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Molecular Enzymology and Drug Targets
Evaluating the Impact of Boosting on-site
Enzymes produced by Trichoderma reesei
RUT C30 and Aspergillus saccharolyticus with
Commercial Enzymes in lowering the use of
Commercial Cellulases
2015
Vol. 1 No. 1:3
Vandana, Rana.,
Birgitte K., Ahring.
Bioproducts, Sciences and Engineering
Laboratory (BSEL), Washington State
University Richland, WA 99354, USA
Corresponding author: Vandana, Rana.

[email protected]
Abstract
In-house production of cellulases from filamentous fungi is widely used, but their
hydrolytic efficiency compared to commercial enzymes is limited. We studied
the effect of supplementing in-house cellulases produced by Trichoderma reesei,
RUT C30 and a novel strain, Aspergillus saccharolyticus with different types of
commercial enzymes for the efficient hydrolysis of wet exploded loblolly pine.
Cellic®Ctec 2, Cellic®Htec2 and Novozym 188 were used as the commercial base
enzymes for supplementing the in-house produced enzymes. Compared to nonsupplemented in-house enzymes preparation, commercial enzymes (Cellic®Ctec2)
added in the same amount as FPU and CBU, resulted in 68% higher glucose yield
using wet exploded loblolly pine (WELP) at a 20% DM concetration. The highest
saccharifcation yield was achieved by supplementation of the in-house produced
cellulases with Cellic®Htec2 compared to Cellic®Ctec2 and Novozym 188. Optimal
glucose, xylose and mannose yields, 85%, 92% and 86%, respectively were achieved
by using in-house enzymes (15 FPU/g cellulose) supplemented with commercial
hemicellulase (7.5 FPU/g cellulose). These results showed that supplementing
in-house enzymes with commercial enzymes can be advantageous and work for
lowering the overall cost of enzymes in a biorefinery.
Keywords: Wet explosion; loblolly pine; Trichoderma reesei ; Aspergillus
saccharolyticus ; Cellic®Ctec2; Novozym 188
Introduction
Efficient conversion of lignocellulosic polysaccharides to
fermentable sugars is the key for commercial production of
biofuels [1,2]. Despite many technological improvements,
pretreatment and utilization of commercially available
lignocellulolytic enzymes to break the recalcitrant nature of
lignocellulosic biomass is limiting the viability of producing
biofuels [3-7]. Efficient and economically viable ethanol
production requires that all sugars produced from cellulose and
hemicellulose be converted to ethanol or other bioproducts [8,9].
Enzymatic hydrolysis plays a pivotal role in efficient conversion of
the cellulose and hemicelluloses fractions of pretreated biomass
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materials. The efficiency of enzymatic hydrolysis depends on the
pretreatment and right kind and proportions of enzyme cocktails
that are used.
Numerous studies to investigate the potential of softwood
conversion to ethanol have been done in the last two decades
[6,10,11]. Pretreatment conditions affect the requisite enzyme
mixture employed for polysaccharide hydrolysis. Previously,
acid and SO2 catalyzed steam explosion was extensively used to
pretreat softwood for enhancing the enzymatic hydrolysis [1217]. Among different pretreatment methods developed to date,
wet explosion (WEx) is considered one of the most appropriate
and cost effective methods for deconstruction of softwood and
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Molecular Enzymology and Drug Targets
has demonstrated unparalleled performance for fermentable
sugar production as discussed by Rana et.al. [18].
In-house enzyme production has recently gaining interest for
reducing the cost associated with enzymatic hydrolysis but
the efficiency of those crude enzymes is limited compared to
commercial enzymes. Still commercial enzymes are expensive
and have been problematic for the exploitation of lignocellulosic
biomass for production of biofuels [3,19]. In the present study
we examine the possibility of supplementing in-house produced
enzymes with small amounts of commercial enzymes to obtain
sufficient hydrolytic affects with reduces dosages of commercial
enzymes and thereby lowering the overall cost of enzymatic
hydrolysis.
Fungal derived cellulolytic enzymes were investigated extensively
in the last few decades for the hydrolysis of lignocelluloses
[20,21]. Lignocellulolytic enzymes can be produced by a
diverse group of fungi including ascomycetes (e.g. T. reesei),
basidiomycetes including white-rot fungi (e.g. P. chrysosporium),
brown-rot fungi (e.g. Fomitopsis palustris) and some anaerobic
species (e.g. Orpinomyces sp.) which degrade cellulose in the
gastrointestinal tract of ruminant animals [22]. Of these strains,
aerobic fungal strains, specifically T. reesei, is of interest as it
produces large amounts of extracellular cellulolytic enzymes
when grown in liquid culture. Lignocellulose degradation
requires three enzymatic components/domains: (endoglucanase,
cellobiohydrolase and β-glucosidase), however none of the fungal
strains including the best mutants described are able to produce
all three required enzyme components at the same time [23]. T.
reesei exhibits high Cellobiohydrolase (CBH) and Endoglucanase
(EG) activities but lack sufficient β-glucosidase activities [24,25].
Therefore, in order to achieve good cellulose hydrolysis, T. reesei
cellulases are typically supplemented with β-glucosidase from
Aspergillus niger.
Fungal strains displaying cellulolytic activity are capable of
degrading cellulose and have great potential to be used in a
consolidated biorefinery for cost-effective biofuels production
but their efficiency is generally low compared to commercial
enzymes. A substantial amount of research has been done
to investigate the potential benefit of accessory enzymes to
supplement commercial cellulases such as commercial xylanase,
arabinase, mannanase, pectinase, and other auxiliary enzymes
which can reduce the amount of enzymes required for efficient
biomass hydrolysis of acid or alkaline pretreated biomass. Alvira et
al. [26] observed that endoxylanase and a-L-arabinofuranosidase
supplementation during enzymatic hydrolysis of steam exploded
wheat straw increased the enzymatic hydrolysis yield by 10%.
According to Kumar et al. [27] an incremental increase in glucose
release was observed with xylanase supplementation. Varnai
et al. [28] observed that the addition of endo-β-mannanase
increased the overall hydrolysis yield by 20-25% of cellulose.
