Acknowledgements
The authors thank the postgraduate students and laboratory technicians
who contributed to the work and the BIOPLAN-DBQ-UEM and the FPB/B group,
Wood Science Department-UBC. Graphical abstract was created with
BioRender.
Introduction
Climate change, resulting from the increasing emission of greenhouse
gases, has a great impact on all living organisms. The
CO2 atmospheric concentration
([CO2]) is predict to increase to 550 μmol
mol-1 by 2050 while the average global temperature has
risen by 0.85 °C comparing to pre-industrial era (IPCC 2019). In this
way, lignocellulosic biomass (LCB) is a promising and sustainable method
to reduce our carbon footprint meanwhile improving the efficiency of
energy production to meet our ever-increasing energy demand (Fatmaet al. 2018). LCB consist of approximately 75% of
polysaccharides that can be used, via fermentation, for second
generation ethanol production; thus, it represent a renewable and
abundant resource that is relatively cheap, highly accessible and
expands the energy matrix (Singhvi & Gokhale 2019).
However, agroecosystems growth, productivity and development might be
strongly impact by the climate changes expected for the next decades
(Gamage et al. 2018; Ruiz-Vera, Siebers, Jaiswal, Ort &
Bernacchi 2018; Ziaco, Truettner, Biondi & Bullock 2018). Several
studies have been conducted in order to predict the effect of increased
[CO2] and temperature on crop yields, growth and
development (Ainsworth & Ort 2010; Asseng et al. 2015; Zhaoet al. 2017). Nevertheless, much less is known about its effect
on lignocellulosic biomass composition for bio-energy solutions and
since they are one of the most potential alternative to obtain clean
energy, their acclimation responses to climate change must be
investigated.
Moreover, the changes in climate conditions are not expected to be
isolated events i.e. both temperature and atmospheric
[CO2] are anticipated to increase simultaneously and
studies interacting climate variables have not been adequately explored
under field conditions. Plants response and vulnerability to isolated
stresses are different from those resulted from combined stresses and
will depend on their tolerance and mitigation ability to combined stress
situations (Borjas-Ventura, Alves, de Oliveira, Martínez & Gratão
2019). Therefore, in this study we characterize the lignocellulosic
biomass composition and enzymatic hydrolysis of Panicum maximumperennial C4 grass (grown in elevated CO2 and
temperature future conditions), which ultimately influence its ability
to provide for a sustainable and cost-effective feedstock for biofuel
production.
Among diverse LCB classification, fast-growing perennial C4 grasses have
remarkable potential as a lignocellulosic feedstock for cellulosic
ethanol production due to their high biomass yield, broad geographic
adaption and low mineral-nutrient and pesticides inputs (van der Weijdeet al. 2013). Feedstocks with these characteristics contribute to
the production of second-generation ethanol because they do not compete
with our food resources, do not require extensive capital investment,
and also can be used to avoid currently intermittent biofuel production,
which make the final product more feasible specially during idle periods
(Sosa et al. 2019a). P. maximum (Jacq. cv. Mombaça) is one
of the most important C4 grass used as pasture in South America,
especially Brazil, and also has significantly high efficiency in
converting solar energy into biomass through photosynthesis (EMBRAPA
2014). Moreover, P. maximum grass represents an important
alternative to sugarcane cellulosic ethanol, since it has
lignocellulosic composition similar to sugarcane bagasse, as well as a
bioethanol yield of 8571,0 L/ha compared to 8478,6 L/ha for sugarcane
bagasse (Lima et al. 2014).
Enzymatic hydrolysis depends on the cellulose accessibility to cellulase
enzymes. Therefore, it is utmost important to characterize
lignocellulosic components’ (i.e. cellulose and hemicellulose)
accessibility and its impact on enzymatic hydrolysis. For this purpose,
we used carbohydrate-binding modules (CBMs) that corresponds to a
non-catalytic polysaccharide-recognition module of carbohydrate-active
enzymes, such as glycoside hydrolases acting on glycosidic bonds. CBMs
contributes to optimize the catalytic activity of plant cell wall
hydrolases by increase the enzyme concentration on the carbohydrate
surface (i.e. proximity effect) and by preferentially binding to
specific substrate characteristics (i.e. substrate targeting) (Boraston,
Bolam, Gilbert & Davies 2004; Khatri, Hébert-Ouellet, Meddeb-Mouelhi &
Beauregard 2016). The high specificity of CBMs regarding to
lignocellulosic polymers makes them attractive as probes to study the
chemistry and structure variations on lignocellulosic biomass, cellulose
accessibility to cellulase enzymes and verify the effect of applied
treatment.(Cultures; Knox 2012; Oliveira, Carvalho, Domingues & Gama
2015) Specifically, four distinctive fluorescent protein tagged CBMs
(FP-CBM) were construct to detect alterations in amorphous cellulose,
crystalline cellulose, xylan and mannan hemicellulose, respectively
(Bombeck et al. 2017; Khatri, Meddeb-Mouelhi & Beauregard 2018b;
Gatt et al. 2019a).
