Lignin and glucose content in the
fiber fraction increased with increasing pretreatment-severity, while the
xylose, phosphorus, calcium, zinc, and manganese content decreased linearly
with pretreatment-severity. The arabinose, potassium, and magnesium content
showed an exponential decrease with increasing pretreatmentseverity (Figures 7
and 8), whereas silicon, iron, aluminum, and copper levels were insensitive to
variation in the pretreatment-severity, as they produced insignificant response
models in the design. As observed for the cpH values presented in Figures 7 and
8, the constituents could be divided into two groups: less pH-sensitive
constituents (cpH between 0.044 and 0.069) (Figure 7) and more pH-sensitive
constituents (cpH between 0.201 and 0.330) (Figure 8). The first group
consisted of potassium and the main structural components of wheat straw, that
is, glucose, xylose, arabinose, and lignin. The remaining elements (phosphorus,
magnesium, calcium, zinc and manganese) constituted the more pH-sensitive
group.
Discussion
Composition
and pretreatment factor analysis
The changes in composition of
wheat straw after hydrothermal pretreatment were as expected in terms of
xylose, arabinose, and glucose content (Figure 1) [14]. Xylose and arabinose
content decreased with high pretreatment-severity, whereas glucose content
increased with pretreatment-severity because its recovery was not dependent on
pretreatment conditions, and hence, it constituted a larger proportion of the
fiber fraction when hemicellulose was solubilized. Ammonium hydroxide has a
milder effect than other alkaline solutions (NaOH and KOH) on lignin [15], so
recovery of lignin did not increase with increasing pH. As the objective of
this study was to investigate the behavior of the mineral components during
hydrothermal pretreatment, and to learn about their interactions with the
biomass, thus retaining lignin in the fiber fraction was intended. Retention of
lignin in the fiber fraction caused minimal variation between samples in terms
of the structural components, so that these variations did not overshadow the
variations of the less abundant mineral components. The mineral composition of
wheat straw was, in general, in agreement with literature (8850 to 17320 ppm
silicon, 50 to 560 ppm aluminum, 70 to 350 ppm iron, 3090 to 4870 ppm calcium,
440 to 660 ppm magnesium, 90 to 190 ppm Na, 4120 to 20720 ppm potassium, 270 to
760 ppm phosphorus) [16]. Some elements were above the stated ranges, but this
was expected, because of seasonal and geographical variations. Potassium was the
only element solubilized from the fiber fraction under all pretreatment conditions,
yielding a low recovery range (Table 1, Figure 3f ). This was also as expected,
because potassium is exclusively present in the aqueous phase of plant cells,
so it is easily leached from the biomass during pre-soaking and pretreatment.
Potassium is known to be highly abundant in wheat straw, especially in the
cytoplasm and aqueous environments of the vacuole, where it stabilizes the
ionic strength of enzymes and osmotic pressure of the cells [17]. Magnesium, in
spite of being 70% freely diffusible and present at fairly high concentrations
in the cytoplasm, [17] required pre-soaking in acid before it could be solubilized
from the fiber fraction (Figure 3d). The same effect of acid pre-soaking was
observed for calcium (Figure 3e). Magnesium and calcium are deposited in wheat
straw cell walls, where they are associated with carboxyl and phenolic hydroxyl
groups of organic components, making them resistant to solubilization [18].
Neither calcium nor magnesium was leached from the relatively intact cell walls
(for example, see high recoveries at high pH and low temperatures in Figure
3d). The similarity in results from response surface modeling of calcium and
magnesium (Figure 3) to some structural components of the biomass, especially
arabinose but also to some degree xylose, indicates that the integrity of the
cell wall influences the solubilization of calcium and magnesium.
Correlation
between biomass constituents
Using PCA, it was possible to
group the wheat straw constituents into two main groups: water-soluble and
water-insoluble constituents (Figure 4). In the water-insoluble group, lignin
and glucose were clustered together. This was not surprising considering that
these components interact strongly in lignocellulosic fibers, and are both
insoluble across the range of pretreatment conditions tested in this study.
Silicon formed a separate cluster, reflecting its unique properties relative to
the other elements. Silicon is deposited as SiO2 · nH2O, either in intimate
association with the organic components of plant cell walls or in silica bodies
formed within the lumen of specialized cells [19-21]. Owing to the insoluble
nature of SiO2, even releasing it from the organic material would not remove
silicon from the insoluble fraction. Coupled with the high recovery range for
silicon in the fiber fraction (Table 1), the implications of these findings are
that the vast majority of the silicon is likely to remain associated with
lignin and thus accumulate in the lignin residue stream during the further processing
of the biomass. Aluminum, iron, and copper were also clustered together. These
are all toxic elements for plants if they are accumulated at high
concentrations in their free form [18,22,23]. The plants therefore need to
control and immobilize these elements to protect themselves from the toxic
effects.