Despite a large research effort within this field there is still a
lack of understanding of the effect of supplementing low-cost inhouse enzymes with small amounts of commercial enzymes for
hydrolyzing pretreated biomass materials such as softwood.
The overall objective of this study is to evaluate the effect and
potential application of supplementing in-house produced
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Vol. 1 No. 1:3
enzymes with commercial enzymes for reducing the overall cost of
enzymatic hydrolysis without sacrificing the hydrolytic efficiency.
The proposed research is innovative as we use hydrolysis of
softwood which has proven to be more difficult than pretreating
agricultural residues. In this study, sugar yields from hydrolyzing
wet exploded loblolly pine under three different scenarios are
compared. Scenario 1- In-house produced enzymes, Scenario 2commercial enzymes and Scenario 3- in-house enzymes added
with commercial enzymes with the objective of finding the lowest
amount of commercial enzymes needed to achieve optimal
saccharification yields when using in-house produced enzymes.
Materials and Methods
Enzymes
Cellulolytic enzymes produced in-house from mutant fungi,
Trichoderma reesei RUT-C30, and novel fungal strain, Aspergillus
saccharolyticus (CBS 127449) were studied. These fungi were
used for cellulase and β-glucosidase production, respectively.
Three commercial enzymes were used to evaluate the effect
of supplementation or boosting. Cellic®Ctec2 and Cellic® Htec2
from Novozymes and Novozym188 from Sigma were employed.
Cellic®Ctec2, Cellic® Htec2 and Novozym 188 were used as a
source of cellulase, hemicellulase and β-glucosidase, respectively.
Measurement of enzymatic activities was performed using
different substrates. Cellulase activity was determined using
filter paper (Ghose 1987) and β-glucosidase activity using pNPG
(Flachner 1999) as substrates.
Cellulase activity (FPase) was assayed with glucose as the
standard. The assay mixture comprising 0.5 mL fermentation
broth sample, 1.0 mL citrate buffer (50 mM, pH 4.8) and Whatman
No. 1 filter disc was incubated at 50 oC for 60 min followed by
addition of 3 ml DNS reagent for color development and boiling
for 5 min. Absorbance was measured at 550 nm using UV
spectrophotometer. One unit of FPase activity was defined as the
amount of enzyme that released 1 µmol of glucose equivalent
per min per mL enzyme.
β-glucosidase activity was measured using 4-Nitrophenyl b-Dglucopyranoside, pNPG (Sigma Aldrich) as the substrate. 500 µl of
50 mM pNPG was mixed with 50 µl of enzyme solution in 50 mM
sodium citrate buffer (pH 4.8). After 10 min of incubation at 50oC,
the reaction was quenched by adding 1mL of 1 M ice-cold sodium
carbonate. Subsequently, the absorbance of 4-nitrophenol
produced during the reaction was measured at 405 nm using UVspectrophotometer. One unit (U) of BGL activity was defined as 1
µmol of 4-nitrophenol released per minute.
Raw material and wet explosion pretreatment
Representative samples of softwood (loblolly pine) chipped
to approximately quarter inch were obtained from Iowa State
University. The samples were screened for uniform chip size and
equilibrated to a moisture content less than 10% (w/w) prior
to compositional analysis and pretreatment. Wet explosion
pretreatment of loblolly pine was carried out in a 10L reactor
described in (Rana et al., 2012) at 170oC for 24 min and 5.5 bar
oxygen. These conditions were selected according to previous
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Molecular Enzymology and Drug Targets
optimization studies (data not published) and based on optimal
sugars recovery and hydrolysis yield.
For analytical purposes, representative samples of the wet
exploded loblolly pine slurry was filtered to obtain the solid
fraction which was washed multiple times with deionized water
and dried to obtain water insoluble solids (WIS). The filtered
liquid was used for soluble sugars and degradation products
determination. Chemical composition of both raw and
pretreated solids (WIS) was determined according to Sluiter et
al. [29]. The liquid fraction was hydrolyzed with 4% (v/v) H2SO4
for total sugars and sugar degradation products determination
according to the standardized methods of Laboratory Analytical
Procedures provided by National Renewable Energy Laboratory
(NREL, Golden, CO, USA) [30].
Preparation of inoculum
Inoculum cultures for fungal strain T. reesei RUT-C30 were
prepared from −80◦C glycerol stocks on agar plates containing 39
g L−1 yeast extract potato dextrose (YPD) and incubated static at
30◦C for 120 h. The resultant spore colonies from the plates were
covered with 10 mL of sterile distilled water and the suspensions
were made by gently probing the surface of the plate with the tip
of an inoculating loop. The composition of the medium was 30
g/l wheat bran, 25 g/l corn steep liquor (CSL), 5 g/l Avicel, 50 g/l
glucose, 30 g/l peptone and 5 g/l yeast extract in a flask with a
total volume of 150 ml. 50 ml of autoclaved mineral solution was
added to the media with the composition: 0.3 g/l of MgSO4.7H2O,
4 g/l of KH2PO4, 2 g/l (NH4)2SO4 and 0.3 g/l of CaCl2.2H2O. Media
was inoculated with 3 ml of spore’s suspension, cooled, and the
cultures were grown for 48 h at 30◦C in a rotary shake at 140 RPM
[31].
Inoculation media for A. saccharolyticus was prepared in 1000 ml
shake flasks with the active volume of 200 ml. The composition
of inoculation media was 40 g/l wheat bran, 40 g/l corn steep
liquor (CSL), 4 g/l peptone, 2 g/l yeast extract, 2 g/l casamino
acids, 12 g/l NaNO3, 1 g/l KCl, 1 g/l MgSO4 7H2O, 3 g/l KH2PO4,
0.1 g/l Na4 EDTA, 4.5 mg/l ZnSO4 7H2O, 22 mg/l H3BO3, 10 mg/l
MnCl2 4H2O, 10 mg/l FeSO4 7H2O, 3.4 mg CoCl2 6H2O, 3.2 mg/l
CuSO4 5H2O, 0.17 mg/l Na2MoO4 2H2O. The media was sterilized
by autoclave at 121°C, for 15 min. The inoculation media was left
to equilibrate in a shake incubator set to operate at 28°C, 140
RPM. After equilibration the media was inoculated using 3 ml
spore suspension and the cultures were grown for 48 h at 30◦C
and pH 4.8 in a rotary shaker.
