The use of cellulose solvents is considered the most facile method to disrupt highly order hydrogen bonding of crystalline cellulose structure, increasing surface accessibility to enzymes, facilitating hydrolysis.
Acknowledgement
5 FPUs + 10 units β-glucosidase
Enzymatic hydrolysis of corn stover (CS)
Avicel + H3PO4 RACCOSLIF
Avicel + IL RACIL
Why cellulose solvents?
SummaryWe compared two cellulose solvent-based lignocellulose pretreatments: COSLIF based on concentrated phosphoric acid and ionic liquid (IL) based on [BMIM]Cl. Enzymatic glucan digestibilities of COSLIF- and IL-pretreated corn stover were 96% and 52% by 5 filter paper units of cellulase per gram of glucan at hour 72, respectively. Regenerated amorphous (pure) cellulose from Avicel by COSLIF and IL had digestibilities of 100% and 92%, respectively. Our systematic experiments suggested that these large differences in enzymatic glucan digestibility of corn stover and Avicel pretreated by two cellulose solvents were attributed to the combinatory causes: (i) IL did not pretreat cellulose as efficiently as COSLIF based on enzymatic hydrolysis and cellulose accessibility to cellulase; (ii) the residual ionic liquid in the pretreated biomass inhibited cellulase activity; and (iii) the residual lignin on the cellulosic materials after the pretreatments decreased cellulase hydrolysis performance.
Fig. 3. Hydrolysis of Avicel and RAC
Fig. 2. Hydrolysis of corn stover
Intact CS showed fibril structure (Fig. 2B), but COSLIF- and IL-pretreated CS did not show any fibril structure. CSCOSLIF was hydrolyzed fast, and glucan digestibilities reached 93% at hour 72, while CSIL reached 55%.
What caused such a large difference?
(C)
(B)
before after
before after
2nd Hypothesis: IL inhibition
(B) Intact (C) CSCOSLIF (D) CSIL
0 12 24 36 48 60 720
20
40
60
80
100
Glu
can
dige
stib
ility
(%)
Time (h)
CSCOSLIF
CSIL
untreated (15 FPU)
(A)
0 12 24 36 48 60 720
20
40
60
80
100
Glu
can
dige
stib
ility
(%)
Time (h)
RACCOSLIF
RACIL
Avicel (15 FPU)
(A)
0 15 30 45 60 750
20
40
60
80
100
Glu
can
dige
stib
ility
(%)
Time (h)
0 g/L 1 gL 2 g/L 5 g/L 10 g/L
(A) RACCOSLIF
0 12 24 36 48 60 720
20
40
60
80
100
0 g/L 1 g/L 2 g/L 5 g/L 10 g/L
Glu
can
dige
stib
ility
(%)
Time (h)
(B) Avicel
0 12 24 36 48 60 72
10
100 0 g/L 1 g/L 2 g/L 5 g/L 10 g/L
(D) Avicel
Nor
mal
ized
hyd
roly
sis
rate
(%)
Time (h)0 12 24 36 48 60 72
10
100 0 g/L 1 g/L 2 g/L 5 g/L 10 g/L
(C) RACCOSLIF
Nor
mal
ized
hyd
roly
sis
rate
(%)
Time (h)
Fig. 4. Influence of [BMIM]Cl on hydrolysis of RAC and Avicel
1st Hypothesis: low pretreatment efficiency
3rd Hypothesis: competitive lignin adsorption
Material TSACm2/g biomass
CACm2/g biomass
NCACm2/g biomass
Pure substrate AvicelRACCOSLIFRACIL
2.3 ± 0.119.1 ± 1.3
NDLignocellulose Corn stover (CS)
CSCOSLIFCSILCSDA
1.13 ± 0.0114.4 ± 1.15.8 ±0.37.7 ± 0.6
0.42 ± 0.0111.6 ± 0.85.0 ± 0.25.9 ± 0.3
0.71 ± 0.012.9 ± 0.2
0.73 ± 0.091.8 ± 0.6
Avicel can be completely dissolved in both cellulose solvents. Cellulose solutions appeared to be transparent (Figs. 3B & C). But RACCOSLIF yielded higher glucan digestibility than RACIL.
