Subscriber access provided by UNIV OF NEWCASTLE
Article
Cellulase adsorption during the hydrolysis of organosolv- and organocat-pretreated beech wood Yumei Wang, Nico Anders, and Antje C. Spiess Energy Fuels, Just Accepted Manuscript • DOI: 10.1021/acs.energyfuels.7b01454 • Publication Date (Web): 26 Jul 2017 Downloaded from http://pubs.acs.org on July 27, 2017
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Energy & Fuels is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 33
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
Energy & Fuels
1
Cellulase adsorption during the hydrolysis of
2
organosolv- and organocat-pretreated beech wood
3
Yumei Wang1,2, Nico Anders1, Antje C. Spieß1,2,3,*
4 5
1
6
2
7 8
3
Aachener Verfahrenstechnik - Enzyme Process Technology, RWTH Aachen University, Forckenbeckstraße 51, 52074 Aachen, Germany DWI - Leibniz Institute for Interactive Materials, 52074 Aachen, Germany
Institute of Biochemical Engineering, Technische Universität Braunschweig, Rebenring 56, 38106 Braunschweig
9
10
*Corresponding author. Tel: +49 (0)531/391-55310
11
E-Mail:
[email protected] 12
13
Abstract
14
In a bio-refinery, only a complete raw material cellulose saccharification, at high consistencies,
15
leads to high sugar concentrations and thereby, to high yields in the subsequent fermentation.
16
However, enzymatic hydrolysis is significantly hampered by a strong slowdown of the reaction
17
after its start, which may be explained e.g. by enzyme inactivation, inhibition, or compositional
18
and structural changes of the biomass. Effective pulp enzymatic hydrolysis strongly depends on
19
the biomass pretreatment. We studied enzyme adsorption to three cellulosic materials and its
20
effect on the reaction rate during hydrolysis. The maximal cellulase adsorption decreased from
21
137.7 mg protein/g biomass via 106.1 mg protein/g biomass to 88.8 mg/g biomass for α-cellulose, 1 ACS Paragon Plus Environment
Energy & Fuels
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 2 of 33
1
organocat and organosolv material, respectively. The initial reaction rates directly correlated to
2
the amount of adsorbed enzyme as predicted by the adsorption isotherms. However, the adsorbed
3
cellulase loading increased dramatically during the reaction and drifted away from the adsorption
4
isotherm. One reason was the changes in substrate composition, e.g. lignin content increased
5
from 22% to 42% for organosolv beech wood during hydrolysis. Most importantly, the cellulases
6
irreversibly adsorbed to the pulp with proceeding hydrolysis, such that they were inactivated.
7
8
Key words
9
Enzymatic hydrolysis; Adsorption isotherm; (Ligno)cellulosic biomass; Cellulase activity; Pore
10
size; Particle size
11
12
2 ACS Paragon Plus Environment
Page 3 of 33
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
Energy & Fuels
1
1. Introduction
2
The conversion of renewable plant biomass to fuels and chemicals is considered to be an
3
alternative for the decreasing fossil resources 1. Forestry residues such as beech wood or poplar,
4
as well as agricultural residues constitute possible lignocellulosic raw materials for a bio-refinery
5
process 2-4. Such a bio-refinery process includes the pretreatment of lignocellulosic biomass to
6
reduce its recalcitrance to enzymes, the enzymatic hydrolysis of the pulp to produce high sugar
7
concentrations, and the fermentation of the sugars to the desired fuels and chemicals 5. The
8
enzymatic hydrolysis of pretreated lignocellulosic biomass can be operated under moderate
9
conditions without by-products, thereby potentially leading to high sugar concentrations 6, but it
10
is significantly hampered after its start.
11
The reason for the slowdown of the multi-enzyme catalyzed heterogeneous hydrolysis is still
12
under debate 6. First, cellulases are inactivated by thermal and mechanical influences, with the
13
inactivation degree varying from marginal to significant depending on the hydrolysis
14
conditions 7-10. Second, the products glucose and cellobiose as well as the xylooligomers are
15
known to be cellulase inhibitors 11-14; the same is true for some pretreatment by-products of
16
lignocellulosic biomass, such as phenols 15. Third, biomass composition and structural properties
17
change as hydrolysis progresses: since amorphous fraction of the cellulose is hydrolyzed faster
18
than the recalcitrant crystalline fraction, the specific surface area decreased 16. In addition,
19
cellulase diffusion into the core of larger particles leads to a preferential hydrolysis of surficial
20
cellulose before internally buried cellulose, which results in a decrease of the hydrolyzability 17.
21
Besides this, the lignin fraction in lignocellulosic biomass keeps on increasing during hydrolysis
22
of the cellulose and hemicellulose fractions of the lignocellulosic biomass and is considered an
23
additional reason for the slowdown of the reaction rate 18. 3 ACS Paragon Plus Environment
Energy & Fuels
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 4 of 33
1
The majority of studies on substrate accessibility and enzyme adsorption only investigate the
2
enzyme adsorption in relation to the initial lignocellulosic biomass 19, 20. Some studies monitored
3
the development of the free protein concentration in the liquid during the hydrolysis of model
4
celluloses or pretreated lignocellulosic biomass 21, 22, but only one study correlated the enzyme
5
adsorption during hydrolysis with the adsorption isotherm for bacterial microcrystalline cellulose
6
23
7
the (ligno)cellulose type 24, two lignocellulosic materials with promise for bio-refinery
8
application were chosen, namely ethanol organosolv-pretreated and organocat fractionated beech
9
wood 25, 26.
. Since the hydrolysis rate, its slow down, and the final biomass conversion depend strongly on
10
In this work, we investigated the adsorption of a commercial cellulase preparation to organosolv
11
and organocat pretreated beech wood as well as to α-cellulose during hydrolysis under different
12
cellulase dosages. The cellulase adsorption during hydrolysis was correlated to the cellulose
13
adsorption isotherm to understand the rate slowdown during the hydrolysis. The composition,
14
pore size and particle size distribution, as well as the cellulase activity and protein recovery from
15
the hydrolysis residue were reported to further dissect the mechanism of rate slowdown.
16
Understanding the hydrolysis rate decrease is essential for designing a biorefinery process.
17
18
2. Materials and methods
19
2.1 Materials
20
α-cellulose C8002-1 KG, lot No. 006K0076, was purchased from Sigma (Taufkirchen, Germany).
21
Beech wood type 1-4 with particle size ranging from 2.5 mm to 3.5 mm from JRS (J. Rettenmaier
22
& Söhne Group, Rosenberg, Germany) was milled with a laboratory blender (Waring, Torrington, 4 ACS Paragon Plus Environment
Page 5 of 33
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
Energy & Fuels
1
USA) and sieved to obtain particles < 1.5 mm. The moisture content was determined via drying
2
at 105 °C for 24 h to be 8.6% (w/w). The composition of cellulose (36% w/w), hemicellulose (15%
3
w/w), acid insoluble lignin (ASL, 6% w/w), and acid soluble lignin (ASL, 25% w/w) was
4
determined using the NREL protocol 27. The liquid cellulase preparation from Trichoderma
5
reesei ATCC 26921, Celluclast Novozymes (Sigma-Aldrich, Taufkirchen, Germany) used for
6
hydrolysis had a protein content of 122 g/L and an activity of 34.3 FPU/mL.
7 8
2.2 Pretreatment
9
Within the typical range of organosolv pretreatment conditions 3 the pretreatment parameters
10
were chosen to be comparable to the organocat pretreatment 28. To this end, 16.6 g beech wood
11
was pretreated using 100 mL 50% (v/v) aqueous ethanol (solid/liquid ratio of 1:6 w/v) at 180 °C
12
for 2 hours in a high-temperature and high-pressure Parr reactor 4848 (Parr Instruments,
13
Frankfurt am Main, Germany). After pretreatment, the cellulose pulp was recovered by
14
centrifugation. Afterwards, the pretreated biomass was washed only with 600 mL water.
