Lignin-First Approach to Biorefining: Utilizing Fenton's Reagent and

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A Lignin-First Approach to Biorefining: Utilizing Fenton’s Reagent and Supercritical Ethanol for the Production of Phenolics and Sugars William J. Sagues, Hanxi Bao, John L Nemenyi, and Zhaohui Tong ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.7b04500 • Publication Date (Web): 13 Feb 2018 Downloaded from http://pubs.acs.org on February 15, 2018

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ACS Sustainable Chemistry & Engineering

1

A Lignin-First Approach to Biorefining: Utilizing

2

Fenton’s Reagent and Supercritical Ethanol for the

3

Production of Phenolics and Sugars

4

William J. Sagues, Hanxi Bao, John L. Nemenyi, Zhaohui Tong*

5

Department of Agricultural & Biological Engineering, University of Florida, 1741 Museum Rd

6

Gainesville, Florida 32611, United States

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*Corresponding Author

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[email protected]

9

KEYWORDS

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Biomass, Lignin depolymerization, Pretreatment, Phenolic monomers, Carbohydrates

11

ABSTRACT

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Selective lignin depolymerization (SLD) has emerged as a value-added method of pretreatment

13

for lignocellulosic biorefining, in which lignin is depolymerized into valuable phenolic

14

monomers prior to utilization of the hemicellulose and cellulose. Herein, we report a biomimetic

15

Fenton catalyzed SLD process, converting sweet sorghum bagasse into an organic oil that is rich

16

in phenolic monomers and a solid carbohydrate that is favorable for enzymatic hydrolysis into

17

sugars. Initially, the feedstock’s molecular structure was modified through iron chelation and

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free radical oxidation via Fenton’s reagent (Fe3+, H2O2 ). The lignin component of the modified

2

feedstock was then selectively depolymerized in supercritical ethanol (250°C, 6.5MPa) under

3

nitrogen to produce a phenolic oil, with a maximum yield of 75.8 wt %. Six valuable phenolic

4

monomers were detected in this oil, with a maximum cumulative yield of 19.1 wt %. The solid

5

carbohydrate obtained after the SLD process was enzymatically hydrolyzed to liberate 62.7 wt %

6

and 79.9 wt % of the initial 5-carbon and 6-carbon polysaccharides within 24 hours, respectively,

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indicating the majority of the hemicellulose and cellulose were preserved during the SLD

8

process. Fenton modification not only increased the yields of phenolic monomers, particularly

9

ethyl-p-coumarate and ethyl-ferulate, but also enhanced enzymatic hydrolysis.

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INTRODUCTION

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In a traditional biochemical approach to lignocellulosic biorefining, the biomass feedstock

12

undergoes a form of energy-intensive pretreatment to partially destruct the cell wall matrix

13

comprised of lignin (15-30%), hemicellulose (25-30%), and cellulose (35-50%). Then, the

14

holocellulose (hemicellulose and cellulose) is enzymatically converted into sugars, leaving lignin

15

as a waste product.1 Lignin is the most abundant renewable aromatic polymer on the planet, and

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it is of particular interest to apply lignin-derived phenolic monomers in high value applications

17

such as antioxidant supplements, UV scavenging sunscreens, anti-inflammatory medications,

18

flavors, and fragrances.2,3,4 Valuable polymers can also be synthesized from lignin-derived

19

monomers, such as plastics and resins2. The majority of lignin residues are termed “technical

20

lignins”, due to the undesirable condensation reactions during pretreatment and other unit

21

operations in which naturally present carbon-oxygen bonds (C-O) are cleaved, followed by rapid

22

carbon-carbon (C-C) bond formation. Technical lignins are difficult to depolymerize due to the

23

high abundance of C-C bonds, which have a higher bond-dissociation energy than C-O bonds.5,6


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There exist many methods for depolymerization of various technical lignins using organic

2

solvents, such as methanol and ethanol, with or without catalysts, and at temperatures ranging

3

from 200 – 380 °C.7,8,9,10,11 However, there are relatively few methods that selectively

4

depolymerize native lignin from intact biomass. The selective lignin depolymerization (SLD)

5

method, also termed as early-stage catalytic conversion of lignin (ECCL), is a “lignin-first”

6

approach to biorefining in which lignin is selectively depolymerized into valuable phenolic

7

monomers as a first step, without degrading the holocellulose.5 Zhai et al. (2017) developed a

8

SLD process that obtained a 39.5 wt % yield (relative to initial lignin content) of phenolic

9

monomers from birch using a bimetallic Ni-Fe/activated carbon catalyst in the presence of

10

hydrogen (2MPa H2 at ambient temperature) and methanol at 200 °C.12 Notably, Zhai et al.’s