It was difficult to wash IL from RACIL. ~0.1-1.0 g [BMIM]Cl remained on RACIL. It was found that a small amount of [BMIM]Cl decreased hydrolysis rate and glucan conversion. Instantaneous hydrolysis rates of RAC and Avicel decreased drastically over time.
0 12 24 36 48 60 720
20
40
60
80
100
Glu
can
dige
stib
ility
(%)
Time (h)
RACIL
RACIL + lignin RACIL + ligninIL
Avicel Avicel + lignin
(B) IL
0 12 24 36 48 60 720
20
40
60
80
100
Glu
can
dige
stib
ility
(%)
Time (h)
RACCOSLIF
RACCOSLIF + lignin RACCOSLIF + ligninCOSLIF
Avicel Avicel + lignin
(A) COSLIF
Fig. 5. Effect of isolated lignin on cellulose hydrolysis
0 12 24 36 48 60 720
20
40
60
80
100
Glu
can
dige
stib
ility
(%)
Time (h)
RACCOSLIF
(Avicel + lignin)COSLIF
RACCOSLIF + ligninCOSLIF
(A) COSLIF
0 12 24 36 48 60 720
20
40
60
80
100
Glu
can
dige
stib
ility
(%)
Time (h)
RACIL
(Avicel + lignin)IL
RACIL + ligninIL
(B) IL
4th Hypothesis: lignin redistribution
Fig. 6. Effects of lignin redistribution
Overview of COSLIF pretreatment procedure using PA and IL (BMIMCl)
Precipitation Tank
ethanol
Washer -1
BlackLiquor
Washer -2
LightLiquor
Water
lignin
Cornstover
Digester
cellulosesolvent
CSTR
amorphous cellulosic material
ethanol
water-soluble hemicellulose
ethanolethanol
cellulosesolvent
Fig. 1. Cellulose solvent-based lignocellulose pretrement procedueFig. 1. Cellulose solvent-based lignocellulose pretrement procedue
H3PO4
The negative effects of lignin addition were larger on RACCOSLIFthan RACIL.
Lignin can redistribute on the surface of cellulose during pretreatment, which can block cellulose accessibility. We mixed lignin and Avicel prior to COSLIF and IL. there were no significant differences in hydrolysis rates and digestibilities between (Avicel+lignin)COSLIF and (Avicel+lignin)IL.
Both H3PO4 and [BMIM]Cl can dissolve cellulose and corn stover by disrupting ordered hydrogen bonds in cellulose fibers. A large difference in glucan digestibilities of CSCOSLIF (96%) and CSIL (53%) was attributed to the combinatory effects: (1) IL did not pretreat cellulose as efficient as COSLIF, (2) residual IL inhibited cellulase activity, and (3) residual lignin on pretreated substrates decreased hydrolysis performance. But it was not due to lignin redistribution on the surface of cellulose.
Cellulose accessibility to cellulase (CAC) was measured by a fusion protein, TGC. CSCOSLIF had the highest CAC value of 11.6 m2/g biomass compared to those pretreated by IL and dilute acid (DA). At an enzyme loading of 5 FPU/g glucan, the enzymatic digestibilities increased as follows: COSLIF (96%) > DA (60%) > IL (53%) at hour 72, which is in good agreement with the CAC values in Table I.
Discussion
Table I. Surface accessibility assay
Promisingcandidates
Noppadon Sathitsuksanoh, Zhiguang Zhu, and Percival Zhang*Biological Systems Engineering Department, Virginia Tech, Blacksburg, VA, [email protected]
Cellulose solvent-based lignocellulose pretreatment comparison: Concentrated phosphoric acid vs Ionic liquid
Top Related