15
Organocat pretreatment was performed according to vom Stein et al. 2011 28. 100 mL of the
16
biphasic solvent system 0.1 M aqueous oxalic acid/2-MTHF (1:1 v/v) at a solid/liquid ratio of 1:6
17
(w/v) was used for separating beech wood cellulose from hemicellulose and lignin at 140 °C for
18
3 h in the Parr reactor. After pretreatment, the solid were first recovered by centrifugation, were
19
then washed with 200 mL ethanol, and finally, with 800 mL water to thoroughly remove all
20
solvents from the pretreatment process.
21
The pretreatment was performed in six batches. The pretreated beech wood was freeze dried
22
batch-wise and then mixed to obtain homogenous material, which was stored at room
5 ACS Paragon Plus Environment
Energy & Fuels
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
temperature for further use. Two aliquots of the mixture were dried at 105 °C for composition
2
analysis.
Page 6 of 33
3
4
2.3 Enzymatic hydrolysis and protein desorption
5
The enzymatic hydrolysis was performed by modifying the protocol of Engel et al 2012 29.
6
100 mg freeze dried pretreated beech wood was hydrolyzed in 1 mL 0.1 M sodium acetate buffer
7
pH 4.8 using cellulase at different enzyme dosages (21 mg protein/g biomass, 84 mg protein/g
8
biomass and 336 mg protein/g biomass) to investigate the hydrolysis over a wide range of
9
enzyme dosages. The mixture was incubated in 2 mL Eppendorf tubes at 45 °C and 900 rpm in a
10
thermomixer MHR 23 (HLC Biotech, Bovenden, Germany). At defined time intervals, each two
11
Eppendorf tubes were withdrawn and centrifuged at 4 °C, 10,000 × g for 5 min. 200 µL liquid
12
supernatant was stored at 4 °C for protein measurement. 500 µL liquid supernatant was incubated
13
at 100 °C for 10 min to deactivate the enzyme and was subsequently centrifuged for 10 min at
14
10,000 × g. The supernatant was diluted appropriately for monosaccharide analysis and filtered
15
through 0.2 µm PVDF filter. The solid residue of the enzymatic hydrolysis was thoroughly
16
washed with Millipore water and then freeze dried to obtain the solids weight, which is available
17
for enzyme adsorption during hydrolysis.
18
In a large-scale experiment, each 2.5 g pretreated beech wood was incubated with 84 mg
19
protein/g biomass in 25 mL 0.1 M sodium acetate buffer under the same conditions for 6 h, 24 h
20
and 72 h. After centrifugation at 4 °C, the supernatant was analyzed for protein and
21
monosaccharide concentration. The hydrolysis residues were re-buffered with 5 mL 0.1 M
22
sodium acetate buffer pH 4.8 and were incubated at 45 °C for 1 h. After centrifugation, the 6 ACS Paragon Plus Environment
Page 7 of 33
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
Energy & Fuels
1
supernatant that contained the desorbed protein was collected and stored at 4 °C for protein
2
determination. Protein desorption was repeated with 0.1 M sodium acetate buffer pH 4.8. For the
3
final desorption, 10 mL buffer containing 0.5% (v/v) Tween 80 was added to desorb the rest of
4
the cellulases. Protein recovery refers to the total protein concentration found in the supernatant
5
and both wash fractions relative to the initial cellulase dosage.
6
The hydrolysis residue after protein desorption was further washed with 100 mL Millipore water,
7
filtrated, and stored at 4 °C for determining the enzyme adsorption isotherm, for composition
8
analysis, as well as the for particle and pore size measurement. An aliquot was dried at 105 °C for
9
the water content determination.
10
11
2.4 Enzyme adsorption isotherm
12
The enzyme adsorption isotherm on different types of biomass was measured by varying the
13
enzyme dosage between 5.25 mg/g biomass and 1344 mg/g biomass. Appropriate enzyme
14
amounts in 1 mL 0.1 M sodium acetate buffer pH 4.8 were incubated with 100 mg of biomass in
15
2 mL Eppendorf tubes, resulting in solids loading of 10% (w/v), each in two experimental
16
replicates. The mixture was incubated at 4 °C and 1,000 rpm in the thermomixer for 2 h to
17
achieve adsorption equilibrium. After centrifugation for 5 min at 4 °C and 10,000 × g, the
18
supernatant was collected. The free enzyme concentration in the supernatant Efree (mg/mL) was
19
determined using the BCA assay. The adsorbed protein amount was calculated from the
20
difference of the initial and the free protein amount. The adsorption loadings Eads (mg/g biomass)
21
were fitted to the Langmuir isotherm (Equation 1) using non-linear regression by Excel 2010
7 ACS Paragon Plus Environment
Energy & Fuels
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
solver, obtaining the maximum enzyme adsorption capacity Emax (mg/g biomass) and the
2
Langmuir constant K (mL/mg enzyme) 30.
3
Page 8 of 33
= ( ∙ ∙
)/(1 + ∙
) (1)
4
2.5 Protein concentration and enzyme activity assay
5
The Pierce™ BCA (bicinchoninic acid) Protein Assay Kit (Thermo Scientific, Rockford, IL,
6
USA) was used to quantify the protein concentration during hydrolysis, as well as the
7
concentration of desorbed protein from the hydrolysis residue. Each analysis was performed in
8
duplicate. The BCA assay signal was corrected for the respective sugar content by subtraction.
9
The specific cellulase activity was measured in duplicate at the same conditions as the enzymatic
10
hydrolysis using 2% (w/v) α-cellulose as substrate. The rate of sugar formation in the first 10 min
11
was used for calculating the cellulase activity. In contrast, the sugar formation over 1 h was used
12
to determine the specific cellulase activity of Tween 80-extracted cellulase. 1 U cellulase activity
13
is defined as the amount of enzyme needed for releasing 1 µmol sugars in 1 minute in 0.1 M
14
sodium acetate buffer pH 4.8 at 45 °C. The sugars refer to the sum of sugars determined by
15
HPAEC-PAD chromatography as outlined below.
16
17
2.6 Saccharide and composition analysis
18
The saccharides in the enzymatic hydrolysis supernatant were measured in duplicates using High
19
Performance Anion Exchange Chromatography coupled to Pulsed Amperometric Detection
20
(HPAEC-PAD, ICS-5000+, Thermo Scientific, Rockford, IL, USA), using a CarboPacTM
21
PA100 column operating at 40 °C for saccharide separation at a flow rate of 1 mL/min. The
22
gradient was set according to Anders et al. 31. The yield after 72 h hydrolysis was calculated by 8 ACS Paragon Plus Environment
Page 9 of 33
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
Energy & Fuels
1
dividing the total saccharide concentration by the initial total cellulose and hemicellulose content
2
of the biomass materials. The total saccharide concentration after 2 h was used to calculate the
3
initial reaction rate.
4
For composition analysis, the polysaccharide fraction of the dry solid lignocellulosic materials
5
was hydrolyzed in duplicates using the NREL two-step acid hydrolysis procedure 27. The
6
monosaccharide concentration of both hydrolysate sample was analyzed individually by
7
HPAEC-PAD 31 and used for calculating the cellulose and hemicellulose content of the
8
corresponding lignocellulosic material. The lignin content was determined gravimetrically as
9
Klason ligin 27.
10
11
2.7 Particle size distribution
12
The particle size distribution was analyzed via light scattering using Mastersizer 2000 (Malvern
13
Instruments Ltd., Malvern, Worcestershire, UK). Biomass was suspended in water and
14
appropriately diluted to obtain an obscuration of 2-5%. Measurements were performed in
15
duplicate for each sample, and the Mie light scattering model was used for quantification with a
16
cellulose refractive index of 1.54 32. Since the relative standard deviation amounted to less than
17
3%, only one measurement was reported.