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process obtained a significantly lower yield (20.3 wt %) when using organosolv lignin instead of

12

birch, demonstrating the significant advantages of the lignin-first SLD approach. Van den Bosch

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et al. (2017) demonstrated a SLD process using a Ni-Al2O3 catalyst in the presence of hydrogen

14

(3MPa H2 at ambient temperature) and methanol at 250 °C to selectively depolymerize the lignin

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component of birch reaching total phenolic monomer yields of 44 wt %.13 Parsell et al. (2015)

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demonstrated a SLD process using a Zn/Pd/C catalyst in the presence of hydrogen (3.45 MPa H2

17

at ambient temperature) and methanol at 225 °C to selectively depolymerize the lignin

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component of several feedstocks into two phenolic monomers with yields ranging from 19 – 54

19

wt %.4 The aforementioned SLD processes that produced high yields of phenolic monomers

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from whole biomass involved costly operations such as high initial pressures of hydrogen gas,

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rare metal catalysts, or long reaction times.

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In this research, we demonstrated the ability of Fenton’s reagent (Fe3+, H2O2) to catalyze the

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selective lignin depolymerization of sweet sorghum bagasse in supercritical ethanol under an


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inert (N2) atmosphere. Fenton’s reagent has been proven to be an effective low-cost, earth-

2

abundant, and biomimetic catalyst for the oxidation of organic polymers, such as lignin.11,14

3

Ethanol was chosen as the solvent because it is classified as a green solvent and has beneficial

4

physiochemical properties at the supercritical state.15,16 Our previous work used Fenton’s reagent

5

to modify organosolv hardwood lignin prior to its depolymerization in ethanol at 250°C.11 In this

6

study, we hypothesized the low-cost Fenton’s reagent coupled with supercritical ethanol could

7

selectively depolymerize lignin from the intact cell wall of whole biomass through the cleavage

8

of the alkyl-aryl ether β-O-4 (C-O) bond, the most prevalent bond in native lignins. Due to the

9

high abundance of β-O-4 bonds in native lignins and low abundance in technical lignins, we

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expected this SLD process for whole biomass to be more effective than our previous study in

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which organosolv lignin (a technical lignin) was used. In addition, we hypothesized the SLD

12

process could preserve the holocellulose for further enzymatic hydrolysis. Sweet sorghum

13

bagasse was chosen as the feedstock because it is a C4 grass that contains a high abundance of

14

esterified ferulate and p-coumarate end groups (hydroxycinnamates) in its lignin fragments.17

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Therefore, the opportunity exists for the monomerization of ferulic and p-coumaric acids and

16

related derivatives as target high value monomers for human nutrition applications.17,18 For the

17

first time, a comparatively mild selective lignin depolymerization process was demonstrated in

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which Fenton’s reagent catalyzed the conversion of native lignin into low molecular weight oils

19

rich in phenolic monomers, as well as the conversion of holocellulose into free 5- and 6-carbon

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sugars. The lignin-first process presented herein fills the aforementioned gaps in knowledge by

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using an inert atmosphere, earth abundant catalyst, and relatively short reaction time.

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EXPERIMENTAL

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Materials


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The sweet sorghum was grown and harvested by Agricenter LLC in Memphis, TN. After

2

juicing via an industrial roller press, the bagasse was washed to remove soluble sugars and stored

3

dry. Prior to experimentation, the bagasse was sieved through mesh size 20 to ensure uniform

4

particle size. Compositional analysis of the sweet sorghum bagasse was conducted according to

5

NREL’s analytical procedure (see Table S1). Iron (III) nitrate, citric acid monohydrate, and

6

trisodium citrate dihydrate were purchased from Fisher. Aqueous hydrogen peroxide (35 wt %)

7

and 4-vinylphenol (10 wt % in propylene glycol) were purchased from Sigma-Aldrich.

8

Anhydrous ethanol was purchased from Decon Labs. 4-vinylguaiacol, isoeugenol, and ethyl

9

ferulate were purchased from Sigma-Aldrich, and ethyl-p-coumarate was purchased from Ark

10

Pharm, all in high purity. Novozyme Cellic® CTec 3 enzyme solution was generously provided

11

by Novozymes®.

12

Fenton Modification (FM)

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In a general reaction, the bagasse underwent a Fenton modification (FM) in which a 50 mL

14

solution was prepared in ethanol with 2.5% biomass (w/v solution), 2.5% Fe3+ (w/w biomass),

15

and either 5 or 20% hydrogen peroxide (w/w biomass). The biomass was milled and extractives

16

were removed using the same method for biomass compositional analysis (see SI for details).