Enzyme fermentation
For enzyme production from T. reesei RUT-C30, 150 mL of the
pre-culture was used to inoculate a 3 L stirred reactors. The
airflow was kept at 1.2 L min−1 and the stirrer speed at 800 rpm.
The culture conditions were maintained at 28◦C and at pH 3.75
by automatic addition of 5 N (NH4)OH. The medium composition
was as follows: 2.5% (DM) wet exploded-alkali pretreated corn
stover 2.5 % corn steep liquor, 2.5% wheat bran, 0.05 % yeast
extract, 0.3% peptone, 5% corn mash(liquefied with α-amylase
to contain dextrin). The final volume was 1.8 L and the cells were
cultured for 24 h until the ethanol was consumed (seen on the
carbon dioxide drop in the off-gas), after which the corn mash
© Copyright iMedPub
2015
Vol. 1 No. 1:3
(with 33% M.C.) was fed into the reactor in a fed-batch mode with
an average initial dilution rate of 0.007 h-1. For saccharification
of maltodextrin to D-glucose in corn mash, 300 µl of glucoamylase per liter of mash was mixed in the feed. To prevent from
bacterial growth during fermentation, 0.1% v/v of kanamycin was
introduced to both of the reactor and feed bottle.
β-glucosidase production from A. saccharolyticus was conducted
in a stirred reactor (5 L) equipped with online control system for
adjustment of pH, temperature, antifoam, agitation and dissolved
oxygen level (DO). The fermentation was performed in a feed
batch setup. 800ml of inoculation media was used as a startup
media, the media was added to the 5L reactor for sterilization
by autoclave, 121°C for 15 min. After sterilization the reactor
was stabilized for 4 hours at 28 °C, pH 4.8, 800 RPM, aeration
set-point was 0.7L air/L/m. The reactor was seeded using 200ml
of inoculum as seed culture and, at 24 hours of operation the
feed was started at 22 ml/h, total fermentation time was 8 days.
The enzyme containing liquid was filtered and consequently
concentrated 10 times by rotary vacuum evaporation.
Enzymes and activities
Celluclast 1.5 L and Novozyme 188, were obtained from Sigma
Aldrich. Filter-paper and Carboxymethyl cellulose activities were
used as a measure of cellulase activity. FPA 4.49 FPU/mL and
CMCase to 20.6 U/mL was measured after 7 days of fermentation.
β-glucosidase activity on pNPG was measured as 4.77 U/mL
and xylanase activity as 6.61 nkat/mL. Commercial cellulase
(celluclast 1.5 L) showed 81.8 FPU/mL filter paper activity,
β-glucosidase activity of 58.66 U/mL and xylanase activity 107.3
nkat/mL. PNPG activities (β-glucosidase) in A. saccharolyticus and
in commercial enzyme Novozym 188 were 339.9 U/mL and 698.3
U/mL, respectively.
Enzymatic hydrolysis of wet exploded loblolly
pine (WELP)
Enzymatic hydrolysis assays were carried out in 125 mL shake
flasks at 50 oC with agitation at 150 rpm at pH 5 in a shaking
incubator. The total working volume was 50 mL and all the
experiments were performed in quadruplicate with standard
deviations less than ±2. WELP was mixed with citrate buffer (50
mM, pH 4.8) and appropriate amount of enzymes to achieve total
solids 20%. All the enzymatic hydrolysis assays were conducted
at 20% (w/w) total solids at the above mentioned conditions
for 72 h. After 72 h, enzymatic hydrolysis was stopped. Samples
were withdrawn periodically at 24, 48 and 72 h, centrifuged using
bench-top centrifuge (Eppendorf, Model 5804, 8000 rpm, and 10
min) and analyzed for sugars and sugar degradation compounds.
Constant amounts of in-house produced cellulase (15 FPU/g
cellulose from T. reesei RUT C30 and 30 CBU/g cellulose from
A. saccharolyticus) was used in all the experiment with varying
amounts of commercial enzymes supplementation, ranging
between 1% to 100% of in-house enzymes (FPU or CBU).
Two controls were used, control-1, in-house produced enzymes
without any supplementation with loading of 15 FPU + 30 CBU per
gram cellulose and control-2 commercial enzyme, Cellic®Ctec2
with loading of 45 FPU per gram cellulose. 45 FPU/g cellulose
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Molecular Enzymology and Drug Targets
of Cellic®Ctec2 represents the sum of both filter paper as well
as β-glucosidase activities. For the supplementation assays,
commercial cellulase, hemicellulase and β-glucosidase were
used. Cellic®Ctec2, Cellic®Htec2 were used in a dosage ranging
from 1% to 100% of the total FPU (15 FPU) of the in-house
enzymes. Novozym 188 was used with dosage ranging from 1%
to 100% of total CBU (30 CBU) of in-house enzymes.
Analytical methods and calculations
Sugars concentration were measured by high-performance liquid
chromatography using Bio-Rad (Hercules, CA, USA) Aminex HPX
87P column with RI detector, operating at 83 0C with a flow rate
of 1.0 ml/min with Milli-Q water (Barnstead Nanopure, USA) as
mobile-phase.
Sugars degradation compounds (furfural, 5-hydroxymethylfurfural
(HMF) and acetic acid) and ethanol were analyzed using an
Aminex ion exclusion HPX-87H cation-exchange column (BioRad, Hercules, CA) at 60oC equipped with 1050 photodiode-array
detector (Agilent Technologies, Santa Clara, CA). 89% 5 mM/L
H2SO4 and 11% acetonitrile were used as mobile phase at a flow
rate of 0.6 mL/min were used.