18
19
2.8 Pore size distribution
20
The wet porosity and distribution of pore sizes between 1.32 nm and 396 nm of wet biomass
21
samples was measured in duplicates by thermoporosimetry using a differential scanning 9 ACS Paragon Plus Environment
Energy & Fuels
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 10 of 33
1
calorimeter DSC 1 STARe System (Mettler Toledo, Schwerzenbach, Switzerland). The
2
measurement principle is based on the melting point depression (∆T) of water confined in pores
3
that depends on the pore diameter , where = −2 / (Gibbs-Thomson equation) with
4
= 19.8 ∙ , which was calculated according to Park et al 33.
5
The samples were washed with Millipore water, filtrated through a cellulose filter paper, and then
6
transferred into 100 µL aluminum pans in duplicate. Thermoporosimetry was performed
7
according to Park et al. 33 by freezing the samples down to -50 °C and then heating up according
8
to the temperature step program adapted from Pihlajaniemi et al. 34 (Supplementary material
9
Table S1).
10
The mass of melting water in each temperature step was analyzed from the absorbed heat, and the
11
sample heat capacity was corrected by using the suggested value for the specific heat capacity of
12
cellulose of 0.0067 J g-1 K-1 35. This mass of the melting water was divided by the ice density
13
(917 g dm-3) to obtain the pore volume for each temperature. The cumulative pore volume was
14
thus obtained as a function of pore diameter.
15
16
3. Results and discussion
17
3.1 Enzymatic hydrolyzability of pretreated lignocellulosic biomass
18
Since the biomass fractionation method and the cellulase dosage strongly affect the efficiency of
19
enzymatic hydrolysis 11, we compared the rate and yield of enzymatic hydrolysis and the
20
cellulase adsorption of different pretreated biomass materials. To this end, organosolv or
21
organocat pretreated beech wood, as well as α-cellulose were hydrolyzed using varying cellulase
10 ACS Paragon Plus Environment
Page 11 of 33
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
Energy & Fuels
1
dosages of 21, 84, and 336 mg protein/g biomass. Fig. 1 shows the resulting total sugar yield as
2
well as the initial reaction rate of the enzymatic hydrolyses.
3 4
Fig. 1. Initial rates and yields after 72 h of enzymatic hydrolysis for α-cellulose as well as
5
organosolv and organocat pretreated beech wood at varying enzyme dosages of 21, 84, and 336
6
mg protein/g biomass. (A) Sugar yield after 72 h; (B) Initial reaction rate. Sugar yield was
7
calculated by dividing the total saccharide concentration at 72 h by the initial total cellulose and
8
hemicellulose content of the biomass materials. The total saccharide concentration after 2 h was
9
used to calculate the initial reaction rate. Error bars indicate the standard deviation of two
10
experimental replicates. Enzymatic hydrolysis was performed in 0.1 M sodium acetate buffer, pH
11
4.8, 45 °C at 10% (w/v) solid loading.
12
13
The sugar yield increased for all cellulosic biomasses with increasing enzyme loading (Fig. 1A).
14
A fourfold increase in total enzyme dosage from 21 mg protein/g biomass to 84 mg protein/g
15
biomass increased the yield by the factor of 1.5 to 2.2. However, a further fourfold increase of the
16
enzyme dosage to 336 mg protein/g biomass did not improve the hydrolysis yield significantly. 11 ACS Paragon Plus Environment
Energy & Fuels
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 12 of 33
1
This behavior is consistent with the hydrolysis of corn stover at varying cellulase dosages 22.
2
Interestingly, organosolv pretreated beech wood showed the same hydrolysis yield within
3
experimental error as α-cellulose. In contrast, the hydrolysis yield of organocat pretreated beech
4
wood was only half of that of α-cellulose or organosolv. While organosolv and organocat
5
pretreatments both fractionate lignocellulosic biomass 25, 28, the different enzymatic
6
hydrolyzability of the pretreated beech wood may be anticipated to result from different
7
delignification degrees, as well differences in pretreated biomass structure, such as pore size
8
distribution and crystallinity 32, 36.
9
Compared to the yield after 72 h hydrolysis, the initial reaction rate depended even stronger on
10
the type of biomass. The initial reaction rate for α-cellulose increased significantly with
11
increasing enzyme dosage, that for organosolv pretreated beech wood only slightly, whereas that
12
for organocat pretreated beech wood remained stable (Fig. 1B). These differences in hydrolysis
13
yield and rate may be due to structural differences, which would be reflected in the enzyme
14
adsorption properties 19.
15
16
3.2 Adsorption isotherms for cellulosic biomass
17
The adsorption equilibrium was investigated for α-cellulose, organosolv and organocat pretreated
18
beech wood using different total cellulase dosages (5.3 mg/g biomass to 1344 mg/g biomass) at
19
4 °C for 2 h (Fig. 2). The equilibration temperature of 4 °C was chosen instead of hydrolysis
20
temperature at 45 °C to avoid enzymatic cellulose degradation 37, and an equilibration time of 2 h
21
was shown to be sufficient for reaching the equilibrium in a preliminary kinetic study using α-
22
cellulose (data not shown). The experimental data of equilibrium protein loading vs. equilibrium 12 ACS Paragon Plus Environment
Page 13 of 33
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
Energy & Fuels
1
free protein concentration was fitted using the Langmuir equation with R2 >0.9. Equilibrium
2
protein loading refers to the protein mass over the biomass mass at adsorption equilibrium. The
3
maximum cellulase adsorption capacity (Emax) and the adsorption affinity (K) for the different
4
pretreated biomasses are summarized in Table 1.
5
α-Cellulose, which has similar crystallinity and porosity as wooden biomass 38, had the highest
6
maximum cellulase adsorption capacity of 137.7 mg protein/g biomass, which is 1.5 - 4 times
7
that of the maximum cellulase adsorption capacity of 34.9 – 95.2 mg/g reported for Avicel using
8
T. reesei cellulases at 4 °C 38, 39. The maximum cellulase adsorption capacity for organosolv
9
pretreated beech wood was 88.8 mg protein/g biomass and, thus, lowest. The maximal cellulase
10
adsorption capacity for pretreated beech wood had the same order of magnitude as the one for α-
11
cellulose, indicating a good accessibility to the cellulases. Maximum cellulase adsorption
12
capacities on hard wood were reported to be 87.69 mg cellulase/g for ethanol-pretreated
13
Lodgepole pine 40 and between 56 mg cellulase/g solid and 195 mg cellulase/g solid for
14
differently pretreated poplar 41, which confirms the range of values for organosolv and organocat
15
pretreated beech wood found in this study.
16
13 ACS Paragon Plus Environment
Energy & Fuels
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 14 of 33
1 2
Fig. 2 Adsorption isotherms of cellulase on cellulosic biomasses at 4°C before and after 72 h
3
enzymatic hydrolysis. (A) α-cellulose, (B) organosolv pretreated beech wood, (C) organocat
4
beech wood. Symbols represent each one data of one experiment with two analytical replicates.
5
Lines represent the best fit by the Langmuir equation (eq. 1). Adsorption was performed in 0.1 M
6
sodium acetate buffer, pH 4.8, 4 °C at 10% (w/v) solid loading. 14 ACS Paragon Plus Environment
Page 15 of 33
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
Energy & Fuels
1
Table 1 Maximum cellulase adsorption capacity Emax, adsorption affinity K and binding strength
2
S for different pretreated biomass before and after 72 h hydrolysis Adsorption was performed in
3
0.1 M sodium acetate buffer, pH 4.8, 4 °C at 10% (w/v) solid loading. The adsorption loadings
4
Eads (mg/g biomass) were fitted to the Langmuir isotherm, obtaining the maximum enzyme
5
adsorption capacity Emax (mg/g biomass) and the Langmuir constant K (mL/mg enzyme).