17

The solution was vigorously mixed at 60 °C for 1h. For the control experiments, neither Fe3+ or

18

hydrogen peroxide was added. The Fe3+ loading was chosen to be in excess for this study since

19

the cost of hydrogen peroxide contributes significantly in techno-economic analyses of organic

20

oxidation processes that use Fenton’s reagent.14 After mixing for 1h, the solids were separated

21

from the solution via vacuum filtration, washed with deionized water, and dried.

22

Selective Lignin Depolymerization (SLD)


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A 100 mL Parr reactor was used to prepare a 50 mL solution in ethanol with 2.5% Fenton

2

modified biomass (w/v solution). The reactor was purged with nitrogen gas at 0.7 MPa prior to

3

raising the temperature to 250°C (~ 6.5°C /min) at autogenous pressure (6.5MPa), under 700 rpm

4

agitation rate, and holding for either 0.5, 1, 3, or 12h. Upon completion, the reactor was cooled to

5

room temperature within 30 min using a custom-built air cooling device. The solids were

6

separated via vacuum filtration and washed with excess ethyl acetate, which was collected as

7

filtrate. The liquid filtrate then underwent a threefold liquid-liquid extraction using deionized

8

water to separate the organic and aqueous fractions. The organic fraction was dried with

9

magnesium sulfate prior to extended vacuum evaporation to obtain the “phenolic oil”.

10

Depolymerized Lignin Characterization

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The phenolic oil yields were calculated on a mass basis using Equation 1. The lignin mass used

12

for all yield calculations refers to the initial Klason lignin content of the sweet sorghum bagasse.

13

The phenolic monomers present in the phenolic oil were analyzed by gas chromatography-mass

14

spectroscopy (GC-MS, GC - Agilent 7820 and MS - Agilent 5977). Six phenolic monomers

15

present in the phenolic oil were chosen for analysis due to their relatively high concentrations

16

and valuable applications. The yields of phenolic monomers were calculated using Equation 2.

17

See Figures S1-S9 for more details regarding monomer quantification and qualification, as well

18

as statistical analyses (Table S5).

19

ℎ =

20

6 =

21

The weight average (Mw) molecular weights of the phenolic oils were determined by gel

22

permeation chromatography (GPC) on an Agilent 1260 infinity HPLC system with a refractive

23

index detector (RI-G1362A) and a multiple wavelength detector (MWD). Heteronuclear single

∑$ # "#

(1) (2)


6

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1

quantum coherence (HSQC) nuclear magnetic resonance (NMR) spectra for the phenolic oil

2

were generated using a Bruker Avance II 600 MHz equipped with a 5mm TXI cryoprobe at

3

27°C.

4

Residual Holocellulose Characterization

5

To characterize the holocellulose (hemicellulose and cellulose), the “solid carbohydrate”

6

retained from the SLD process underwent enzymatic hydrolysis. The enzymatic hydrolysis

7

aimed to quantify the polysaccharides available for liberation to free sugars after lignin

8

depolymerization. A 20 mL solution was prepared in citrate buffer (pH ~5.0) with 1 wt % solid

9

carbohydrate and 0.5 wt % Novozyme Cellic® CTec 3 enzyme solution. Each experiment was

10

maintained at 50 °C with moderate mixing for 24h. The 5- and 6-carbon sugars liberated during

11

enzymatic hydrolysis were quantified and then used to calculate the percentage of functional

12

holocellulose retained, as shown in Equation 3. The 5-carbon sugars that were quantified include

13

xylose and arabinose, and the 6-carbon sugars include glucose and galactose. The monomeric

14

sugars were analyzed according to our previous report24. % ℎ& '( =

15

∑#( *+# ,")(..00)1 ∑#( *2# ,")(..3.) ,

(3)

16

RESULTS & DISCUSSION

17

Production of Low Molecular Weight Phenolic Oil

18

In the first step of the lignin-first process, sweet sorghum bagasse was modified via

19

Fenton’s reagent (Fe3+, H2O2) in ethanol. Fenton’s reagent involves the reaction of hydrogen

20

peroxide with transition metals (i.e. iron) or ions to generate free radicals as reactive oxidants.

21

After the modification was complete, the lignin component of the modified bagasse was

22

selectively depolymerized in supercritical ethanol under nitrogen gas to produce a low molecular


7


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weight phenolic oil while leaving the holocellulose as a “solid carbohydrate” (See Table S2 for

2

mass balance). The solid carbohydrate was then enzymatically converted to 5- and 6-carbon

3

sugars in high yields, indicating the previous two steps preserved the holocellulose.