In this study, P. maximum perennial C4 grass were grew under
expected future climate conditions (i.e. elevated CO2and temperature) under field conditions through a Trop-T-FACE system and
characterized to study the change in their chemical composition, cell
wall-bound phenolics, cellulose crystallinity, and lignocellulosic
component accessibility to enzymes. Following this, enzymatic hydrolysis
was performed to study the impact of elevated CO2 and
temperature on potential production of bioethanol. Since the composition
of the plant’s organic matter, transpiration, photosynthesis, and
metabolism can be influenced by climate temperature and
[CO2], we hypothesize that the climate change
expected in the next years will influence the set of characteristics
studied here, and thus affect P.maximum use for bioenergy
production. We strongly believe that this work will enable more accurate
projections of the responses of this important C4 crops species
regarding to climate and productivity models and will also contribute
for future decision-making in efficient production of bioenergy.
Materials and methods
Growth study site and Trop-T-Face system
We evaluated the effect of two different climate change factors expected
to impact on environment in the upcoming decades (i.e. elevate
CO2 atmospheric concentrations [CO2]
and global average temperature increase) in the tropical grasslandPanicum maximum cv. Mombaça by using a Trop-T-FACE facility,
located at the campus of the University of São Paulo, Ribeirão Preto,
SP, Brazil. The following treatments were used: (a) ambient atmospheric
[CO2] and temperature (named here as Control: C) (b)
ambient atmospheric [CO2] and +2 °C above ambient
temperature (annotated as elevate temperature: eT), (c) 600 ppm
atmospheric [CO2] and ambient temperature (termed as
elevate CO2: eC) and (d) 600 ppm atmospheric
[CO2] and +2 °C above ambient temperature (designate
as eT+eC). The Trop-T-Face system are described in details elsewhere (De
Assis Prado, De Camargo-Bortolin, Castro & Martinez 2016; Habermannet al. 2019b).
After 2 months, plants were clipped at 30 cm above the ground followed
by the treatments with different levels of [CO2] and
temperature. After 24 days of experiment, at 12 P.M., P. maximumleaves were collected, milled at 20 mesh and storage in free humidity
environment until analyses. Meteorological data of total solar radiation
(Rad), air temperature (Tair), and relative air humidity (Rh) during the
whole growing season is discussed elsewhere (Habermann et al.2019b).
Lignocellulosic preparation and
chemical composition
Lignocellulosic substrate preparation and the chemical composition ofP. maximum under different climate conditions were determined
using a modified Klason lignin method derived from the TAPPI standard
method T222 om-88, as described elsewhere (Nakagame, Chandra & Saddler
2010). The P. maximum extractives quantification was performed
following the protocol proposed by National Renewable Energy
Laboratory’s (NREL) (Sluiter, Ruiz, Scarlata, Sluiter & Templeton
2008).
Profile of cell wall-bound phenolics
The cell wall bound phenolics analysis was done as described elsewhere
(Oliveira et al. 2019a). Quantification of cell wall-bound
phenolics was accomplished by a HPLC system (Shimadzu® Liquid
Chromatograph, Tokyo, Japan).
Quantification of lignin and composition of monomers
To accurately quantify the lignin content in its monomer composition it
is crucial to obtain a protein-free cell wall aiming to exclude protein
and other UV-absorbing materials. The dry matter contained protein-free
cell wall fraction as obtained according to a defined protocol
(Ferrarese, MLL; Zottis, A; Ferrarese-Filho 2002). Lignin was quantified
by the acetyl bromide assay described elsewhere (Oliveira et al.2019a) and alkaline nitrobenzene oxidation was used to determine lignin
monomeric composition (Salvador et al. 2013).
Cellulose crystallinity
XRD analyses were performed on a Bruker D8-Advance X-ray diffractometer
(Bruker, Germany), with Cobalt Kα radiation (λ= 0.178892 nm) as
described elsewhere (Jiang et al. 2020). Individual crystalline
peaks were extracted by a curve-fitting method using the diffraction
intensity profiles.
Non-structural carbohydrate content
Non-structural carbohydrates were quantified on leaves at 24 days of
experiment collected at 12 P.M. of the respective day. The procedure
used for reducing sugars content was based on (Somogyi 1945) and starch
quantification was performed using an enzymatic assay (Amaral, Gaspar,
Costa, Aidar & Buckeridge 2007).
Enzymatic Hydrolysis
To avoid interference of soluble sugar during hydrolysis experiments,P. maximum samples were washed by immersion in 80ºGL ethanol six
times and then washed with water until the soluble sugars in the
material were completely removed. All samples were then dried in an oven
at 50ºC. The enzymatic hydrolysis of P. maximum (non-treated) was
carried out applying enzyme cocktails Cellic CTec2 and Cellic HTec
(Novozymes®) in the proportion of 9:1 (Ctec2:Htec ratio) (Jung, Yu, Eom
& Hong 2013) with a total amount of 20 mg of protein per gram of
cellulose. The hydrolysis experiments were carried out at 2% (w/v)
solids loading in sodium acetate buffer (50 mM, pH 4.8) at 50°C and 150
rpm. Subsequently to 72 hours hydrolysis, the enzymes were inactivated
by heating the hydrolysis mixture for 10 minutes at 100 °C. After
centrifugation at (16,000 x g /10 minutes) the supernatants were
collected and stored at -20 °C for further analyses.