α-cellulose
Organosolv
Organocat
Time
[mg/g]
[mL/mg]
S = ×
[mL/g]
R
0h
137.7±12.8
0.040±0.008
5.5
0.94
72 h
129.0±17.8
0.033±0.009
4.26
0.92
0h
88.8±4.9
0.133±0.020
11.8
0.95
72 h
135.9±4.9
0.043±0.007
5.84
0.96
0h
106.1±6.2
0.068±0.009
7.2
0.97
72 h
180.7±20.4
0.028±0.006
5.06
0.95
2
6 7
In reverse order to the maximum cellulase adsorption capacity, the cellulase affinity of cellulases
8
to organosolv pretreated beech wood is the highest (0.133 mL/mg protein), followed by the one
9
to organocat pretreated beech wood and the one to α-cellulose. The binding strength S can be
10
expressed as " = × and describes the relative affinity of the cellulases to the biomass
11
solids at low cellulose concentration
12
strength to organosolv pretreated beech wood than to organocat pretreated beech wood and to α-
13
cellulose. The difference in maximal adsorption capacity and binding strength may be due to the
14
biomass composition and to structural differences, including particle size and pore size
15
distribution, as will be discussed below. As a general expectation, a higher proportion of
16
adsorbed cellulases on the biomass should lead to increased initial reaction rates. Therefore, the
30, 42
. Table 1 shows that cellulases had a stronger binding
15 ACS Paragon Plus Environment
Energy & Fuels
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 16 of 33
1
initial observed reaction rates of the enzymatic hydrolysis were plotted over the protein loadings
2
predicted by the adsorption isotherms in Fig. 3.
3 4
Fig. 3 Initial hydrolysis rate at 45 °C as function of the equilibrium protein loading calculated
5
from the adsorption isotherms at 4 °C. Initial reaction rate was the average rate of duplicate
6
hydrolyses during the first 2 h. The equilibrium protein loading refers to the protein mass over the
7
biomass mass at adsorption equilibrium. Enzymatic hydrolysis was performed in 0.1 M sodium
8
acetate buffer, pH 4.8, 45 °C at 10% (w/v) solid loading.
9 10
The initial reaction rates rise linearly with the equilibrium enzyme loading, but the slope depends
11
strongly on the type of cellulosic biomass. With cellulase loading increasing by a factor of 10, the
12
initial reaction rate rises by a factor of 2 for α-cellulose, but only slightly for organosolv
13
pretreated beech wood and negligibly for organocat pretreated beech wood. Apparently, the high
14
cellulase adsorption to organocat pretreated beech wood does not lead to high initial hydrolysis
15
rate, but organocat pretreated beech wood still remains recalcitrant towards enzymatic hydrolysis.
16
Before digging deeper into the reason for Organocat pretreated beech wood’s recalcitrance by 16 ACS Paragon Plus Environment
Page 17 of 33
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
Energy & Fuels
1
charactering its composition, particle size and pore size distribution in comparison to α-cellulose
2
and organosolv pretreated beech wood, the development of the reaction rate and of enzyme
3
adsorption isotherm during the course of enzymatic hydrolysis was of interest.
4
5
3.3 Enzyme adsorption during hydrolysis
6
In order to investigate the development of enzyme adsorption during 72 h progress of enzymatic
7
hydrolysis at 45 °C, both the sugar formation and the protein in the supernatant was measured
8
with substrate loading of 10%, and a cellulase loading between 21 mg protein/g biomass to 336
9
mg protein/g biomass. Fig. 4A, B, and C shows typical enzymatic hydrolysis curves for α-
10
cellulose, organosolv and organocat pretreated beech wood.
11
For all biomass materials, the measured biomass weight decreased in accordance with the
12
asymptotic increase of the total sugar amount (Fig. 4A, B, C). The hydrolysis progress can be
13
divided into three phases: Within the first 6 h, the reaction proceeded fastest, followed by a
14
reaction rate decrease between 12 and 30 h. Finally, only small amounts of sugars were produced
15
until the end of the hydrolysis after 72 h.
16
17 ACS Paragon Plus Environment
Energy & Fuels
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 18 of 33
1 2
Fig. 4. Progress of enzymatic hydrolysis and of enzyme adsorption during the hydrolysis.
3
Hydrolysis of α-cellulose (A), organosolv (B) and organocat (C) pretreated beech wood under an
4
enzyme dosage of 84 mg protein/g biomass. Enzyme adsorption onto α-cellulose (D), organosolv
5
(E) and organocat (F) pretreated beech wood during hydrolysis as well as related to adsorption
6
isotherm. Symbols represent each one experiment with two analytical replicates. Enzymatic
7
hydrolysis was performed in 0.1 M sodium acetate buffer, pH 4.8, 45 °C at 10% (w/v) solid
8
loading. 18 ACS Paragon Plus Environment
Page 19 of 33
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
Energy & Fuels
1
Additionally, the protein concentration in the liquid phase decreased rapidly in the first 6 h, and
2
remained almost stable afterwards as shown in Fig. 4 A, B and C. Tai and Keshwani observed a
3
similar behavior where first, the enzyme adsorption approaches the equilibrium, and afterwards,
4
the free enzyme concentration remains during the whole enzymatic hydrolysis of corn stover 22.
5
In contrast, Pribowo et al. reported a rapid protein adsorption to the pretreated corn stover in the
6
first 30 min followed by a constant concentration to 24 h, followed by an increase in protein
7
concentration in the liquid phase due to desorption 21. In the present study however, desorption of
8
the enzyme could not be observed.
9
The adsorbed enzyme loading during hydrolysis was calculated from the measured protein
10
concentration in the liquid phase during hydrolysis and the initial protein dosage. Accordingly,
11
the adsorbed protein loading per biomass weight was calculated from the adsorbed enzyme
12
amount and the measured residual solid during enzymatic hydrolysis. Due to the decrease of the
13
biomass, the adsorbed enzyme increased over hydrolysis time in a pattern similar to the total
14
sugar concentration increase (Fig. 4A, B, C). This observation qualitatively conforms to our
15
expectation. We assume that the enzymes are stable throughout the 72 h hydrolysis, such that the
16
total protein amount is constant. In addition, we assume that the enzyme adsorption throughout
17
the hydrolysis is reversible according to Langmuir adsorption isotherm. Based on these two
18
assumptions, the decreasing biomass mass over hydrolysis progress as shown in Fig. 4A, B and C
19
should lead to decreasing protein amount on the decreasing surface area, and by this to an
20
increasing protein loading. However, in contrast to our expectations, the adsorbed protein loading
21
does increase more than expected, which might be explained by morphological changes of the
22
observed biomasses.
23
Lee et al. reported the specific surface area (SSA, specific surface area) of partially crystalline
24
cellulose decreases significantly with the progress of enzymatic hydrolysis, due to a rapid 19 ACS Paragon Plus Environment
Energy & Fuels
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 33
1
hydrolysis of amorphous regions having a high surface area 16. In contrast, the SSA of also
2
partially crystalline cellulose Solka Floc SW40 dramatically increased from 3.9 to 11.6 m2/g,
3
subsequently levelling off, which is consistent with our results 43. Recently, Eibinger et al. 44 used
4
atomic force microscopy (AFM) to directly image the enzymatic hydrolysis progress of specially
5
prepared cellulose and of Avicel. The initially smooth surface became completely rugged after
6
3 h incubation with cellulases. This morphological change was accompanied by an increase in
7
specific enzyme adsorption capacity from 1 to 10 µg protein/mg cellulose. Besides the
8
morphological changes, also compositional changes, i.e. the hemicellulose and in particular,
9
lignin content, should be considered in the cellulase adsorption on lignocellulosic biomass, since
10
lignin is a strong cellulase adsorbent 30.