4

As shown in Figure 1C, the phenolic oil yields increased with longer SLD reaction times,

5

ranging from 50.1% for 0.5h to 75.8% for 3.0h. For comparison, 12.0h SLD reactions were

6

performed, with a maximum oil yield of 84.8%, but the data for such are not discussed in detail

7

due to relatively low yields of the desired phenolic monomers and the impractical reaction time.

8

During the 12h reaction, the desired phenolic monomers generated during hours 1-3 must have

9

become degraded or repolymerized during the remaining hours 4-12. The effect FM had on

10

phenolic oil yields is evident from the increase and decrease in yields with 5 and 20% hydrogen

11

peroxide loadings, respectively, particularly for 1.0 and 3.0h SLD reactions. The phenolic oil

12

yields decreased with the 20% hydrogen peroxide loading, when compared to the 5% loading,

13

because of excessive oxidation during the FM step that led to undesirable lignin degradation. The

14

preference for a relatively low loading of hydrogen peroxide for high yields of phenolic oil

15

increases the economic feasibility of this process.

16

We propose that Fenton’s reagent performed two beneficial modifications to the

17

lignocellulosic structure, supported by our previous research (see the first reaction step in

18

Scheme 1.B). First, free radicals (e.g. •OH, •OOH) generated by Fenton’s reagent led to

19

oxidation of lignin’s pendant groups and aromatic substituents via hydroxylation and

20

demethoxylation.11 Second, an iron-lignin complex formed via chelation of iron with aromatic

21

substituents. These two structural modifications lowered the energy barrier for subsequent lignin

22

depolymerization via ß-O-4 bond cleavage. The SLD reactions presented herein did not involve

23

an acid catalyst in order to prevent the formation of reactive intermediates from degradation of


8

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the holocellulose and to improve the process feasibility since expensive materials are often

2

needed in acidic environments at elevated temperatures and pressures.19,20 The pH values of

3

solutions after SLD were within the range of 5.75 – 6.75, with or without FM.

4

Six valuable phenolic monomers of relatively high yield were consistently detected in the

5

phenolic oils (Figure 1A). Other monomeric products were present as well, however, they are not

6

discussed due to their low concentrations, non-phenolic structures, or the inability for accurate

7

identification. SLD reactions were performed at supercritical (250°C) and subcritical (180°C)

8

temperatures to compare yields of phenolic oil and the six phenolic monomers (Figure 1B), as

9

well as phenolic oil molecular weight (Figure 1D).

A

C 5

2

3

4

Phenolic Oil Yield (w/w Klason lignin)

1

6

B

10

80% 70% 60% 50% 40% 30% 20% 10% 0%

0.5h SLD Reaction Time



75% 60% 45% 30% 15% 0%

D

0%

5%

20%

0%

5%

20%

0%

5%

20%

Hydrogen Peroxide Loading (w/w biomass)

10000

3h, 180C 0.5h, 250C 3.0h, 250C 1.0h, 250C 12.0h, 250C

8000

Supercritical Subcritical

Intensity

Yield (w/w Klason lignin)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


6000 4000 2000 0

Phenolic Oil

6 Phenolic Monomers

20

22

24

26

28

Time (min)

11

Figure 1. (A) The six major phenolic monomers detected in the phenolic oil are as follows: 1.)

12

4-vinylphenol, 2.) 4-vinylguaiacol, 3.) trans-isoeugenol, 4.) 4-propenylsyringol, 5.) ethyl-p-

13

coumarate, and 6.) ethyl ferulate. (B) Yields of phenolic oil and the six phenolic monomers after

14

selective lignin depolymerization (SLD) at supercritical (250 °C) and subcritical (180 °C)

15

reaction temperatures. The SLD reaction time was 3h and the Fenton modification (FM)

16

hydrogen peroxide loading was 20% for both supercritical and subcritical reactions. (C) Phenolic


9


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oil yields obtained from SLD of sweet sorghum bagasse with varying FM hydrogen peroxide

2

loadings and reaction times. The 0% hydrogen peroxide loadings represent the control

3

experiments. (D) Gel permeation chromatogram showing the molecular weight distribution of

4

phenolic oils with varying SLD reaction times and temperatures. The phenolic oils were

5

generated using a 20% FM hydrogen peroxide loading. The peaks shifted to the right have a

6

lower average molecular weight (Mw) relative to those shifted to the left.