Sugar and protein assay
The quantitative analysis of glucose and xylose concentration in the
hydrolysate was performed using a high-performance anion exchange
chromatography (Dionex DX-3000, Sunnyvale, CA). The total glucan or
xylan conversion of the substrate was calculated as a percentage of the
theoretical glucan/xylan available in the substrate.
Holocellulose (cellulose + hemicellulose) accessibility
using FTCM-depletion assay
The FTCM (F luorescent protein-T aggedC arbohydrate-binding modules M ethod) -
depletion assay was used to evaluate the cellulose and hemicellulose
surface accessibility in untreated and un-hydrolyzed P. maximumfibers grown in four different climate conditions (C, eT, eC and eT+eC).
The assay was proceed as described elsewhere (Khatri et al. 2016;
Khatri, Meddeb-Mouelhi, Adjallé, Barnabé & Beauregard 2018a; Khatriet al. 2018b; Bombeck et al. 2017; Hébert-Ouellet et
al. 2017; Gatt et al. 2019b; Mboowa, Khatri & Saddler 2020)
with four different FTCM probes: eGFP-CBM3a (GC3a) (fluorescent protein
eGFP genetically linked with CBM3a), mCherry-CBM17 (CC17) (construct of
the fluorescent protein mCherry and CBM17), mOrange2-CBM15 (OC15) (a
chimera made of mOrange2 fluorescent protein and CBM15) and eCFP-CBM27
(CC27) (composed of eCFP and CBM27), targeting crystalline cellulose,
amorphous cellulose (non-crystalline), xylan and mannan hemicellulose,
respectively. The affinities and specificities of the FP-CBM probes had
been previously characterized by a solid-state depletion assay (SSDA),
isothermal titration calorimetry (ITC) and affinity gel electrophoresis
(AGE) (Khatri et al. 2016, 2018a b) Prior to FTCM-depletion
assay, all substrates were incubated with bovine serum albumin (BSA) to
block the lignin in order to prevent the non-specific binding of CBM
probes to lignin (Khatri et al. 2018a) The difference between the
binding isotherm of substrates without BSA blocking and with BSA
blocking is summarized in Figure S1.
Statistics and numerical analysis
All tests were performed in triplicates, and the means and standard
deviations were analyzed and indicated as the mean of three replicates ±
standard error of the means (SD). A principal component analysis (PCA)
was performed to identify patterns in the dataset, the analysis was
carried out in past (version 2.14). Analysis of variance (ANOVA) was
performed to test the significance of the observed differences using the
Graph-Pad Prism 8.0 software (San Diego, EUA). Differences among
parameters were assessed by the Tukey test, and p values ≤ 0.05
were considered statistically significant.
Results
Biomass chemical composition
For all four studied energy crops (Figure S2), the biomass composition
was analyzed for soluble extractives, ash, cellulose, hemicellulose and
lignin contents and the results are summarized in Table 1. As
hemicellulose from grasses is manly composed by Arabinoxylan (AXs)
residues, the glucose from hemicellulose sources represents low
quantities of the total glucan content, thus the cellulose content was
considered for further discussions as the total glucan content of each
group. The highest glucan content was found for eT (30.2%) and eT+eC
(29.7%), followed by eC (27%) and control (25.8%) (Table 1).
Significant differences (p≤0.05) in glucan content were observed for eT
and eT+eC when compared to C and eC groups. There was no significant
(p≥0.05) difference between C and eC substrates. Lignin contents (Table
1) were quite similar for all studied climate conditions and no
significant (p≥0.05) difference was found among them, showing that the
expected futuristic climate condition seems not to affect total lignin
content on the studied forage grass. The hemicellulose content (Table 1)
was calculated based on the monomeric sugars (xylose, arabinose,
galactose and mannose) released after acid hydrolysis and was slightly
(p≤0.05) higher for eC conditions when compared with C and eT (Table 1).
Furthermore, grasses with different treatments exhibited somewhat
similar hemicellulose monosaccharide profiles, primarily composed of
xylose, arabinose, and galactose (Figure 1). Moreover, mannose sugars
were below detectable levels, which suggests that very low or no mannan
was present in the hemicellulose and pectin fractions of the cell wall.
A significant (p≤0.05) difference was found in the monosaccharide
profiles for eC treatment which exhibited more xylan (Fig. 1A) compared
to C and more arabinose compared to other treatments (Figure 1B). On the
other hand, no significant difference was found for galactose sugar in
all the studied conditions (Fig. 1C). Figure 1D illustrate the
xylan/arabinosil ratios that indicate the degree of arabinosyl
substitution on grass’s xylan backbone. In this work, we observed that
the grasses in different climate conditions have Xyl:Ara ratio about
5:1, with significant difference among them (p≤0.05). eT grasses
exhibited the highest Xyl:Ara ratio and eC the lowest.