11
In order to explain the decreased reaction rate during the enzymatic hydrolysis progress from the
12
view of adsorption, cellulase adsorption equilibrium during the enzymatic hydrolysis at 45°C was
13
plotted together with the cellulase adsorption isotherm at 4°C (Fig. 4D, E and F). The calculated
14
adsorbed enzyme loading during hydrolysis first approached the adsorption isotherm from below,
15
and then left the isotherm with increasing time, as shown in Fig. 4D, E and F. This phenomenon
16
applies not only for α-cellulose, but also for organosolv and organocat pretreated beech wood for
17
all three enzyme dosages. In reversible adsorption and for a homogeneous substrate, the plot of
18
the adsorbed enzyme loading as function of the free enzyme concentration should move upward
19
along the isotherm during hydrolysis. During hydrolysis, the decreasing amount of biomass
20
should lead to an increase in free enzyme and consequently, to an increase both of the free
21
enzyme concentration and the adsorbed enzyme loading. Palonen reported the same deviation
22
from the isotherm as in this study during the hydrolysis of bacterial microcrystalline cellulose 23,
23
however starting on the adsorption isotherm, which was taken at the same temperature as
24
hydrolysis. In contrast, we measured the adsorption isotherm at 4°C to avoid changes to the 20 ACS Paragon Plus Environment
Page 21 of 33
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
Energy & Fuels
1
lignocellulosic substrate, and performed the enzymatic hydrolysis at 45 °C, which disfavors
2
protein adsorption and results in a starting point of the adsorbed protein loading below the
3
equilibrium value at 4°C.
4
In order to pinpoint what causes the deviation of the protein loading from the adsorption isotherm,
5
we analyzed the composition of the (ligno-) celluloses, their structural changes, enzyme recovery
6
after desorption and activity before and after enzymatic hydrolysis.
7
8
3.4 Compositional changes during enzymatic hydrolysis
9
The composition of the respective celluloses before and after enzymatic hydrolysis was analyzed
10
using the NREL protocol 27 and is shown in Table 2. After organosolv and organocat
11
pretreatment of beech wood, the cellulose content is 63% and 60% respectively, which is much
12
higher than for native beech wood (36%). The hemicellulose content in organocat biomass is
13
6.2%, half of that in the organosolv pretreated beech wood (12.5%). However, Klason lignin
14
content is higher in organocat- than in organosolv pretreated beech wood (Table 2). Since lignin
15
adsorbs more cellulase than cellulose 30, the high lignin content for organocat pretreated beech
16
wood may explain the a higher maximum enzyme adsorption capacity compared to organosolv
17
beech wood.
18
The composition of α-cellulose remained the same before and after enzymatic hydrolysis, due to
19
the simultaneous hydrolysis of both cellulose and hemicellulose (Table 2). For organosolv and
20
organocat pretreated beech wood, the lignin content increased from 22% to 42% and from 29% to
21
35%, respectively, due to the hydrolysis of cellulose and hemicellulose. The relative increase in
22
the lignin content increases cellulase adsorption and reduces the binding of cellulases to cellulose.
23
This is one additional reason why enzyme adsorption increased during hydrolysis, whereas the 21 ACS Paragon Plus Environment
Energy & Fuels
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 22 of 33
1
reaction rate decreased. Ko et al. reported that lignin from liquid hot water pretreated hardwoods
2
adsorbs a large amount of cellulase, in particular β-glucosidase, resulting in a dramatic decrease
3
in hydrolysis efficiency 30. Lignin in poplar solids was likewise reported to have a high cellulase
4
maximum adsorption loading 41. In contrast, delignification of corn stover did not affect the
5
accessibility of cellulose for purified CBHI, but improved the effectiveness of cellulases and
6
xylanase significantly 45.
7
Table 2 Composition of the investigated (ligno)cellulosic biomasses and standard deviation
8
before (0 h) and after (72 h) enzymatic hydrolysis Time
Cellulose % (w/w)
Hemicellulose % (w/w)
AIL % (w/w)
ASL % (w/w)
0h
82.8 ± 1.83
22.5 ± 0.35
0.3 ± 0.17
4.1 ± 0.12
72 h
82.0 ± 0.96
20.9 ± 0.36
0
3.8 ± 0.02
0h
63.0 ± 2.19
12.5 ± 0.72
21.9 ± 3.09
3.4 ± 0.06
72 h
50.1 ± 1.60
8.8 ± 0.06
42.0 ± 0.52
3.0 ± 0.03
0h
60.0 ± 3.61
6.2 ± 0.55
28.9 ± 0.41
2.4 ± 0.01
72 h
50.6 ± 2.02
5.4 ± 0.37
35.5 ± 0.65
2.2 ± 0.02
α-cellulose
Organosolv
Organocat 9
ASL - Acid Soluble Lignin; AIL - Acid Insoluble Lignin.
10
The composition was analyzed through two-step sulfuric acid hydrolysis using the NREL
11
protocol of two experimental replicates.
12
13
3.5 Particle size distribution changes during enzymatic hydrolysis
14
Besides the composition of the selected lignocellulosic biomasses, their particle size distribution
15
was measured (Fig. 5). In the beginning of the enzymatic hydrolysis, organosolv and organocat
16
pretreated beech wood showed similar particle size distribution. Both had a wide size distribution,
17
and the particles were larger than those of α-cellulose. The highest volume frequency for α22 ACS Paragon Plus Environment
Page 23 of 33
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
Energy & Fuels
1
cellulose was at the diameter of 69 µm, whereas that for organosolv and organocat beech wood
2
was at 479 µm.
3 4
Fig. 5 Particle size distribution before and after 72 hours of enzymatic hydrolysis. Closed
5
symbols represent the samples before enzymatic hydrolysis; open symbols represent the samples
6
after enzymatic hydrolysis. Each curve represented one measurement, which had a relative
7
standard deviation less than 3%. Wet samples were suspended in water and measured using light
8
scattering.
9 10
After enzymatic hydrolysis, as indicated in Fig. 5 by the open symbols, the particles were smaller
11
than in the beginning for all three (ligno)cellulosic biomasses. The particle size distribution of α-
12
cellulose still showed only one major peak at 35 µm, which corresponds to half of the initial
13
particle size. The particle size distributions of organosolv and organocat pretreated beech wood
14
exhibited three discernable peaks after enzymatic hydrolysis. One peak is at the same particle size
15
as in the beginning of the enzymatic hydrolysis. However, two more peaks appeared, one at 158 23 ACS Paragon Plus Environment
Energy & Fuels
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 24 of 33
1
µm and one at the very small particle size of 11 µm. organosolv pretreated beech wood had a
2
higher content of very small particles after enzymatic hydrolysis compared to organocat
3
pretreated beech wood. This difference coincides with the high sugar yield after 72 h hydrolysis
4
of organosolv pretreated beech wood.
5
Since the particle size decreased after enzymatic hydrolysis, the external surface area per gram
6
biomass should increase accordingly. This might partially explain the increase of the adsorbed
7
enzyme loading during hydrolysis. Lee et al. suggested that this increase may originate from the
8
water swelling of cellulose 16. However, in our study, we observed that samples swollen in water
9
for 24 hours did not exhibit changed particle size distributions (data not shown). For porous
10
particles, the internal specific surface is expected to contribute more significantly to the total
11
surface area, and thereby, to cellulase adsorption. Therefore, the pore size distribution was
12
analysed next.