7

Supercritical ethanol significantly increased the yields of phenolic oil (145% increase) and the

8

six phenolic monomers (477% increase), relative to subcritical ethanol (Figure 1B). As ethanol

9

reaches the supercritical state (241°C, 6.14MPa), its polarity drops while maintaining its

10

hydrogen donating ability, and its density becomes near gas-like.16 We hypothesized these

11

physiochemical changes would improve the SLD process for several reasons. The reactivity of

12

lignin with ethanol increases at the supercritical state due to lignin’s non-polar nature, whereas

13

the reactivity of holocellulose with supercritical ethanol decreases due to the polar nature of

14

hemicellulose and cellulose. The hydrogen donating ability of ethanol at the supercritical state

15

allows for a solvolytic cleavage of the interunit bonds in lignin, primarily the ß-O-4 bonds. The

16

gas-like density of ethanol at the supercritical state dramatically increases the rate of diffusion

17

into the biomass cell wall, which subsequently increases the rate of lignin depolymerization. The

18

subcritical temperature of 180 °C was chosen for comparison because we believe this

19

temperature is well outside of the fluid structural transition zone for ethanol, and it’s a common

20

temperature used in organosolv pretreatment methods.21,22

21

The molecular weight distribution of the phenolic oils differed with varying reaction times and

22

temperatures (Figure 1D). In subcritical ethanol, the molecular weight was 1283 g/mol for the

23

3.0h SLD reaction time. In supercritical ethanol, the molecular weights of the phenolic oils


10

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1

ranged from 1085 to 668 g/mol (Mw) for 0.5 and 12h SLD reaction times, respectively. The

2

molecular weight of phenolic oil for the 1h reaction in supercritical ethanol was 873 g/mol,

3

whereas that of the 3h reaction was 1032 g/mol. Therefore, the average molecular weight of the

4

phenolic oils appeared to decrease during the 1st hour of SLD in supercritical ethanol, increase

5

during the 2nd and 3rd hours, and then decrease during hours 4 – 12. We propose that during the

6

1st hour of SLD in supercritical ethanol, the exposed portions of lignin were readily

7

depolymerized, particularly through cleavage of ferulate and p-coumarate end groups. During the

8

2nd and 3rd hours of SLD, the bulk of the lignin began depolymerizing, particularly through

9

cleavage of ß-O-4 bonds interconnecting lignin subunits. The resulting depolymerized fragments

10

of lignin during the 2nd and 3rd hours of SLD were primarily oligomeric, hence why the average

11

molecular weight of the oil increased.

12

Analysis of the HSQC 2D NMR plots (Figure 2) revealed that the phenolic oil from the SLD

13

process was predominantly aromatic in structure, consisting of syringyl (S) and guaicyl (G) type

14

lignin structures. Therefore, the phenolic oil was predominantly derived from lignin. Figure 2

15

shows spectra corresponding to the aromatic region (4 C/ 4 H at 150 – 90/6.0 – 8.0) and aliphatic

16

region (4 C/ 4 H at 90 – 50/3.0 – 5.0) for two different phenolic oils. There was no notable

17

difference in the HSQC spectra between phenolic oils produced in supercritical ethanol with and

18

without FM (Figures 2B/C & S11F/G). The aliphatic region of the spectra for the phenolic oil

19

produced in subcritical ethanol (Figure 2E) showed increased evidence of polysaccharides

20

(4 C/ 4 H at 60/3.6ppm, 65/3.4ppm, 70/3.5ppm), relative to the spectra for phenolic oil produced in

21

supercritical ethanol (Figure 2C).23 The increased detection of lignin-carbohydrate complexes in

22

the subcritical oil supported our initial hypothesis that subcritical ethanol is more reactive with

23

the holocellulose (polysaccharides) relative to supercritical ethanol. Evidence of ß-O-4 bonds in


11


Page 12 of 27

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the subcritical oil (Figure 2.E) was obtained via strong signals for L-5 and L-6 (4 C/ 4 H at

2

72/4.85ppm, 84/4.4ppm), indicating these bonds had not been thoroughly cleaved.23,24 The L-5

3

and L-6 signals were however not present in spectra for supercritical oil (Figure 2.C), indicating

4

that the majority of the ß-O-4 bonds in lignin were cleaved in the supercritical state. The

5

aromatic region of the spectra for phenolic oil produced in subcritical ethanol (Figure 2.D)

6

lacked strong evidence of p-coumarate (pCA 2/6 - 4 C/ 4 H at 127.5/7.1ppm, 127.5/7.0ppm,

7

122.5/7.1ppm) and X1 (4 C/ 4 H at 131/6.3ppm) moieties.23,24 Considering the majority of p-

8

coumarate moieties exist as end groups to lignin fragments, they were likely depolymerized and

9

then repolymerized via condensation in subcritical ethanol. Whereas in supercritical ethanol

10

(Figure 2.B), the p-coumarate monomers were preserved via the stabilizing effect of ethanol in

11

the supercritical state.10


12

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1 2

Figure 2. Heteronuclear single quantum coherence (HSQC) 2D Nuclear Magnetic Resonance

3

(NMR) spectra of phenolic oil produced from 3h selective lignin depolymerization (SLD)

4

reactions of varying temperature and Fenton modification (FM) hydrogen peroxide loadings: A.)

