidation to valorize biorefinery HL. For the first time, the treatment of high-purity industrially fraction-ated birch HL with enzymes was used in this work. Beside crude HL, three solubilized HL fractions and acid precipitated lignin was used. Multiple reaction conditions were tested in-cluding different buffers and NaOH solutions at specific pH values. Four different laccases as well as a peroxidase were tested in these conditions. All enzymes showed activity in most conditions for different HL fractions. The optimal enzyme treatment conditions were found, which encompass a range of pH values, high lignin solubility and enzyme activity. Although, when the enzymes showed activity the lignin repolymerized. This leads to new challenges, which require our attention in order to resolve the repolymerization issue. These first steps are still promising as the obtained knowledge in terms of the analysis and treatment methods, which allows us to further progress in this field. A positive result was the observation of enzyme activity in conditions with several times higher lignin content than previously de-scribed.
38
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Appendix
Figure 1. APL treated with ScLac at pH 6 and 8 in B&R buffer.
Figure 2. APL treated with AmLac and SvLac in Trsi-HCl buffer.
0
10 100 1000 10000 100000
Area
42
10 100 1000 10000 100000
Area
Molecular Weight (Da) APL(pH-10)
AmLac- pH10 C-pH10
c
43
Figure 4. APL treated with SvLac at three different pH. 6 , pH 8, pH 10 in B&R buffer.
Table 1. Mn, Mw and PDI values of APL treated with AmLac(red), SvLac(green), Sclac(blue) and Control (black) at pH 6(a), 8(b), 10(c) in B&R and Tris-HCl(d) buffers.
Laccases Buffer pH Mn
10 100 1000 10000 100000
Area
Molecular Weight (Da) APL(pH-10)
SvLac-pH10 C-pH10
44
Table 2. Mn, Mw and PDI values of HL treated AmLac(red), ScLac(green), Svlac(blue) and Control (black) at pH 6(a), 8(b), 10(c) in B&R and Tris-HCl(d) buffers.
Laccases Buffer pH Mn
(Da)
Mw
(Da) PDI
AmLac B&R
6 213 728 3.4
8 244 691 2.8
10 268 856 3.1
Tris-HCl 7.75 241 665 2.7
ScLac B&R
6 241 1624 6.7
8 245 1205 4.9
10 265 1322 4.9 Tris-HCl 7.75 253 1594 6.3
SvLac B&R
6 232 1020 4.4
8 238 950 3.9
10 258 958 3.7
Tris-HCl 7.75 246 871 3.5
Control B&R
6 192 469 2.4
8 208 437 2.0
10 215 492 2.2
Tris-HCl 7.75 211 448 2.1
45
Figure 5. HL treated with SvLac at three different pH. 6 , pH 8, pH 10 in B&R buffer.
Figure 6. HL treated with ScLac at two different pH. 6 , pH 8 in B&R buffer.
0
46
Figure 7.HL treated with AmLac and SvLac in Tris-HCl buffer.
Figure 8. De- and Repolymerization of HL with DYP enzyme in B&R buffer
Table 3. Mn, Mw and PDI values of HL treated with ScLac (blueat pH 6, 8, 10 in B&R and Tris-HCl buffers.
47
Figure 9. APL with Sclac in 1% NaOH solution at pH 11(a) and 10(b).
Figure 10. HL with ScLac in 1% NaOH solution at pH 11(a) and 10(b).
0
10 100 1000 10000 100000
Area
10 100 1000 10000 100000
Area
10 100 1000 10000 100000
Area
10 100 1000 10000 100000
Area
10 100 1000 10000 100000
Area
10 100 1000 10000 100000
Area
Molecular Weight (Da) APL(pH-10)
APL(0.4%)_10 N(0.4%)_10
b
48
Figure 11. APL with ScLac in 0.4% NaOH solution at pH 11(a) and 10(b).
Figure 12. HL with Sclac in 0.4% NaOH solution at pH 11(a) and 10(b).
Table 4. Mn, Mw and PDI values of APL and HL with ScLac at 1% and 0.4% NaOH solu-tion where APL(red), HL(green) and Control (black) at pH 12, 11, 10 and 9.
Lignin Solution pH Mn
10 100 1000 10000 100000
Area
10 100 1000 10000 100000
Area
Molecular Weight (Da) HL(pH-10)
Crude(0.4%)_10 N(0.4%)_10
b
49
Table 5. Mn, Mw and PDI values of HL(red) and APL(blue) in presence of mediator with Dyp in B&R (pH 6, 8, 10), NaOH solution and control (black).
Lignin
Table 6. Mn, Mw and PDI values of HL in NaOH solution.
Time Mn(Da) Mw(Da) PDI
Figure 13. HL treated with L371 in NaOH solution at pH 10.
0
10 100 1000 10000 100000
Area
Molecular Weight (Da) HL(pH-10)
22h Start
50
Figure 14. >3kDa fractions treated with L371 in NaOH solution at pH 10.
Table 7. Mn, Mw and PDI values treated >3kDa with L371 fractions in NaOH solution at pH 10.
Time Mn (Da) Mw (Da) PDI
S 148 218 1.47
S+E 150 220 1.5
1H 150 230 1.5
3H 160 250 1.6
5H 160 280 1.7
22H 300 290 1.8
0 0.005 0.01 0.015 0.02 0.025 0.03 0.035
10 100 1000 10000 100000
Area
Molecular Weight (Da)
>3 kDa (pH 9)
22 h Start
b
51
NON-EXCLUSIVE LICENCE
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20/05/2021