13
14
3.6 Pore size distribution changes during enzymatic hydrolysis
15
Pore size distribution is an additional important factor affecting the substrate accessibility in
16
cellulose hydrolysis. Therefore, the wet porosity was measured for a hydrolysis time of 0 and
17
72 h. Initially, organosolv pretreated beech wood showed an overall higher porosity than both α-
18
cellulose and organocat pretreated beech wood (Fig. 6). Before hydrolysis, α-cellulose showed a
19
pore size distribution similar to organosolv pretreated beech wood until a pore diameter of 20 nm,
20
but less macro pores larger than 50 nm. Thus, organosolv pretreated beech wood had the
21
cumulative pore volume at 396 nm of 357 µL/g, compared to 264 µL/g for α-cellulose. Based on
22
the internal pore volume and the accompanying specific surface area, cellulase adsorption to 24 ACS Paragon Plus Environment
Page 25 of 33
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
Energy & Fuels
1
organosolv pretreated beech wood should in principle be preferred, since the cellulase affinity of
2
cellulases to organosolv pretreated beech wood was the highest. However, this does not lead to
3
the highest cellulase adsorption capacity, but the larger particle size of organosolv pretreated
4
beech wood may result in some pore volume being inaccessible to cellulases, thereby limiting the
5
maximum cellulase adsorption capacity. In contrast, organocat pretreated beech wood had the
6
lowest porosity; the cumulative pore volume at 396 nm is only 221 µL/g. The low porosity of
7
organocat pretreated beech wood might be explained by a lack of hemicellulose, which serves as
8
the upholder of the pores 34.
9 10
Fig. 6. Pore size distribution before and after 72 hours of enzymatic hydrolysis. Closed symbols
11
represent the samples before enzymatic hydrolysis; open symbols represent the samples after
12
enzymatic hydrolysis. Error bars indicate the standard deviation of two experimental replicates.
13
Wet samples were measured using DSC thermoporosimetry.
14 15
After enzymatic hydrolysis for 72 h, the overall porosity increased for all three biomasses. The
16
porosity of organosolv pretreated beech wood increased most, followed by organocat pretreated 25 ACS Paragon Plus Environment
Energy & Fuels
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 26 of 33
1
beech wood and α-cellulose. For organosolv pretreated beech wood, the cumulative volume at 56
2
nm increased to 462 µL/g, which is even higher than that of 357 µL/g at 396 nm before
3
hydrolysis. Due to the hydrolysis of cellulose and hemicellulose, the structure disintegrates
4
leading to an increase in pores. In addition, lignin in organosolv and organocat pretreated beech
5
wood may preserve the original porosity. Thus, the pore size increase would entail the increase of
6
enzyme adsorption during hydrolysis. However, Pihlajaniemi et al. observed the decrease of pore
7
size distribution for sodium hydroxide pretreated wheat straw during enzymatic hydrolysis; and
8
no trend was seen for auto hydrolyzed straw 36. These contrasting results might be explained by
9
the lignin content and the hemicellulose composition. Auto-hydrolyzed straw contained 30%
10
lignin, whereas sodium hydroxide delignified straw had only 3.8% lignin compared to organosolv
11
pretreated beech wood in this study with 22% Klason lignin.
12
13
3.7 Adsorption isotherm at 72 h
14
Based on the changed biomass composition, the decreased particle size, and the increased pore
15
volume, one would also expect a changed adsorption isotherm after enzymatic hydrolysis at 72 h
16
relative to the initial one. The residual material after enzymatic hydrolysis was thoroughly
17
washed with buffer to desorb cellulase, followed by washing with water and filtration to obtain
18
wet solids for adsorption isotherm measurement. The adsorption isotherms of α-cellulose,
19
organosolv and organocat pretreated beech wood after 72h are shown in Fig. 2.
20
For α-cellulose, the maximum enzyme adsorption capacity and the cellulase affinity decreased
21
slightly, but this change was far from being significant. This nearly unchanged adsorption
22
isotherm contradicts the increase in protein adsorption during hydrolysis and also the expected 26 ACS Paragon Plus Environment
Page 27 of 33
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
Energy & Fuels
1
increase of protein adsorption due to the particle size decrease and porosity increase. For
2
organosolv and organocat pretreated beech wood, the maximum enzyme adsorption capacity
3
increased significantly (Table 1), whereas the adsorption affinity and the binding strength
4
decreased. These changes to the adsorption isotherm were in accordance with the compositional
5
and structural changes of the biomass materials, since increased lignin and decreased
6
hemicellulose content accompanied the increased pore size, and the decreased particle size should
7
favor cellulase adsorption. However, the adsorbed protein loading during enzymatic hydrolysis
8
was still significantly higher than predicted by the adsorption isotherm. Thus, the structural and
9
composition changes during hydrolysis only partially explained the increase of the enzyme
10
adsorption during hydrolysis. Additional unknown factors presumably affect the cellulose
11
adsorption and the reaction rate, e.g. the specific enzyme activity.
12
13
3.8 Protein balance and activity
14
The enzyme concentration during larger scale hydrolysis at 84 mg/g or 8.4 mg/mL cellulose
15
dosage was measured and is shown in Table 3. After 6, 24 or 72 h hydrolysis, the protein
16
concentration in the supernatant was measured to be only 52% - 67% of the originally added
17
amount. Two rounds of buffer extraction recovered additional 9% - 14% protein. Tween 80 was
18
found to be a surfactant that improved the cellulase desorption from the solid biomass to the
19
liquid phase 40. Even with 0.5% (v/v) Tween 80 to extract the protein from the solid fraction, only
20
72% - 86% of the initially supplied protein amount could be recovered. Presumably, the
21
unrecovered protein might be irreversibly adsorbed to the biomass. For lignocellulose, this
22
protein loss most probably results from non-productive and irreversible adsorption of the enzyme
23
to lignin. In addition, during enzymatic hydrolysis, cellulases get progressively confined to parts 27 ACS Paragon Plus Environment
Energy & Fuels
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 28 of 33
1
of the substrate rendering a desorption impossible 44. This phenomenon of irreversible adsorption
2
may explain why adsorption during hydrolysis was increased in comparison to the adsorption
3
isotherm, which is based on equilibrium assumptions.
4
Table 3 Enzyme recovery during the hydrolysis from the supernatant, two consecutive buffer
5
extractions and a detergent extraction. 1st buffer
2nd buffer
Detergent
Total
Liquid Biomass
Time
Recovery extraction
extraction
extraction
protein.
[mg/mL]
α-cellulose
Organosolv
Organocat
[%] [mg/mL]
[mg/mL]
[mg/mL]
[mg/mL]
6h
5.44
0.71
0.43
0.29
6.87
82
24 h
5.57
0.66
0.34
0.16
6.73
80
72 h
4.68
0.55
0.22
0.53
6.01
72
6h
5.62
0.72
0.44
0.43
7.20
86
24 h
5.20
0.56
0.33
0.36
6.45
77
72 h
4.36
0.67
0.43
0.34
5.80
69
6h
5.47
0.69
0.46
0.56
7.18
85
24 h
5.64
0.62
0.45
0.46
7.16
85
72 h
5.11
0.63
0.41
0.39
6.55
78
6
The results are the average of two replicates, and the standard deviation is less than 1%. Total
7
protein refers to the total calculated protein amount i.e. the sum of the enzyme amount in the
8
liquid, 1st and 2nd buffer extraction, and detergent extraction. Recovery relates the total calculated
9
protein amount to the initial protein dosage of 84 mg/g or 8.4 mg/mL.
10
In addition, the specific activity of the extracted enzyme was much lower than the activity in the
11
liquid supernatant (Table 4), indicating the irreversibly adsorbed enzyme to be less active than
12
the free enzymes. It must be mentioned that the enzyme activity assay used for quantifying the 28 ACS Paragon Plus Environment
Page 29 of 33
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
Energy & Fuels
1
activity of the recovered proteins used α-cellulose instead of filter paper for the initially dosed
2
enzymes, which amounted to 0.28 FPU/mg at 0 h. In contrast, enzyme extracted by the detergent
3
Tween 80 showed comparable residual enzyme activity to the enzyme in the liquid supernatant.
4
This indicates Tween 80 could reconstitute the cellulase activity, which agrees with the finding
5
that Tween 80 improved enzymatic hydrolysis yield of steam exploded and ethanol pretreated
6
Lodgepole pine by 46% 40.