In addition, the PCA analysis shows that warming is the main driver of
changes in P. maximum cell wall composition. It shows a clear
separation of the warmed plots (eT and eT+eC) from the non-warmed plots
(C and eC) along the PC1 axis, explaining 62.31% of the variance in the
cell wall composition and carbon storage (Figure 2A). It also indicates
that the glucan content was the main responsible for these differences.
No pattern was observed for the PC2 axis, which explained 19.61% of the
distribution. The residual variation observed can be explained by the
[CO2] (18.08%), as shown by the PC3 (Figure 2B).
Extractives are organic and inorganic molecules extracted from biomass
by a polar or nonpolar solvent. They have a particular importance due to
their low degradability, which can cause problems during lignocellulosic
biomass processing (i.e. deconstruction and/or modification). Soluble
ethanol extractives were of similar values, approximately 4%, showing
no significant difference among the tested groups (p≥0.05).
Lignin profile
Besides measuring the lignin content (Table 1), we also analyzed the
guaiacyl (G), syringyl (S) and p -hydroxyphenyl (H) monomeric content
for lignin, for all climate conditions (Figure 3). The results show that
H and G content is similar among the treatments (Figure 3A-B). However,
we found higher (p≤0.05) syringyl (S) content for eT and lower content
for eT+eC treatment when compared to the other groups (Figure 3C). The S
content observed was 5.12 and 3.5 µg/mg protein free cell for eT and
eT+eC, respectively, compared to about 4.5 µg/mg protein free cell wall
for C and eC treatments. Interestingly, differences were found in the
S/G ratio (Figure 3D) among the studied climate conditions, in which eT
treatment presented higher S/G ratio (0.58) than C and eC conditions
(p≤0.05).
Cell wall-bound phenolics
The ferulic acid (FA) and p -coumaric (p -CA) have an
important role in cell wall structure and is described as an influencing
factor in hydrolysis yields (Oliveira et al. 2019b), therefore
the FA and p -CA content was analyzed to verify if it could
interfere on hydrolysis of the different climate conditions studied
(Figure S3). It was apparent that FA varied between 2.26 and 2.38 mg/g
of biomass and p -CA between 3.04 and 3.41 mg/g of biomass, with
no significant difference found among the groups for both FA andp -CA (p ≥ 0.05). Hence, FA and p -CA should not be
an aspect that lead to differences in hydrolysis yields among the
climate conditions studied in this work.
Cellulose Crystallinity
Crystallinity index (CI) is a quantitative representation that symbolize
the relative amount of amorphous and crystalline regions of cellulose.
As cellulose crystallinity is believed to have a role in its biological
conversion, we then evaluated the CI for all climate conditions studied.
The XRD graphs with the analysed peaks are represented in supplementary
material (Figure S4). The CIs for C, eT, eC and eT+eC were 71.7; 53.2;
64.7 and 57.8%, respectively. The elevated temperature treatments (eT
and eT+eC) were the groups with lower crystallinity.
Non-structural carbohydrate content
Due to the importance of non-structural carbohydrates to the plant
physiology, we estimated the total content of soluble reducing sugar and
starch content after 24 DOE (Days of experiment), are shown in Figure 4.
Soluble reducing sugars decreased by 21.45% and 27.4% (p≤0.05) under
eT and eT+eC, respectively, related to C (Fig. 4A). Regarding to the
leaf starch content we found a 52.9 and 41.4% reduction (p≤0.05) for eT
and eT+eC, respectively, when compared to C (Fig. 4B) and a
non-significant increase (16%) in the leaf starch content for eC
compared to C (Fig. 4B).
Enzymatic saccharification
Further the enzymatic saccharification of P. maximum grew under
the four climate conditions (C, eT, eC and eT+eC) was performed to
identify whether there would be any impact of climatic conditions on
sugars releases for ethanol production (Figure 5). For this purpose, we
used two commercial enzyme cocktails containing all the essential
enzymes for lignocellulosic biomass deconstruction. We quantified the
sugars released (glucose and xylose) by enzymatic hydrolysis in grasses
without any pretreatment, as a way to avoid interference in biomass
recalcitrance between forage grasses except climate conditions.
Glucan conversion (%) at 72 h of hydrolysis from non-treated biomasses
(2% solid loading) was significantly higher (p≤0.05) for warmed
treatments (eT and eT+eC) with 43.9 and 43.2% of glucan conversion,
whilst control (41%) and eC (41.2%) groups showed lower values of
glucan hydrolysis (Fig. 5A). Moreover, these values represent a relative
increment of 7.07 and 5.37% in glucan conversion for eT and eT+eC
treatments, respectively when compared to C group. Furthermore, the
percentage of xylan conversion for eT treatment (11.05%) was also
higher (p≤0.05) than C (9.4%), eC (9.6%) and eT+eC (9.5%) (Figure
5B), which represent a relative increment of 16.31% for eT when
compared to the control group.