Enzymatic hydrolysis yields were calculated based on the amount
of the reducing sugars released in the hydrolysate divided by the
respective carbohydrate present in raw loblolly pine multiplied
with correction factor of 0.9 for hexose and 0.88 for pentoses (to
correct the increased weight from hydrolysis), as shown in the
following equation:
Enzymatic hydrolysis yield%=(Reducing sugar (g) × 0.9 (or 0.88) ×
100)/Polysaccharide (g)
Mean values and standard deviations were calculated from
quadruplicates.
Results and Discussion
Enzyme activities
Cellulase preparations from in-house produced enzymes
preparations were characterized by measuring different
enzymatic activities. Filter paper activity of 4.49 FPU/mL and
activity on PNPG (as a measure of β-glucosidase activity) was
measured as 4.77 U/mL from cellulase produced in-house from
T. reesei RUT C30. A. saccharolyticus presented practically no FPA
activity and 339.9 U/mL of β-glucosidase activity. It was observed
that T. reesei RUT C30 exhibited insignificant β-glucosidase
activity and that A. saccharolyticus has low filter paper activity.
Higher cellulases (FPU) were detected in Cellic®Ctec 2 and
Cellic®Htec2, 100 and 94.3 FPU/mL, respectively. β-glucosidase
activity in these enzymes were 396.3 U/mL and 312.1 U/mL,
respectively. Higher b-glucosidase activity was detected in
Novozym 188, 698.3 U/mL
Raw material and Wet explosion pretreatment
of loblolly pine
Table 1 shows the composition of WIS and liquid fractions. Wet
explosion pretreatment resulted in solubilization of hemicellulose
into the liquid stream and a higher proportion of cellulose (47.2%)
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Vol. 1 No. 1:3
was left in pretreated WIS. 2.4 g/L glucose and 8.2 g/L xylose
were recovered in the liquid fraction; however some sugars were
degraded to HMF (0.7 g/L, derived from hexoses) and furfural (1.3
g/L, derived from pentoses). Acetic acid (3.6 g/L) was released
from acetyl groups contained in the hemicellulose.
Some authors suggest inhibitors released during pretreatment
could have a negative effect on the enzymatic hydrolysis and
subsequent fermentation thus washing after pretreatment
is preferred [32-36]. However, considering the capital cost of
filtration and washing and losses of potential fermentable sugars
due to washing, utilization of the whole slurry for the enzymatic
hydrolysis may be more practical. Our results further show that
very low concentrations of sugar degradation products were
produced, therefore we used whole slurry after wet explosion
pretreatment for the enzymatic hydrolysis to increase the
concentration of fermentable sugars. Higher concentrations of
sugars result in higher ethanol concentration in the subsequent
fermentation step which reduces the downstream distillation and
concentration costs [37,38].
Enzymatic hydrolysis
Effect of Cellic®Ctec2 supplementation on in-house produced
enzymes for WELP hydrolysis
The in-house produced enzymatic preparation was supplemented
with Cellic®Ctec2 in amounts ranging between 1% and 100%
of total FPU/g cellulose of in-house enzyme preparation used.
Figures 1-3 show the glucose, xylose and mannose production
during 72 h of enzymatic hydrolysis with boosting from different
commercial enzymes (Cellic®Ctec2) for hydrolyzing WELP. It was
observed that supplementation with commercial Cellic®Ctec2
improved the sugars released after 72 h hydrolysis compared to
Table 1 Raw and pretreated composition of loblolly pine.
Pretreated Loblolly pine (175 Raw Loblolly
0C, 24 min and 5.5 bar O2)
pine
WIS (% dry weight)
Glucan
47.24 ± 0.06
35.97 ± 0.46
Xylan
3.76 ± 0.01
8.5 ± 0.61
Galactan
0.85 ± 0.02
2.47 ± 0.40
Arabinan
0.85 ± 0.03
1.57 ± 0.16
Mannan
3.36 ± 0.04
8.15 ± 0.23
Lignin
47.1 ± 1.34
30.65 ± 0.78
Ash
0.19 ± 0.07
0.77 ± 0.05
Sugars in prehydrolyzate (g/100g raw material)
Glucose
2.36 ± 0.44
Xylose
8.16 ± 1.00
Galactan
1.61 ± 0.07
Arabinan
2.51 ± 0.19
Mannan
0.28 ± 0.13
Acid soluble
3.98 ± 0.27
lignin
Degradation products (% dry weight)
Acetic acid
3.58 ± 0.98
HMF
0.71 ± 0.37
Furfural
1.29 ± 0.45
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Molecular Enzymology and Drug Targets
Figure 1:
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Vol. 1 No. 1:3
Glucose, xylose and mannose yields after 72 h of hydrolysis for commercial cellulase, Cellic®Ctec2
supplementation of constant in-house produced cellulase loadings for wet exploded loblolly pine.
Figure 2: Glucose, xylose and mannose yields after 72 h of hydrolysis for commercial hemicellulase, Cellic®Htec2
supplementation of constant in-house produced cellulase loadings for wet exploded loblolly pine.
Figure 3
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Glucose, xylose and mannose yields after 72 h of hydrolysis for commercial β-glucosidase, Novozym 188
supplementation of constant in-house produced cellulase loadings for wet exploded loblolly pine.
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Molecular Enzymology and Drug Targets
2015
Vol. 1 No. 1:3
the control mixture (Figure 1). The highest enzymatic hydrolysis
yields were achieved with 100% supplementation; interestingly,
increasing the supplementation beyond 30% only led to modest
increases in yields. Supplementation with commercial cellulase
not only improved cellulose conversion but also hemicellulose
conversion, which demonstrates the ability of the commercial
enzyme in improving the overall hydrolysis performance.
17% in glucose, xylose and mannose yields, respectively. At 100%
supplementation the highest sugars yields were found giving
68%, 21% and 18%, of glucose, xylose and mannose, respectively.
In general, Cellic®Ctec2 (cellulases) supplementation resulted in
higher production of glucose compared to hemicellulose. These
results were statistically supported by Tukey HSD analysis with
95% confidence level (data not shown).