7
Table 4 Specific cellulase activity in the enzymatic hydrolysis supernatant and the extracted
8
buffer and detergent during enzymatic hydrolysis Liquid Biomass
Time
Buffer extraction1
Detergent extraction2
[U/mg]
[U/mg]
supernatant1 [U/mg]
α-cellulose
Organosolv
Organocat
9
1
6h
0.077±0.0018
0.041±0.0013
0.099±0.014
24 h
0.109±0.0162
0.079±0.0054
0.174±0.052
72 h
0.065±0.0112
0.026±0.0008
0.050±0.024
6h
0.084±0.0005
0.032±0.0009
0.069±0.005
24 h
0.073±0.0080
0.048±0.0039
0.104±0.012
72 h
0.052±0.0056
0.023±0.0014
0.085±0.039
6h
0.061±0.0005
0.037±0.0024
0.076±0.000
24 h
0.077±0.0067
0.035±0.0025
0.087±0.003
72 h
0.044±0.0090
0.014±0.0014
0.091±0.005 2
: Specific cellulose activity based on the reaction rate in the first 10 min. : Specific cellulose
10
activity based on the sugar production in 1 h. The assay time was prolonged to counteract the
11
possibly low enzyme activity after detergent extraction. The specific cellulase activity was
12
measured in duplicate using 2% (w/v) α-cellulose as substrate in 0.1 M sodium acetate buffer pH
13
4.8 at 45 °C. 29 ACS Paragon Plus Environment
Energy & Fuels
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 30 of 33
1
4. Conclusions
2
The effect of enzyme adsorption on the reaction rate during hydrolysis of organosolv and
3
organocat pretreated beech wood as well as α-cellulose was studied. Cellulase adsorption affinity
4
to biomass increased in the order of 0.04 mL/mg protein for α-cellulose, 0.07 mL/mg protein for
5
organocat pretreated beech wood and 0.13 mL/mg protein for organosolv pretreated beech wood.
6
The initial reaction rates of the three cellulosic substrates linearly correlated to the adsorbed
7
enzyme loading as predicted by the respective adsorption isotherms.
8
However, the adsorbed cellulase loading increased dramatically during reaction progress, and
9
drifted away from the enzyme adsorption isotherm. The lignin content increased, the particle size
10
decreased, and the wet porosity increased after 72 h enzymatic hydrolysis, leading to a change of
11
the adsorption isotherm during hydrolysis, however less pronounced than suggested by the drift.
12
In addition, the cellulases lost activity when they adsorbed irreversibly to the biomass during
13
proceeding hydrolysis. Even when the cellulases were desorbed from the biomass, their activity
14
was also reduced. Therefore, the hydrolysis rate decreased whereas the cellulase adsorption
15
increased during proceeding hydrolysis.
16
In order to minimize the irreversible enzyme adsorption, a pretreatment method is recommended
17
that reduces the recalcitrance of the lignocellulosic material with a high degree of delignification.
18
Understanding the decrease of reaction rate during enzymatic hydrolysis with respect to
19
irreversible enzyme adsorption and enzyme residual enzyme recovery is one important step
20
forward towards increasing the process efficiency and lowering the enzyme cost in a biorefinery
21
process.
22 30 ACS Paragon Plus Environment
Page 31 of 33
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
Energy & Fuels
1
Acknowledgement
2
This work was performed as part of the Cluster of Excellence "Tailor-Made Fuels from Biomass",
3
which is funded by the Excellence Initiative by the German federal and state governments to
4
promote science and research at German universities. Yumei Wang acknowledges support from
5
Chinese Scholarship Council (CSC) file number 201206740023.
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
References 1. Lynd, L. R.; Cushman, J. H.; Nichols, R. J.; Wyman, C. E., Fuel ethanol from cellulosic biomass. Science 1991, 251, (4999), 1318-1323. 2. Viell, J.; Wulfhorst, H.; Schmidt, T.; Commandeur, U.; Fischer, R.; Spiess, A.; Marquardt, W., An efficient process for the saccharification of wood chips by combined ionic liquid pretreatment and enzymatic hydrolysis. Bioresource Technology 2013, 146, 144-151. 3. Pan, X. J.; Gilkes, N.; Kadla, J.; Pye, K.; Saka, S.; Gregg, D.; Ehara, K.; Xie, D.; Lam, D.; Saddler, J., Bioconversion of hybrid poplar to ethanol and co-products using an organosolv fractionation process: Optimization of process yields. Biotechnology and Bioengineering 2006, 94, (5), 851-861. 4. Zhang, J.; Chu, D. Q.; Huang, J.; Yu, Z. C.; Dai, G. C.; Bao, J., Simultaneous saccharification and ethanol fermentation at high corn stover solids loading in a helical stirring bioreactor. Biotechnology and Bioengineering 2010, 105, (4), 718-728. 5. Jorgensen, H.; Kristensen, J. B.; Felby, C., Enzymatic conversion of lignocellulose into fermentable sugars: challenges and opportunities. Biofuels, Bioproducts and Biorefining 2007, 1, (2), 119-134. 6. Lynd, L. R.; Weimer, P. J.; van Zyl, W. H.; Pretorius, I. S., Microbial cellulose utilization: Fundamentals and biotechnology. Microbiology and Molecular Biology Reviews 2002, 66, (3), 506-577. 7. Caminal, G.; López-Santín, J.; Solà, C., Kinetic modeling of the enzymatic hydrolysis of pretreated cellulose. Biotechnology and Bioengineering 1985, 27, (9), 1282-1290. 8. Ohlson, I.; Trägårdh, G.; Hahn-Hägerdal, B., Enzymatic hydrolysis of sodium-hydroxidepretreated sallow in an ultrafiltration membrane reactor. Biotechnology and Bioengineering 1984, 26, (7), 647-653. 9. Gunjikar, T. P.; Sawant, S. B.; Joshi, J. B., Shear deactivation of cellulase, exoglucanase, endoglucanase, and β-glucosidase in a mechanically agitated reactor. Biotechnology Progress 2001, 17, (6), 1166-1168. 10. Eriksson, T.; Karlsson, J.; Tjerneld, F., A model explaining declining rate in hydrolysis of lignocellulose substrates with cellobiohydrolase I (Cel7A) and endoglucanase I (Cel7B) of Trichoderma reesei. Applied Biochemistry and Biotechnology 2002, 101, (1), 41-60. 11. Gusakov, A. V.; Sinitsyn, A. P.; Klyosov, A. A., Factors affecting the enzymatic hydrolysis of cellulose in batch and continuous reactors: Computer simulation and experiment. Biotechnology and Bioengineering 1987, 29, (7), 906-910. 31 ACS Paragon Plus Environment
Energy & Fuels
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 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
Page 32 of 33