Surface carbohydrate accessibility using FTCM-depletion assay
Since hydrolysis depends on lignocellulosic components accessibility to
the enzymes, we further study the change in the enzymatic hydrolysis
data by evaluating P. maximum fibers accessibility using
fluorescent protein-tagged carbohydrate-binding modules (FP-CBM). via
four highly specific FP-CBM molecular probes (i.e. GC3a, CC17, OC15 and
CC27). The FTCM-depletion assay of P. maximum fibers was well
approximated by a single-site binding model as described elsewhere
(Figure 6) (Khatri et al. 2018a; Gatt et al. 2019b; Mboowaet al. 2020). The parameters derived from these fits
(No (µmoles g-1),K a (µM-1), ΔG (KJ
mol-1)) are summarized in Table S1.
The binding capacity or the total concentration of the available binding
sites (No) on the control (C) fibers were 2.1 ± 0.3 2.2 ± 0.7 and 1.3 ±
0.8 for GC3a (crystalline cellulose accessibility recognition probe),
CC17 (amorphous cellulose accessibility recognition probe) and OC15
(xylan accessibility recognition probe) probes, respectively (Table S1;
Figure 6). It was apparent that there was no significant difference in
the surface exposure/accessibility of crystalline cellulose, amorphous
cellulose and xylan components of the biomass. On the other hand, no
binding of CC27 (mannan recognition probe) probes were observed which
complement the lack of mannan component in the P. maximum fibers.
Furthermore, the total cellulose and hemicellulose accessibility could
be derived by adding crystalline cellulose accessibility (i.e. No of
GC3a) and amorphous cellulose accessibility (i.e. No of CC17), and xylan
accessibility (i.e. No of OC15) with mannan accessibility (i.e. No of
CC27), respectively (Table S1). The total cellulose accessibility was at
least ~3-fold higher than total hemicellulose
accessibility which suggest that cellulose exposure on the surface ofP. maximum fibers were overwhelmingly higher than hemicellulose.
In the case of eT fibers, crystalline cellulose, amorphous cellulose and
xylan accessibility increased by 267%, 100% and 85%, respectively,
when compared to C. This increment also reflected in the 181% and 85%
improvement in the total cellulose and total hemicellulose
accessibility, respectively, when compared to its control counterpart
(Figure 6; Table S1). In contrary, eC fibers did not exhibit any
significant change in the holocellulose accessibility when compared to C
group. Concerning to the combined impact of eT and eC,like eT, this condition improved the GC3a and CC17 bindings by 205% and
64%, respectively, and as a result exhibited significantly higher %
glucan conversion (Figure 5A). However, there was no significant
improvement observed in the xylan accessibility. Furthermore, the lack
of binding of mannan recognition probe (CC27) to eT, eC and eT+eC
biomass suggests that FTCM probes were highly specific towards their
substrate and there was negligible non-specific interaction.
Lastly, in order to examine the robustness of relationship between bound
probes and enzymatic hydrolysis observed here (i.e. type of
correlation and correlation coefficient), a statistical analysis using
all the value pairs (bound probes (µmol/g of biomass) vs glucan/xylan
hydrolysis (%) generated in this study was performed. The results in
the Figure 7 indicate that the total cellulose and hemicellulose
accessibility, as detected by FTCM probes, were positively correlated
with percent glucan and xylan hydrolysis, respectively. The Pearson’s
correlation coefficients for glucan and xylan hydrolysis were R = 0.99,p < .001, and R = 0.99, p < .001,
respectively, which supports the notion of a significant robust positive
correlation.
Discussion
Biomass chemical composition
Lignocellulosic materials are manly composed by cellulose,
hemicelluloses, and lignin. Cellulose is a linear homopolysaccharide
composed of glucose monomers that can be converted into fermentable
sugars through enzymatic hydrolysis (Silveira et al. 2015), which
make feedstocks with higher glucan content likely to be favored in
ethanol biorefinery industries from an economic point of view, since the
cellulose content of the feedstocks is directly proportional to the
ethanol yields under optimal processing conditions (Sosa et al.2019b). Hence, as elevated temperature treatments (eT and eT+eC) led to
an increase in glucan content, warming treatment could have a positive
effect on ethanol yields (Table 1).
In contrary to cellulose, the distribution of both lignin and
hemicellulose in cell wall is described as a physical barrier that
contributes to biomass recalcitrance by encapsulating cellulose
microfibrils obstructing cellulase domains to adsorb on cellulose and
initiate enzymatic hydrolysis (Khatri et al. 2016). Therefore,
the hemicellulose content slightly higher for eC conditions may lead to
lower saccharification yields (Table 1). The findings for hemicellulose
content agrees with a transcriptome study which found that genes of a
number of enzymes involved in hemicellulose and pectin biosynthesis such
as NDP-sugar epimerases, UDP-glucose pyrophosphorylase (UGPase),
glycosyltransferase family 43 (GT43) and others, had increased
transcript levels in response to growth under elevated
CO2 (Wei et al. 2013).