Concentrations of glucose, xylose, and mannose were determined
to be 25.39, 14.33, 13.87 mg/mL (Figures 4-6) corresponding
to 32%, 74% and 77% yields, respectively using our nonsupplemented enzyme in-house enzyme cocktail (control-1).
At 1% supplementation a slight change in yield was observed
compared to control-1, giving an increase in glucose, xylose
and mannose concentrations of 13%, 11% and 9%, respectively.
At 10% supplementation, the glucose, xylose and mannose
concentration increased by 32%, 14% and 13%, respectively after
72 h and the corresponding increases for 30% addition were 46%,
15% and 15% at 30%, respectively. Only small increases in the
sugar release was found between 30% and 50% supplementation
as shown in Figure 1, demonstrating an increase of 57%, 17% and
Cellic®Ctec2 is produced by Trichoderma spp., which secretes an
array of lignocellulolytic enzymes including cellulases, xylanases,
abrabinosidases and β-xylosidase [39]. Mainly they exhibit
cellulase activity while other enzymes are expressed at low levels.
Our results clearly show that high cellulase activity improves
the enzymatic hydrolysis yields during hydrolysis of pretreated
loblolly pine.
6
Effect of Cellic®Htec2 supplementation on in-house produced
enzymes for WELP hydrolysis
Cellic®Htec2 (1% to 100%) was supplemented with our in-house
preparation: cellulases (15 FPU/g cellulose) and β-glucosidase (30
CBU/g cellulose) and results shown in Figure 2 demonstrated that
Figure 4
Sugars (g/L) released after 72 h of enzymatic hydrolysis as an effect of in-house produced cellulase
supplementation with commercial cellulase, Cellic®Ctec2 from wet exploded loblolly pine.
Figure 5
Sugars (g/L) released after 72 h of enzymatic hydrolysis as an effect of in-house produced cellulase
supplementation with commercial hemicellulase, Cellic®Htec2 from wet exploded loblolly pine.
This article is available from: www.medt.com.es
2015
Molecular Enzymology and Drug Targets
hemicellulase supplementation improved the enzymatic hydrolysis
yield of WELP. Glucose production increased by 23%, 44%, 50%,
63% and, 68% with 1, 10, 30, 50 and 100% supplementation of
in-house enzymes with Cellic®Htec2 respectively, compared
to the non-supplemented control. For xylose production,
supplementation resulted in an increase of 8%, 14%, 8%, 20% and,
25% with 1, 10, 30, 50 and 100% supplementation, respectively,
compared to the non-supplemented control. Mannose
production increased by 5%, 7%, 11%, 11% and, 18% with 1, 10,
30, 50 and 100% supplementation, respectively, compared to nonsupplemented control. Cellic®Htec2 supplementation resulted in
higher concentration of glucose, xylose and mannose compared
to the non-supplemented control, and these differences were
Figure 6
Vol. 1 No. 1:3
significant at 95% confidence level of statistical analysis. With
lower level of supplementation, lower yields were obtained;
this clearly shows that the recalcitrance of loblolly pine unlike
agricultural biomass such as corn stover demands a very active
enzyme cocktail for good hydrolysis of pretreated softwood [40].
The hemicellulose degradation due to synergistic effect of
xylanases, arabinases and mannanase prevents the blocking
effect of pentose sugars. Arabinan supposedly, blocks the
access to β-1,4 xylan chain for xylanases [41]. Furthermore,
xylooligomers has previously been found to have an inhibitory
effect on cellulase action [27,42,43]. Therefore, removal of
oligomers could improve production of xylose, arabinose and
mannose and also minimize the inhibitory action on cellulase.
Sugars (g/L) released after 72 h of enzymatic hydrolysis as an effect of in-house produced cellulase
supplementation with commercial β-glucosidase, Novozym 188 from wet exploded loblolly pine.
Table 2. Cellulose conversions from hydrolysis of wet exploded loblolly pine by in-house cellulase preparations supplemented with
commercial enzymes.
Enzyme
In-house cellulase-Control-1
Cellic®Ctec2 supplementation
Cellic®Htec2 supplementation
Novozym 188 supplementationa
Commercial cellulose-Control-2
(Cellic®Ctec2)
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Code
Supplementation
100
1
2
3
4
5
11
12
13
14
15
21
22
23
24
25
No
1%
10%
30%
50%
100%
1%
10%
30%
50%
100%
1%
10%
30%
50%
100%
200
No
Enzyme loading Enzyme loading
(FPU)
(CBU)
15
30
15.2
30
16.5
30
19.5
30
22.5
30
30
30
15.2
30
16.5
30
19.5
30
22.5
30
30
30
15
30.3
15
33
15
39
15
45
15
60
45
0.00*
CCb
CCc
0.76
2.41
2.82
3.02
3.27
3.32
2.71
3.43
3.24
3.79
3.3
2.24
2.43
2.48
2.53
2.64
0.38
0.44
0.56
0.71
0.88
1.19
0.49
0.68
0.76
1.02
1.19
0.4
0.4
0.34
0.3
0.24
Total celluloseb
conversion (g)
11.44
13.14
16.72
21.17
26.46
35.79
14.76
20.35
22.7
30.71
35.6
12.11
13.1
13.37
13.64
14.23
2.2
-
35.63
7
Molecular Enzymology and Drug Targets
It has been observed that supplementation of cellulases with
xylanases enhances the glucose release from pretreated biomass
solids [44-46]. Moreover, xylanase supplementation is more
efficient when xylan is present in lower amounts in pretreated
material [45] which is true for the present study. Overall, our
results would suggest that supplementation of the cellulases with
hemicellulase has a great impact on enzymatic hydrolysis yields
and process efficiency.
Effect of Novozym 188 supplementation on inhouse produced enzymes for WELP hydrolysis
Figure 3 shows glucose, xylose and mannose yields after
enzymatic hydrolysis of WELP supplemented with Novozym
188. Novozym 188 contains high amounts of β-glucosidase and
helps in cellobiose conversion to glucose and also prevents endproduct inhibition of cellulase due to accumulation of cellobiose.