12. Hsieh, C. W.; Cannella, D.; Jorgensen, H.; Felby, C.; Thygesen, L. G., Cellulase inhibition by high concentrations of monosaccharides. Journal of Agricultural and Food Chemistry 2014, 62, (17), 38003805. 13. Lee, Y.-H.; Fan, L. T., Kinetic studies of enzymatic hydrolysis of insoluble cellulose: (II). Analysis of extended hydrolysis times. Biotechnology and Bioengineering 1983, 25, (4), 939-966. 14. Qing, Q.; Yang, B.; Wyman, C. E., Xylooligomers are strong inhibitors of cellulose hydrolysis by enzymes. Bioresource Technology 2010, 101, (24), 9624-9630. 15. Ximenes, E.; Kim, Y.; Mosier, N.; Dien, B.; Ladisch, M., Deactivation of cellulases by phenols. Enzyme and Microbial Technology 2011, 48, (1), 54-60. 16. Lee, S. B.; Kim, I. H.; Ryu, D. D. Y.; Taguchi, H., Structural properties of cellulose and cellulase reaction mechanism. Biotechnology and Bioengineering 1983, 25, (1), 33-51. 17. Luterbacher, J. S.; Parlange, J. Y.; Walker, L. P., A pore-hindered diffusion and reaction model can help explain the importance of pore size distribution in enzymatic hydrolysis of biomass. Biotechnology and Bioengineering 2013, 110, (1), 127-136. 18. Rahikainen, J.; Mikander, S.; Marjamaa, K.; Tamminen, T.; Lappas, A.; Viikari, L.; Kruus, K., Inhibition of enzymatic hydrolysis by residual lignins from softwood—study of enzyme binding and inactivation on lignin-rich surface. Biotechnology and Bioengineering 2011, 108, (12), 2823-2834. 19. Grethlein, H. E., The effect of pore size distribution on the rate of enzymatic hydrolysis of cellulosic substrates. Nature Biotechnology 1985, 3, (2), 155-160. 20. Carrillo, F.; Lis, M. J.; Colom, X.; López-Mesas, M.; Valldeperas, J., Effect of alkali pretreatment on cellulase hydrolysis of wheat straw: Kinetic study. Process Biochemistry 2005, 40, (10), 3360-3364. 21. Pribowo, A.; Arantes, V.; Saddler, J. N., The adsorption and enzyme activity profiles of specific Trichoderma reesei cellulase/xylanase components when hydrolyzing steam pretreated corn stover. Enzyme and Microbial Technology 2012, 50, (3), 195-203. 22. Tai, C.; Keshwani, D. R., Enzyme adsorption and cellulose conversion during hydrolysis of diluteacid-pretreated corn stover. Energy & Fuels 2014, 28, (3), 1956-1961. 23. Palonen, H.; Tenkanen, M.; Linder, M., Dynamic interaction of Trichoderma reesei cellobiohydrolases Cel6A and Cel7A and cellulose at equilibrium and during hydrolysis. Applied and Environmental Microbiology 1999, 65, (12), 5229-5233. 24. Sun, Y.; Cheng, J., Hydrolysis of lignocellulosic materials for ethanol production: a review. Bioresource Technology 2002, 83, (1), 1-11. 25. Zhao, X.; Cheng, K.; Liu, D., Organosolv pretreatment of lignocellulosic biomass for enzymatic hydrolysis. Applied Microbiology and Biotechnology 2009, 82, (5), 815-827. 26. Viell, J.; Harwardt, A.; Seiler, J.; Marquardt, W., Is biomass fractionation by Organosolv-like processes economically viable? A conceptual design study. Bioresource Technology 2013, 150, 89-97. 27. Sluiter, A., B. Hames, R. Ruiz, C. Scarlata, J. Sluiter, D. Templeton, D. Crocker, Determination of structural carbohydrates and lignin in biomass. LAP-002 NREL Analytical Procedure. National Renewable http://www.nrel.gov/docs/gen/fy13/42618.pdf, 2008 (accessed Energy Laboratory Golden, CO. 16.07.01). 28. vom Stein, T.; Grande, P. M.; Kayser, H.; Sibilla, F.; Leitner, W.; Dominguez de Maria, P., From biomass to feedstock: one-step fractionation of lignocellulose components by the selective organic acidcatalyzed depolymerization of hemicellulose in a biphasic system. Green Chemistry 2011, 13, (7), 17721777. 29. Engel, P.; Krull, S.; Seiferheld, B.; Spiess, A. C., Rational approach to optimize cellulase mixtures for hydrolysis of regenerated cellulose containing residual ionic liquid. Bioresource Technology 2012, 115, 27-34. 30. Ko, J. K.; Ximenes, E.; Kim, Y.; Ladisch, M. R., Adsorption of enzyme onto lignins of liquid hot water pretreated hardwoods. Biotechnology and Bioengineering 2015, 112, (3), 447-456. 32 ACS Paragon Plus Environment
Page 33 of 33
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 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
Energy & Fuels
31. Anders, N.; Humann, H.; Langhans, B.; Spieß, A. C., Simultaneous determination of acid-soluble biomass-derived compounds using high performance anion exchange chromatography coupled with pulsed amperometric detection. Analytical Methods 2015, 7, (18), 7866-7873. 32. Bragatto, J.; Segato, F.; Cota, J.; Mello, D. B.; Oliveira, M. M.; Buckeridge, M. S.; Squina, F. M.; Driemeier, C., Insights on how the activity of an endoglucanase is affected by physical properties of insoluble celluloses. The Journal of Physical Chemistry B 2012, 116, (21), 6128-6136. 33. Park, S.; Venditti, R. A.; Jameel, H.; Pawlak, J. J., Changes in pore size distribution during the drying of cellulose fibers as measured by differential scanning calorimetry. Carbohydrate Polymers 2006, 66, (1), 97-103. 34. Pihlajaniemi, V.; Sipponen, M. H.; Liimatainen, H.; Sirvio, J. A.; Nyyssola, A.; Laakso, S., Weighing the factors behind enzymatic hydrolyzability of pretreated lignocellulose. Green Chemistry 2016, 18, (5), 1295-1305. 35. Driemeier, C.; Mendes, F. M.; Oliveira, M. M., Dynamic vapor sorption and thermoporometry to probe water in celluloses. Cellulose 2012, 19, (4), 1051-1063. 36. Pihlajaniemi, V.; Sipponen, M. H.; Kallioinen, A.; Nyyssola, A.; Laakso, S., Rate-constraining changes in surface properties, porosity and hydrolysis kinetics of lignocellulose in the course of enzymatic saccharification. Biotechnology for Biofuels 2016, 9, 18. 37. Medve, J.; Ståhlberg, J.; Tjerneld, F., Isotherms for adsorption of cellobiohydrolase I and II fromtrichoderma reesei on microcrystalline cellulose. Applied Biochemistry and Biotechnology 1997, 66, (1), 39-56. 38. Zhang, Y. H.; Lynd, L. R., Toward an aggregated understanding of enzymatic hydrolysis of cellulose: noncomplexed cellulase systems. Biotechnology and Bioengineering 2004, 88, (7), 797-824. 39. Zheng, Y.; Zhang, S.; Miao, S.; Su, Z.; Wang, P., Temperature sensitivity of cellulase adsorption on lignin and its impact on enzymatic hydrolysis of lignocellulosic biomass. Journal of Biotechnology 2013, 166, (3), 135-143. 40. Tu, M.; Chandra, R. P.; Saddler, J. N., Recycling cellulases during the hydrolysis of steam exploded and ethanol pretreated Lodgepole pine. Biotechnology Progress 2007, 23, (5), 1130-1137. 41. Kumar, R.; Wyman, C. E., Access of cellulase to cellulose and lignin for poplar solids produced by leading pretreatment technologies. Biotechnology Progress 2009, 25, (3), 807-819. 42. Kyriacou, A.; Neufeld, R. J.; MacKenzie, C. R., Effect of physical parameters on the adsorption characteristics of fractionated Trichoderma reesei cellulase components. Enzyme and Microbial Technology 1988, 10, (11), 675-681. 43. Fan, L. T.; Lee, Y.-H.; Beardmore, D. H., Mechanism of the enzymatic hydrolysis of cellulose: Effects of major structural features of cellulose on enzymatic hydrolysis. Biotechnology and Bioengineering 1980, 22, (1), 177-199. 44. Eibinger, M.; Bubner, P.; Ganner, T.; Plank, H.; Nidetzky, B., Surface structural dynamics of enzymatic cellulose degradation, revealed by combined kinetic and atomic force microscopy studies. The FEBS Journal 2014, 281, (1), 275-90. 45. Kumar, R.; Wyman, C. E., Cellulase adsorption and relationship to features of corn stover solids produced by leading pretreatments. Biotechnology and Bioengineering 2009, 103, (2), 252-67.
41 42
33 ACS Paragon Plus Environment