The lignin content found in this work are consistent with previous
studies and indicates that P. maximum has similar percent
composition of lignin with those reported for sugarcane bagasse, about
27.79% (Lima et al. 2014; Oliveira et al. 2019a).
Therefore, lignin amount present in P. maximum does not represent
a possible inconvenience (in terms of biomass deconstruction and
modification) when compared to sugarcane bagasse (Table 1).
The results concerning to monosaccharide profiles agrees with the
characteristics from type II hemicellulose cell wall of grasses is
primarily composed of xylan with arabinofuranose (Araf)substitution of the b-(1,4)-xylose backbone by α-linked, forming
arabinoxylan (AXs) (Hatfield, Rancour & Marita 2017). Another important
parameter for biomass recalcitrance investigated in this work was the
Xyl:Ara ratios. Substitution patterns on the xylan backbone (described
here as Xyl:Ara ratio) has a robust relation with how strongly
hemicellulose can form hydrogen bond to other cell wall polysaccharides,
mainly lignin and cellulose, affecting structural properties of the cell
wall as well as enzymatic hydrolysis yields (Hatfield et al.2017). In this sense, hydrolysis yields of eT might be favored, since
this group exhibited higher Xyl: Ara, opposite to the eC group that had
the highest degree of arabinosyl substitution (Figure 1).
By analyzing the above observed results for chemical composition ofP. maximum LCB, it is probable to suggest that abiotic stress
induced by climate changes can influence the cell wall composition of
the forage grasses especially, the heat stress (i.e. eT) as showed by
PCA analysis (Figure 2). Cell wall remodeling represents an important
mechanism of stress tolerance, some reports related that important
changes on cell wall might be driven by abiotic stress (Moura, Bonine,
de Oliveira Fernandes Viana, Dornelas & Mazzafera 2010; Tenhaken 2015;
Wang, McFarlane & Persson 2016) as a way to maintain growth and
productivity. However, how much the stress will influence on plant
physiological process depend on heating level and the tolerance
mechanisms employed by the plant.
Heat stress in most cases could results in water stress under modified
environment situations (e.g., high temperatures and midday radiation)
(Olivera Viciedo, de Mello Prado, Martínez, Habermann & de Cássia
Piccolo 2019). Under this scenario, C4 photosynthetic pathway plants
increase their evaporative demands resulting in a temporary water
stress. An evidence of water stress in P. maximum grown under
warmer environment was found by Habermann et al., the author described a
15% increase in transpiration rates for P. maximum cv. Mombaça
under eT environment, a slight reduction on solo water content (SWC), as
well as decreased bulliform cells size, which store water (Habermannet al. 2019b). Furthermore, under eT conditions, P.
maximum cv. Mombaça had an increment in amino acids content derived
from pyruvate and oxaloacetate, reported to be related to the stress
defenses (Wedow et al. 2019)
Lately some studies have been exploring the effect of many abiotic
stresses under plant metabolism and some finds can help us to understand
the higher glucan content for eT and eT+eC (Table 1). Most plants make
physiological adjustments that contribute to the acclimation and
survival under heat and water stress. For example, genes encoding
Cellulose Synthase-Like (CSL) proteins, a family of proteins that are
similar to the Cellulose synthases (CesAs), seems to play some role in
cellulose synthesis and appears to be responsive to osmotic stress and
salt stress, which could be a consequence of water stress.
Interestingly, CSLD1, CSLD2, and CSLD3 genes were induced by increased
salt conditions, leading to a cell wall deposition in Arabidopsis
thaliana (Zhu et al. 2010). Cellulose deposition was also
reported to happen in Zea mays stems under salt stress (Oliveiraet al. 2020). Furthermore, an increased level of expression of
sucrose synthase and UDP-glucose pyrophosphorylase genes was detected in
cotton subject to drought stress, suggesting a possibly higher cellulose
biosynthesis (Wei et al. 2013). Therefore, these results showed
that changes in cell wall composition could play a role in the
acclimation process of P. maximum allowing the plant to survive
to non-lethal temperature through thermotolerance.
Lignin Profile
As the lignin content, the subunits ratio are highly variable between
plant species, tissues, cell types, developmental stage and stress
conditions and also has a role on the contribution of lignin to biomass
recalcitrance (Li et al. 2010). The ratio of syringyl (S) and
guaiacyl (G) units in lignin has been regarded as a factor that
influences on recalcitrance to sugar release from energy crops enzymatic
hydrolysis (Studer et al. 2011). This limit arises from the
notion that during lignin biosynthesis coupling reactions in specific
positions lead to polymers linked via C–O bonds and C–C bonds. The C-O
bonds are the most abundant and the key for lignin depolymerization, due
to their labile nature. The S units are prone to formation of C-O bond
during lignin biosynthesis, which are more susceptible to cleavage
(Anderson et al. 2019)
Therefore, biomass with higher S/G ratio, in this study represented by
eT group, may represent a better choice for biorefinery purpose, since
the lower G content allow that lignin structure to be easily modify
increasing the accessibility to hydrolytic enzymes, and therefore,
greater sugar release (Figure 3). Plants with higher S/G ratios shows
that the negative influence of lignin on glucose release was less
pronounced (Studer et al. 2011). The data found for eT group
agrees with previous results that observed an increase in sinapic acid
abundance under eT treatment, which was pointed as an impact of the eT
on lignin compositions by increasing S-type concentrations (Wedowet al. 2019). Also, Zea mays plants under salt stress also
exhibited higher S/G ratios in roots and stem which could be the case of
eT treatment (Oliveira et al. 2020).