After 72 h, glucose yield increased by 6%, 13%, 14%, 16% and,
20% ; xylose yield increased by 5%, 6%, 9%, 13% and, 17% and
mannose yield increased by 2%, 3%, 6%, 10% and, 14% with 1,
10, 30, 50 and 100% supplementation, respectively, compared to
the non-supplemented control. Glucose increase was supported
statistically at the 95% confidence level. Less difference was
observed in xylose and mannose production. Novozym 188
supplementation resulted in slightly higher concentration
of xylose and mannose compared to the non-supplemented
control, however, these differences were not significant at the
95% confidence level of statistical analysis.
A parallel study of enzymatic hydrolysis with only commercial
cellulases, Cellic®Ctec2 (Control-2) was conducted. An enzyme
loading of 45 FPU/mL was used for hydrolysis. Glucose, xylose
and mannose concentrations after 72 h of hydrolysis were 79.1,
18.5 and 16.0 mg/mL corresponding to 99%, 96% and 89% yields,
respectively. Comparing the yields control-1 (in-house enzymes
without any supplementation) and control-2 (commercial
enzyme only), we found that in-house enzymes supplemented
with commercial enzymes resulted in higher conversion of
polysaccharides after wet explosion pretreatment of loblolly
pine. 15FPU/mL in-house enzymes boosted with 15FPU/mL
commercial cellulase, Cellic®Ctec2 resulted in 99%, 93% and 93%
of glucose, xylose and mannose yields, respectively. When 15FPU/
mL in-house enzymes was boosted with 15FPU/mL commercial
hemicellulase, Cellic®Htec2, glucose, xylose and mannose yields
reached to 99%, 99% and 94%, respectively. Similar cellulose
conversion was achieved with Cellic®Ctec2 and Cellic®Htec2 as
can be explained by similar FPA activities (100 and 94.3 FPU/mL,
respectively), but higher yields of xylose and mannose was found
with hemicellulase, Cellic®Htec2.
Table 2 summarizes cellulose conversions from hydrolysis of
WELP. Cellulose conversion was increased with increasing
supplementation of all three commercial enzymes. Probably due
to insufficient hemicellulolytic activity, higher accumulation of
hemicellulosic oligomers was observed with non-supplemented
enzyme preparations.
The best enzymatic hydrolysis yields were obtained when the
in-house cellulase cocktail was supplemented with Cellic®Htec2.
When 15 FPU/mL of in-house cellulase was boosted with
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Vol. 1 No. 1:3
7.5 FPU/mL of commercial hemicellulase, Cellic®Htec2 (50%
supplementation), 85% glucose, 92% xylose and 86% mannose
were produced. Contrary to some previous studies [15,47,48],
higher yields (>70%) of glucose can be obtained with high
loadings (30-80 FPU/g cellulose) of commercial enzymes,. We
obtained higher glucose yield (85%) with lower FPU/g cellulose.
Increasing the supplementation from 50% to 100% resulted in
a near complete sugars conversion. Furthermore, the enzymes
will remain functional during fermentation and therefore
able to convert remaining sugars to monomers along with the
fermentation which means that any remaining cellulose can be
converted to glucose during subsequent fermentation step [49].
1% supplementation of in-house enzymes with Cellic®Ctec2,
Cellic®Htec2 and Novozym 188 showed glucose yields of 37%,
41% and 34% corresponding to increase in glucose yield from
non-supplemented control-1(in-house enzymes) as 13%,
23% and 6% respectively. 100% supplementation of in-house
enzymes with Cellic®Ctec2, Cellic®Htec2 and Novozym 188
resulted into glucose yield of 99%, 99% and 40% respectively. At
highest supplementation (100%), Cellic®Ctec2 and Cellic®Htec2
resulted into similar yield of 99%, and that could be explained
as, Cellic®Ctec2 primarily focuses on cellulose degradation while
Cellic®Htec2 degrades hemicellulose and thus releases cellulose
for cellulase attack. Also, higher enzymes/substrate ratio
eliminates enzyme inhibition due to substrate characteristics.
50% supplementation, of in-house enzymes with Cellic®Ctec2,
Cellic®Htec2 and Novozym 188 resulted in glucose yield of 74%,
85% and 38%.
Considering the amount of enzymes used and yield, 50%
supplementation (7.5 FPU) with Cellic®Htec2 to in-house produced
enzymes cocktail (15 FPU) was determined as optimal. Thus it is
imperative that in-house produced cellulases from mesophilic
fungi and commercial available cellulase formulations can be
made to work synergistically as a lignocellulolytic enzyme system,
providing better saccharification yields from lignocellulosic
biomass for a lower cost than using expensive commercial
enzymes alone. It is important to note that although the focus in
this study was on monomeric sugars release enzymatic hydrolysis
for longer period (more than 72 h) will likely result in higher sugar
yields. Further studies are needed to evaluate the effect of other
important factors such as pH, temperature and hydrolysis period
on the efficiency of mixed enzymes cocktail.
Conclusion
The supplementation of cellulase mixtures produced in-house
with commercial enzymes was shown to have potential for
improving the enzymatic hydrolysis of softwood. This study
indicated that wet explosion pretreatment can provide good
raw material for enzymatic hydrolysis with in-house produced
cellulases (T. reesei RUT C30 and A. saccharolyticus), however, for
saccharification at high solids loading of recalcitrant biomasses
such as softwood, supplementation with commercial enzymes
can be advantageous. Supplementation with commercial
enzymes and in particular the hemicellulase, Cellic® Htec2
significantly improved the sugar release by enzymatic hydrolysis.
Higher hydrolysis yields (glucose, 85%; xylose, 92%; mannose,
This article is available from: www.medt.com.es
Molecular Enzymology and Drug Targets
86%) were achieved when in-house cellulase was supplemented
with commercial hemicellulase, Cellic®Htec2 in a ratio of
15FPU:7.5FPU per mL. Our findings show ways for potentially
© Copyright iMedPub
2015
Vol. 1 No. 1:3
lowering the cost of enzyme hydrolysis in biorefinery. However,
further studies are needed to fully exploit the use of in-house
enzymes supplemented with commercial enzymes.