5

The different moieties detected, B/C.) phenolic oil from SLD in supercritical ethanol with a 20%

6

FM hydrogen peroxide loading, D/E.) phenolic oil from SLD in subcritical ethanol with a 20%

7

FM hydrogen peroxide loading.

8

Fenton Modification for Improved Production of Phenolic Monomers


13


Page 14 of 27

1

The cumulative yields of the six major phenolic monomers are shown in Figure S10.

2

Considering the six major phenolic monomers constituted a maximum 19.1% of the initial

3

Klason lignin content, and the corresponding phenolic oil constituted 75.8%, there was a fair

4

amount of phenolic oil unaccounted for in the GC chromatograms. The unaccounted oil was

5

primarily composed of lignin-derived oligomeric phenols, supported by the 2D-NMR analysis.

6

The FM step did not affect the cumulative monomer yield for the 0.5h SLD reaction, and it

7

slightly increased the yield for the 1h SLD reaction. This observation supports our

8

aforementioned proposal that exposed pendant and end groups in lignin were readily

9

monomerized during the 1st hour of the SLD reaction, with or without FM. For the 3h SLD

10

reactions, the cumulative monomer yields were sensitive to the FM hydrogen peroxide loading,

11

with 5% and 20% loadings providing high (19.1 wt %) and low (12.8 wt %) yields, respectively.

12

The highest cumulative monomer yields were obtained with a 5% FM hydrogen peroxide

13

loading and 3h SLD reaction time (Table 1).

14

Table 1. Yields (w/w Klason lignin) of phenolic monomers and oils from the selective lignin

15

depolymerization process with varying reaction times. For all entries, the Fenton modification

16

hydrogen peroxide loading was 5%. Yield (wt%)

17

SLD Entry Reaction Time (h) 1 0.5 2 1 3 3

Monomers Monomers Phenolic 5 and 6 1-6 Oil 3.2% 6.1% 8.6%

10.6% 14.9% 19.1%

53.2% 64.5% 75.8%

18

Relative to a 5% FM hydrogen peroxide loading, a 20% loading caused a decrease in

19

cumulative monomer yields during the 2nd and 3rd hours of SLD (Figure S10) since the initial

20

Fenton induced free radical oxidation was excessive and led to undesirable holocellulose


14

Page 15 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

degradation and lignin condensation. It is known that the disruption of holocellulose and

2

condensation of lignin usually occur simultaneously in biomass pretreatment processes that

3

employ high temperatures and/or acid/base catalysts.6,25 We propose that oxidation of the

4

holocellulose during excessive FM increased its reactivity, such that its native structure was

5

degraded during the 2nd and 3rd hours of SLD. The degradation of holocellulose led to

6

undesirable side reactions with the depolymerized lignin, which ultimately led to lower monomer

7

yields. To support this proposal, evidence of holocellulose degradation during the 2nd and 3rd

8

hours of SLD was obtained from the enzymatic hydrolysis experiments, which are explained in

9

the subsequent section. The 5% FM hydrogen peroxide loading selectively targeted the lignin,

10

and therefore the holocellulose remained in its native form and was not degraded during the SLD

11

reaction.

12

The detection of monomers 5 and 6 was of interest due to the abundance of ferulate and p-

13

coumarate moieties in sweet sorghum bagasse. The yields of monomers 5 and 6 increased with

14

FM in all cases (Figure 3). The 3h SLD reaction with a 5% FM hydrogen peroxide loading gave

15

a maximum yield of 8.6 wt % for monomers 5 and 6, which equated to a 56.9% increase

16

compared to the control reaction. As shown in Scheme 1A, two plausible pathways are proposed

17

for the production of monomers 5 and 6, including a solvolytic transesterification of the exposed

18

ferulate and p-coumarate moieties in lignin and a reaction pathway induced by FM in which ß-O-

19

4 bonds are solvolytically cleaved (a detailed mechanism is shown in Scheme 1B). We propose

20

that monomers 1 and 2 were produced from decarboxylation of monomers 5 and 6, and

21

monomers 3 and 4 were produced from direct cleavage of ß-O-4 bonds.26


15

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Yield (w/w Klason lignin)


1

10.0%



Page 16 of 27


8.0%

Ethyl Ferulate

6.0% 4.0%

Ethyl-PCoumarate

2.0% 0.0% 0%

5% 20% 0%

5% 20% 0%

5% 20%

Hydrogen Peroxide Loading (w/w biomass)

2

Figure 3. Yields of ethyl-p-coumarate (monomer 5) and ethyl ferulate (monomer 6) from

3

varying Fenton modification hydrogen peroxide loadings and selective lignin depolymerization

4

reaction times. The 0% hydrogen peroxide loadings represent the control experiments.