Cell wall-bound phenolics
Produced in the phenylpropanoid pathway the hydroxycinnamic acids also
known as ferulic acid (FA) and p -coumaric (p -CA) have a
carboxylic group at the end of their propenyl group in contrast with the
alcohol function of monolignols that are produced later (Tobimatsu &
Schuetz 2019). Due to this fact, feruloyl and p-coumaroyl residues can
further esterified to arabinose from the arabinoxylans (AXs) of the type
II cell wall. The FA esterified with arabinosyl residue of AXs is able
to connect with lignin by ether-linkage or dimerize with other
FA-arabinoxylan acting as a universal connector between cell wall
polymers. These reaction results in cross-linking among the cell wall
polymers, which can reduce enzymatic hydrolysis efficiency, by blocking
the accessibility and attack of hydrolases (Oliveira et al.2019b). Furthermore, these acids performs a key role in cessation of
cell growth, anchoring lignin in cell wall polysaccharides and
restricting the accessibility of plant pathogens (de Oliveira et
al. 2015).
The FA average content for P. maximum is relatively lower
compared with sugarcane bagasse (8.0 to 17.0 mg/g) and Perennial
ryegrass (Lollium perenne ) shoot (Gagic et al. 2010;
Masarin et al. 2011) (Figure S3). The lower FA content ofP. maximum should be an advantage since cell well digestibility
and FA content are negatively correlated. In addition, the FA andp- CA contents agrees with those ones reported by Oliveira et al.
(2019a) that observed a FA content about 2 mg/g AIR (Alcohol Insoluble
Residue) and about 3.4 mg/g AIR for p -CA for cv. Mombaça.
Cellulose Crystallinity
Cellulose structure is generally divided into two regions, the first one
is called amorphous cellulose and is described to have molecular order,
the second one is high level of molecular order and is called
crystalline cellulose. The enzymatic and microbial hydrolysis of regions
are easier and faster than the crystalline regions, since amorphous
region is able to adsorb water (Karimi & Taherzadeh 2016). Although
there are many factors affecting the cellulose hydrolysis as available
surface area, and degree of polymerization, the cellulose crystallinity
is reported to be a limiting step on cellulose conversion due to its
recalcitrance, which means that lower CI results in higher bioconversion
rates for lignocelluloses feedstocks (Poornejad, Karimi & Behzad 2013;
Ostovareh, Karimi & Zamani 2015). In addition, many studies showed that
enzyme adsorption, including the non-catalytic cellulose binding module
(CBM) and catalytic glycosyl hydrolase system, generally declined as
cellulose crystallinity increased (Jeoh, T., Ishizawa, C. I., Davis, M.
F., Himmel, M. E., Adney, W. S., & Johnson 2007). In this way, eT and
eT+eC, due to its lower CI, should be the climate conditions that must
results in higher cellulose conversions (Figure S4).
The effect of temperature on reducing cellulose crystallinity forP. maximum could rely on the thermotolerances mechanisms employed
under water stress experienced in warming treatments. Reactive oxygen
species (ROS) are commonly observed as a plant response to abiotic
stress. In general, they are used as a co-substrate by cell wall
peroxidases, which lead to cross-linking increasement between phenolic
compounds and hemicellulose polymers (Miller, Suzuki, cCiftci-yilmaz &
Mittler 2010). However, if ROS production continues and all
cross-linkable substrates have already been used by peroxidases, this
allows the production of OH-radical that is involved in cell wall
loosing process, by cleaving sugar bonds in plant polysaccharides,
action similar to enzymes like expansins that are reported to promote
amorphogenesis in cellulose microfibrils (Tenhaken 2015). This loosing
process promote by ROS is reported to be a tolerance mechanism employed
by plants to overcome the growth arrest under abiotic stress, and could
also lead to enhancement in hydrolytic performance by, as described in
this study, reducing the CI.
Non-structural carbohydrate content
Carbohydrates are molecules with important role in signaling pathways as
well as in the tolerance of plant species to abiotic stresses (Gangola
& Ramadoss 2018). The intercellular content of sugars regulates
multiple functions in source and sink tissues including photosynthetic
performance, gene expression and enzyme activities (Koch 1996). In this
work, both alcohol soluble reducing sugars and starch content were found
to be reduced in warmed treatments (eT and eT+eC) (Figure 4). These
results agrees with others Panicum maximum studies, the first one
was a metabolome study of intracellular content that reported many
carbohydrates (arabionose, frutose, galactose, maltose, ribose, xylose
and melobiose) with increased abundance under eT (Wedow et al.2019), showing that these carbohydrates are being thansportated as
signaling molecules, which could be a response for the abiotic strees
when the plants are subjected to a warmer environment. The second one
found larger quantity and size on starch grains in chloroplasts of
bundle cells for eC treatment and minor starch grains for eT and eT+eC
(Habermann et al. 2019b a).