9
Molecular Enzymology and Drug Targets
Acknowledgement
National Advanced Biofuel Consortium (NABC) and Department
of Energy, USA Grant ZFT04064401 are gratefully acknowledged
for the financial support.
References
1 Wyman, C. E. What is (and is not) vital to advancing cellulosic ethanol.
Trends Biotechnol 2007; 25: 153-157.
2 Yang, B., Wyman, C. E. Pretreatment the key to unlocking low-cost
cellulosic ethanol. Biofuels, Bioproducts and Biorefining 2008; 2: 26-40.
3 Merino, S. T., Cherry, J. Progress and challenges in enzyme
development for biomass utilization. Adv Biochem Eng Biotechnol
2007; 108: 95-120.
4 Tu, M., Chandra R.P., Saddler J.N. Recycling cellulases during the
hydrolysis of steam exploded and ethanol pretreated Lodgepole pine
Biotechnol Prog 2007; 23: 1130-1137.
5 Wingrer ,A., Galbe, M., Roslander, C., Rudolf, A., Zacchi, G., Effect
of reduction in yeast and enzyme concentrations in a simultaneoussaccharification-and-fermentation-based
bioethanol
process:
technical and economic evaluation. Appl Biochem Biotechnol 2005;
121-124: 485-499.
6 Von Sivers, M., Zacchi, G. A. techno-economical comparison of three
processes for the production of ethanol from pine. Bioresource
Technology 1995; 51: 43-52.
7 Nguyen, QA., Saddler, JN. An integrated model for the technical and
economic evaluation of an enzymatic biomass conversion process.
Bioresource Technol 1991; 35: 275-282.
8 Hahn-Hägerdal, B., Karhumaa, K., Fonseca, C., Spencer-Martins,
I. Gorwa-Grauslund, M.F . Towards industrial pentose-fermenting
yeast strains. Appl Microbiol Biotechnol 2007; 74: 937-953.
9 Tomás-Pejó, E., Oliva J,M., Ballesteros, M., Olsson, L. Comparison of
SHF and SSF processes from steam-exploded wheat straw for ethanol
production by xylose-fermenting and robust glucose-fermenting
Saccharomyces cerevisiae strains. Biotechnology and Bioengineering
2008; 100 :1122-1131.
10 Boussaid, A., Robinson, J., Cai, Yj., Gregg, DJ., Saddler, JN.
Fermentability of the hemicellulose-derived sugars from steamexploded softwood (douglas fir) Biotechnol Bioeng 1999; 64: 284289.
11 Schell, D., Nguyen, Q., Tucker, M., Boynton, B., Pretreatment of
softwood by acid-catalyzed steam explosion followed by alkali
extraction. Appl Biochem Biotechnol 1998; 70-72: 17-24.
12 Boussaid, AL., Esteghlalian, AR., Gregg, DJ., Lee, KH., Saddler, JN.,
Steam pretreatment of Douglas-fir wood chips. Can conditions for
optimum hemicellulose recovery still provide adequate access for
efficient enzymatic hydrolysis Biochem Biotechnol 2000; 84-86: 693705.
13 Yang, B., Boussaid, A., Mansfield, SD., Gregg, DJ., Saddler, JN., Fast
and efficient alkaline peroxide treatment to enhance the enzymatic
digestibility of steam-exploded softwood substrates. Biotechnol
Bioeng 2002; 77: 678-684.
14 Kumar, L., Chandra, R., Chung, PA., Saddler, J., Can the same steam
pretreatment conditions be used for most softwoods to achieve
good, enzymatic hydrolysis and sugar yields Bioresour Technol 2010;
101: 7827-7833.
10
2015
Vol. 1 No. 1:3
15 Wu, M., Chang, K., Gregg, D., Boussaid, A., Beatson, R., Saddler, J.
Optimization of steam explosion to enhance hemicellulose recovery
and enzymatic hydrolysis of cellulose in softwoods. Appl Biochem
Biotechnol 1999; 77: 47-54.
16 Ramos, LP., Breuil, C., Saddler, JN. Comparison of steam pretreatment
of eucalyptus, aspen, and spruce wood chips and their enzymatic
hydrolysis. Appl Biochem Biotechnol 1992; 35: 37-48.
17 Mackie, KL., Brownell, HH., West, KL., Saddler, JN. Effect of Sulphur
Dioxide and Sulphuric Acid on Steam Explosion of Aspenwood.
Journal of Wood Chemistry and Technology 1985; 5: 405-425.
18 Rana, D., Rana, V., Ahring, BK. Producing high sugar concentrations
from loblolly pine using wet explosion pretreatment. Bioresour
Technol 2012; 121: 61-67.
19 Hespell, R., O’Bryan, P., Moniruzzaman, M., Bothast, R. Hydrolysis by
commercial enzyme mixtures of AFEX-treated corn fiber and isolated
xylans. Appl Biochem Biotechnol 1997; 62: 87-97.
20 Saddler, J. N. G., D. J. Ethanol production from forest product wastes.
London Taylor & Francis Ltd 1998.
21 Persson, I., Tjerneld, F., Hahn-Hägerdal, B. Fungal cellulolytic enzyme
production A review. Process Biochemistry 1991; 26: 65-74.
22 Ljungdahl, L. G. The cellulase/hemicellulase system of the anaerobic
fungus Orpinomyces PC-2 and aspects of its applied use. Ann N Y
Acad Sci 2008; 1125: 308-321.
23 Dashtban, M., Schraft, H., Qin, W. Fungal bioconversion of
lignocellulosic residues; opportunities & perspectives. Int J Biol Sci
2009; 5: 578-595.
24 Stockton, B. C., Mitchell, DJ., Grohmann, K., Himmel, ME. Optimum
beta-D-glucosidase supplementation of cellulase for efficient
conversion of cellulose to glucose. Biotechnol Lett 1991; 13: 57-62.
25 Kumar, R., Singh, S., Singh, O. V. Bioconversion of lignocellulosic
biomass: biochemical and molecular perspectives. Ind Microbiol
Biotechnol 2008; 35: 377-391.