5

Ferulate and p-coumarate end groups only comprise a small fraction of mass in grasses.17

6

Therefore, the significant increase in yields of monomers 5 and 6 from the SLD process with FM

7

is believed to result from solvolytic cleavage of ß-O-4 bonds as illustrated in Scheme 1B. We

8

propose that FM increased the yields of monomers 5 and 6 due to the decrease in ß-O-4 bond

9

dissociation energy via hydroxyl and methoxy oxidation, as well as stabilizing effects from iron

10

chelation of aromatic substituents.11

11

Scheme 1B illustrates our proposed mechanism for solvolytic monomerization of lignin to

12

produce ethyl-p-coumarate (monomer 5) and ethyl ferulate (monomer 6) via direct ß-O-4 bond

13

cleavage. During FM, free radicals (•OH, •OOH) oxidized hydroxyl and methoxy groups and

14

iron chelation occurred at sterically unhindered aromatic substituents, as was reported in our

15

previous study.11 Oxidation of hydroxyls at both the alpha (carbon 2) and gamma (carbon 4)

16

positions is possible in the presence of appropriate oxidants, such as Fenton’s reagent, however,

17

the reaction presented herein proceeded only after gamma oxidation.11,27 In the first intermediate

18

reaction during SLD, the solvent (ethanol) reacted with the aldehyde to form a hemiacetal, which

19

then caused the ß-O-4 bond to cleave.28 The oxidized gamma position was favorable to

20

hemiacetal formation due to its lack of steric hindrance, relative to the alpha position. The final


16

Page 17 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

product is in its most stable form with ;-bond conjugation. Compared to our previous study,

2

which used an organosolv lignin from hardwoods, the FM step for the process presented herein

3

led to a higher selectivity for phenolic monomers. The stabilizing effects from iron chelation are

4

similar to those used by Shuai et al. in which formaldehyde reacted with lignin pendant groups to

5

form stable acetals.20 In Scheme 1B, substituting sterically-unhindered aromatic substituents with

6

bulky iron ions acted as a capping agent by preventing repolymerization of the phenolic

7

monomers obtained during the subsequent depolymerization reaction in supercritical ethanol. As

8

was the case for the cumulative monomer yields, the highest yields of monomers 5 and 6 were

9

produced from a 5% FM hydrogen peroxide loading and a 3h SLD reaction time.

10

Scheme 1. (A) The proposed reaction pathways for the production of the six phenolic monomers

11

during selective lignin depolymerization. (B) The proposed mechanism for solvolytic

12

monomerization of ethyl-p-coumarate (monomer 5) and ethyl ferulate (monomer 6) via direct ß-

13

O-4 bond cleavage.


17


Page 18 of 27

1 2

Fenton Modification for Improved Enzymatic Hydrolysis of the Holocellulose

3

The lignin-first approach to lignocellulosic biorefining aims to maximize the utilization

4

of both lignin and holocellulose components. In this study, after the SLD reaction was complete,

5

the holocellulose was separated from the phenolic oil as a “solid carbohydrate”. Enzymatic

6

hydrolysis of the solid carbohydrates was performed to understand how effective the SLD

7

reaction was at pretreating the biomass for liberation of both 5- and 6-carbon sugars. A 20%

8

hydrogen peroxide loading was chosen for the solid carbohydrates that underwent FM at

9

different reaction times. It was of interest to know whether the sugar yields from enzymatic

10

hydrolysis would follow a similar trend as the phenolic monomer yields did from SLD at


18

Page 19 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

different reaction times with this particular FM condition. As shown in Figure 4 and Table S3, a

2

maximum 66.1% of the solid carbohydrate mass was converted into free sugars within 24 hours.

3

The maximum sugar release data indicated that 79.9% of the initial 6-carbon polysaccharides

4

(glucan and galactan) and 62.7% of the initial 5-carbon polysaccharides (xylan and arabinan)

5

were converted into free sugars (See Table S4). For most of the enzymatic reactions, the solid

6

carbohydrates that had undergone FM liberated sugars in a higher abundance and at a faster rate

7

than the controls, indicating FM enhanced enzymatic activity. Scanning electron microscopy and

8

energy-dispersive x-ray spectroscopy were used to confirm chelated iron from FM remained

9

bound to the biomass after the SLD reaction (see Figures S12-S16). The solid carbohydrate from

10

the 1h SLD with FM had the highest catalytic effect, with a 23.7% increase in conversion yield

11

relative to the control after the 1st three hours and a sustained effect over subsequent time points,