The increased starch content in leaves for eC treatment is a response
for the higher carbon assimilation found in eC, showing thatPanicum maximum is not saturated under CO2atmospheric concentration (Habermann et al. 2019b). Concerning to
eT and eT+eC, as previously discussed, warming treatments led to a
stress statement in plants that demands more energy. In this case,
starch is described to be involved in plant’s reactions to abiotic
stress and it is the principal carbohydrate stored in source organs that
is quickly remobilized to provide energy to sink tissues, consequently
reducing its content in source tissues (Thalmann & Santelia 2017).
These results suggest that starch was degraded and transported as
sucrose to sinks organs, therefore, its content in leaves (source
organs) is reduced under eT treatment.
Enzymatic saccharification
The efficiency of enzymatic saccharification which is the key step in
the bioconversion of lignocellulose biomass to ethanol can be influenced
by several factors related to the biomass recalcitrance, such as the
cellulose crystallinity, available surface area, degree of hemicellulose
matrix polymerization, lignin distribution, etc (Novy et al.2019). As discussed earlier, elevated temperature treatments (eT and
eT+eC) could play an important role in improving percent glucan content,
reducing biomass recalcitrance by lowering cellulose crystallinity and
minimizing Xyl/Ara (Figure 1) and S/G ratio (Figure 3), which ultimately
might result in a less polymerized matrix between hemicellulose elements
and also between hemicellulose and lignin. All the above-mentioned
results, led to improved accessibility to the hydrolytic enzymes for
warming treatments, and thus, higher cellulose conversion into glucose
during saccharification process (Figure 5). Even though the differences
in conversion rates were not much pronounced, minimal differences on
sugar release could have an important impact on the biorefineries from
an economic point of view.
Moreover, it is important to note that no pretreatment was applied in
this study that is why the conversion rates observed after enzymatic
hydrolysis were quite low. Thus, by applying efficient pretreatment onP. maximum grasses the glucan and xylan conversion rates could be
further improved, since it facilitates the access of glycoside
hydrolases to polysaccharides (Agbor, Cicek, Sparling, Berlin & Levin
2011).
Surface carbohydrate accessibility using FTCM-depletion assay
Hydrolysis is strongly impacted by the accessibility of enzymes to the
lignocellulosic components. According to the FP-CBM data, it was
apparent that growing P. maximum under elevated temperature
increased the holocellulose accessibility (Figure 6; Table S1) and as a
result the enzymatic hydrolysis of both glucan and xylan component
(Figure 5). This also compliment above reported higher glucan chemical
composition (Table 1), lower crystallinity (Figure S4), elevated
xylan/arabinose (Figure 1) and S/G ratio (Figure 3) for eT condition.
Nonetheless, for eC fibers no difference was found in holocellulose
accessibility compared to C group, which was reflect in the any
significant improvement in the % glucan and % xylan conversion for eC.
Moreover, this is likely due to the observed significantly lower
xylan/arabinose (Figure 1) and S/G ratio (Figure 3), since both
hemicellulose and lignin contribute to biomass recalcitrance. Regarding
to eT+eC, it followed similar trends as eT condition for total cellulose
accessibility, which depicted that elevated temperature plays a dominant
role over elevated CO2 by suppressing the inherent
changes occurred via eC environmental condition. The role of warming
treatments (eT and eT+eC) in increase cellulose accessibility can be
also related to ROS production under abiotic stress, as discussed in
Section 4.4, which opens up the plant cell wall by mediated cleavage of
polymer chains and provides catalytic enzymes with improved
accessibility to the glycosidic linkages within the sugar polymers .
Conclusions
Abiotic stress effects plant cell wall architecture although the impact
depends on the plant genotype, species, age and on the timing and
intensity of the stress. Among all the studied climate conditions eT
exhibited higher percent glucan composition, S/G ratio, higher total
cellulose accessibility, and hence elevated enzymatic hydrolysis. In
contrary, eC showed higher percent total hemicellulose composition
(which was due to higher arabinose content), lower S/G ratio,
significantly lower total cellulose accessibility and therefore lower
enzymatic hydrolysis yield. Furthermore, eT+eC conditions followed
similar trends as eT conditions, which depicted that elevated
temperature plays a dominant role over elevated CO2.
Moreover, state-of-the-art FTCM-depletion assay facilitated to study the
surface exposure/accessibility in real time to better understand the
enzymatic hydrolysis. Our data provide clearly evidences that P.
maximum cell wall has several mechanisms of adjustment under abiotic
stress induced by expected futuristic climate conditions, which could
positively affect its use for bioenergy purpose. However, the work has
gone beyond the concerns over the carbon emissions and climate change
brought by fossil fuels, this work has also come up with a potential
alternative of lignocellulosic biomass for cellulosic ethanol
production, which may contribute to better-informed decisions on energy
options for the future.