26 Alvira, P., Negro, MJ., Ballesteros, M. Effect of endoxylanase and αL-arabinofuranosidase supplementation on the enzymatic hydrolysis
of steam exploded wheat straw. Bioresour Technol 2011; 102: 45524558.
27 Kumar, R., Wyman, C. E. Effect of xylanase supplementation of
cellulase on digestion of corn stover solids prepared by leading
pretreatment technologies. Bioresour Technol 2009; 100: 42034213.
28 Várnai, A., Huikko, L., Pere, J., Siika-Aho, M., Viikari, L. Synergistic
action of xylanase and mannanase improves the total hydrolysis of
softwood. Bioresour Technol 2011; 102: 9096-9104.
29 Sluiter, J.B., Ruiz, R. O., Scarlata, C. J., Sluiter, A. D., Templeton DW
Compositional analysis of lignocellulosic feedstocks. Review and
description of methods. J Agric Food Chem 2010; 58: 9043-9053.
30 Ruiz, R. E. T. Dilute acid hydrolysis procedure for determination of
total sugars in the liquid fraction of process samples. In: Laboratory
analytical procedure. 1996; Golden, CO, USA.
31 Rana, V., Eckard, A. D., Teller, P., Ahring, B. K. On-site enzymes
produced from Trichoderma reesei RUT-C30 and Aspergillus
saccharolyticus for hydrolysis of wet exploded corn stover and
loblolly pine. Bioresour Technol 2014; 154: 282-289.
32 Cantarella, M., Cantarella, L., Gallifuoco, A., Spera, A., Alfani, F. Effect
of inhibitors released during steam-explosion treatment of poplar
This article is available from: www.medt.com.es
Molecular Enzymology and Drug Targets
wood on subsequent enzymatic hydrolysis and SSF. Biotechnol Prog
2004; 20: 200-206.
33 Palmqvist, E., Hahn-Hägerdal, B. Fermentation of lignocellulosic
hydrolysates. II: inhibitors and mechanisms of inhibition. Bioresource
Technology 2000; 74: 25-33.
34 Tengborg, C., Stenberg, K., Galbe, M., Zacchi, G., Larsson, S., et al.
Comparison of SO2 and H2SO4 impregnation of softwood prior
to steam pretreatment on ethanol production. Appl Biochem
Biotechnol 1998; 72: 3-15.
35 Gregg, D. J., Saddler, J. N. Factors affecting cellulose hydrolysis and
the potential of enzyme recycle to enhance the efficiency of an
integrated wood to ethanol process. Biotechnol Bioeng 1996; 51:
375-383.
36 Schwald, W., Breuil, C., Brownell, H. H., Chan, M., Saddler, J. M.
Assessment of pretreatment conditions to obtain fast complete
hydrolysis on high substrate concentrations. Appl Biochem
Biotechnol 1989; 21: 29-44.
37 Zacchi, G., Axelsson, A. Economic evaluation of preconcentration in
production of ethanol from dilute sugar solutions. Biotechnol Bioeng
1989; 34: 223-233.
38 Larsson, M., Galbe, M., Zacchi, G. Recirculation of process water in
the production of ethanol from softwood. Bioresource Technology
60: 143-151.
39 Banerjee, G., Scott-Craig, J., Walton, J. Improving Enzymes for
Biomass Conversion: A Basic Research Perspective. Bioenerg Res
2010; 3: 82-92.
40 Kim, S., Holtzapple, M. T. Effect of structural features on enzyme
digestibility of corn stover. Bioresour Technol 2006; 97: 583-591.
41 Raweesri, P., Riangrungrojana, P., Pinphanichakarn. P. alpha-LArabinofuranosidase from Streptomyces sp. PC22: purification,
© Copyright iMedPub
2015
Vol. 1 No. 1:3
characterization and its synergistic action with xylanolytic enzymes
in the degradation of xylan and agricultural residues. Bioresour
Technol 2008 ; 99: 8981-8986.
42 Qing, Q., Wyman, C. E. Supplementation with xylanase and βxylosidase to reduce xylo-oligomer and xylan inhibition of enzymatic
hydrolysis of cellulose and pretreated corn stover. Biotechnol
Biofuels 2011; 4: 18.
43 Qing, Q., Yang, B., Wyman, C. E. Xylooligomers are strong inhibitors
of cellulose hydrolysis by enzymes. Bioresour Technol 2010; 101:
9624-9630.
44 Tabka, M. G., Herpoël-Gimbert, I., Monod, F., Asther, M., Sigoillot,
J. C. Enzymatic saccharification of wheat straw for bioethanol
production by a combined cellulase xylanase and feruloyl esterase
treatment. Enzyme and Microbial Technology 2006; 39: 897-902.
45 García-Aparicio, M., Ballesteros, M., Manzanares, P., Ballesteros,
I., Gonzálezm, A., et al. Xylanase Contribution to the Efficiency of
Cellulose Enzymatic Hydrolysis of Barley Straw. In: J. Mielenz, K. T.
Klasson, W. Adney,J. McMillan editors. Applied Biochemistry and
Biotecnology: Humana Press 2007 ; p. 353-365.
46 Yu, P., McKinnon, J. J., Maenz, D. D., Olkowski, A. A., Racz, V. J. et
al. Enzymic release of reducing sugars from oat hulls by cellulase,
as influenced by Aspergillus ferulic acid esterase and trichoderma
xylanase. J Agric Food Chem 2003; 51: 218-223.
47 Monavari, S., Galbe, M., Zacchi, G. Impact of impregnation time and
chip size on sugar yield in pretreatment of softwood for ethanol
production. Bioresour Technol 2009; 100: 6312-6316.
48 Cullis, I. F., Saddler, J. N., Mansfield, S. D. Effect of initial moisture
content and chip size on the bioconversion efficiency of softwood
lignocellulosics. Biotechnol Bioeng 2004; 85: 413-421.
49 Rana, V., Rana, D., Ahring, B. Process Modeling of Enzymatic
Hydrolysis of Wet-Exploded Corn Stover. Bioenerg Res 2013; 1-10.
11