12

as shown in Figure 4. In nature, various fungal species feed on woody biomass via transition-

13

metal mediated oxidation of lignin and hemicellulose followed by enzymatic hydrolysis of the

14

polysaccharides.29 Therefore, the biomimetic nature of the initial FM step increased the

15

enzymatic digestibility of the biomass. In addition, the stabilizing effect of the chelated iron

16

during SLD in supercritical ethanol limited char formation, thus enhancing enzymatic activity by

17

allowing for less non-productive binding and more accessibility to the polysaccharides. The solid

18

carbohydrate from the 3h SLD was enzymatically hydrolyzed at a slower rate than both the 0.5

19

and 1h solids, with and without FM. We propose that the relatively low sugar yields from the

20

solid carbohydrates that underwent 3h SLD were caused by different mechanisms for the

21

reactions with and without FM. With FM, the holocellulose was degraded during the 2nd and 3rd

22

hours of SLD due to excessive oxidation, as was explained in the previous section. Without FM,


19


1

the long reaction time and absence of stabilizing iron ions led to char formation, thus reducing

2

the accessibility to the enzymes. SLD Reaction Time (h)

Percent Conversion (w sugar / w solid carbohydrate)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 27

75% 0.5 1.0 3.0

0.5 1.0 3.0

65% Control

55% 45%

Fenton Modified

35% 25% 3

3

0.5 1.0 3.0

0.5 1.0 3.0

6

12

24

Enzyme reaction time (h)

4

Figure 4. Percent conversion of solid carbohydrates to free sugars (5-carbon + 6-carbon) via

5

enzymatic hydrolysis. A 20% hydrogen peroxide loading was used for the solid carbohydrates

6

that had undergone Fenton modification.

7

CONCLUSIONS

8

A new lignin-first biorefining process was developed to effectively utilize all three major

9

components of sweet sorghum bagasse (lignin, hemicellulose, and cellulose). Fenton’s reagent

10

proved to be successful in enhancing both lignin depolymerization and enzymatic hydrolysis of

11

the holocellulose. The supercritical state of ethanol proved to be advantageous for lignin

12

depolymerization and holocellulose preservation, relative to the subcritical state. The benefits of

13

this process in comparison with other SLD processes include the use of an inert atmosphere,

14

earth abundant catalyst, reasonable reaction time, and green solvent. The process presented

15

herein could be improved with further investigation into the purification of the phenolic

16

monomers and their application in high-value products. Overall, the novelty presented herein has

17

contributed to the rapidly growing field of lignin-first biorefining by cost-effectively utilizing


20

Page 21 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

Fenton’s reagent and supercritical ethanol without hydrogen to produce valuable phenolics and

2

free sugars.

3 4

ASSOCIATED CONTENT

5

Supporting Information

6 7 8 9

The Supporting Information is available free of charge on the ACS Publications website at DOI: Contains figures, tables, and additional descriptions of procedures. AUTHOR INFORMATION

10

Corresponding Author

11

*Email: [email protected]

12

AUTHOR CONTRIBUTIONS

13 14

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

15 16 17

ACKNOWLEDGEMENTS This work was financially supported by Biomass Research & Development Initiative

18

Competitive Grant no. 2001-10006-3058 from the USDA National Institute of Food and

19

Agriculture, by the U.S. Department of Energy’s International Affairs under award number DE-

20

PI0000031 from the U.S. Department of Energy’s Office of Energy Efficiency and Renewable


21


Page 22 of 27

1

Energy, Bioenergy Technologies Office; and the Institute of Agricultural and Food Sciences

2

(IFAS) graduate scholarship at the University of Florida. The authors kindly thank Dr. Wilfred

3

Vermerris for supplying the sweet sorghum bagasse and Novozymes® for supplying the enzyme

4

solutions.

5 6 7

REFERENCES

8

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Catalyzed Lignin Depolymerization to High-Value Aromatics and Dicarboxylic Acids.

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16 17 18 19

SYNOPSIS


26

Page 27 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

Reported herein is a new lignin-first approach to lignocellulosic biorefining in which whole

2

biomass is converted to high value phenolics and free sugars, via selective lignin

3

depolymerization prior to holocellulose by use of a cost-effective biomimetic catalyst and green

4

solvent.

5 6 7 8 9

TOC/ABSTRACT GRAPHIC Feedstock: Sorghum Bagasse

Fenton Modification

10

Fe3+/ H2 O2

Product 1: Phenolics

Product 2: C5 and C6 Sugars

Selective Lignin Depolymerization

Holocellulose Enzymatic Hydrolysis


27

Lignin-First Approach to Biorefining: Utilizing Fenton's Reagent and

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