Aluminum is strongly bound to
negatively charged groups in cell walls and is water-insoluble, hence it is not
solubilized from the fiber fraction. Iron and copper are also present in an
insoluble form in plants [17,18], and are believed to be mainly associated with
insoluble cell wall components or phytate [24]. However, some iron in plants is
stored in soluble ferritin complexes [25]. As with aluminum and copper, a
fraction of the iron was solubilized during hydrothermal pretreatment, but the
rest remained in the fiber fraction, regardless of the pretreatment conditions.
These findings signify that in relation to biorefining, iron and copper are
likely to be distributed between both the aqueous and solid fractions, and to
gradually become solubilized during further processing via the enzymatic
cellulose and hemicelluloses hydrolysis and fermentation steps. Whether such
gradual solubilization may function as a nutrient supply during
the fermentation, or exert
negative effects, warrants further in-depth examination. In the water-soluble
group, arabinose and xylose were located close together in the loadings plot,
which was meaningful because in wheat straw they are associated in
arabinoxylan. Magnesium and potassium belonged to the same cluster as xylose
and arabinose. These two mineral elements are present at relatively high
concentrations in the cytoplasm [17], and as the straw matures, they may become
loosely bound to negatively charged components within the straw matrix. The
present results indicate that magnesium and potassium are unlikely to accumulate
in the insoluble fiber streams or in the lignin residue after fermentation in
lignocellulose to ethanol processing. The remaining elements (calcium, phosphorus,
manganese, zinc) in the water-soluble group belonged to another cluster; their
common denominator is that they are all restricted in their movement in plant
cells. Calcium and phosphorus interact in calcium–phosphate,
calcium–phospholipid, and calcium–phytate complexes [17], and this could
explain their similar behavior. Manganese and zinc were found to be present in
the fiber fraction at very low concentrations. The low concentrations of
manganese and zinc were either due to their low initial abundance in wheat
straw, or because they were solubilized during pretreatment, as they exist
either as free ions or bound in protein complexes [17]. Manganese and zinc were
clustered together with calcium and phosphorus, because the remaining manganese
and zinc left in the fiber fraction during hydrothermal pretreatment can
interact with the cell wall matrix in a similar fashion to calcium and phosphorus.
Optimization
of cpH for prediction of fiber fraction composition
It is desirable to be able to
predict the composition of the fiber fraction based on the severity of
hydrothermal pretreatment. The temperature and time dependency of the
composition of the fiber fraction was expected to follow the classic
pretreatment-severity equation [26], where 14.75 is an arbitrary empirical
constant based on the activation energy [27]. The pH dependency varied according
to the constituent. Therefore, including pH in pretreatment-severity merely by
subtracting pH in the classic method [28] did not result in satisfactory fits;
in other words, an additional factor, cpH, was needed. We assumed that there
was an underlying dependency of a given constituent on the combined
pretreatmentseverity, which was linear at low pretreatment-severity, but when
pretreatment-severity was increased to a level where most of the constituent
was solubilized, leaving no or only strongly restricted residual constituents
in the fiber fraction, the dependency was assumed to attain an exponentially
decaying nature. Not knowing if the range of pretreatment conditions chosen in
this study were in the linear or exponential range for the constituents, we had
to fit both a linear and exponential function to the data and choose which of
the two gave the best fit (highest R2) for each constituent. As shown in Figure
7b, an exponential function yielded the best fit for arabinose. This was
because the pretreatment effectively solubilized arabinose from the fiber
fraction, so at high pretreatment-severity the arabinose content approached
zero. By contrast, for xylose (Figure 7a), higher pretreatment-severity was
needed before an exponential decay could be expected. The magnesium and
potassium contents also exhibited an exponential decay, although to a lesser
degree than arabinose. Magnesium and potassium were clustered together with
arabinose and xylose (Figure 5), and are both elements that occur at high
concentrations in the cytoplasm [17]. The remaining magnesium (~10%) and
potassium (~4%) recovered in the fiber fraction after hydrothermal pretreatment
at high pretreatment-severity might be more recalcitrant to solubilization than
the rest, causing exponential decay of their contents at increasing
pretreatment-severity. For potassium in Figure 8b, removing the point of low
pretreatment-severity, which appeared to be an outlier, still resulted in an
exponential decaying function. The pH dependencies of carbohydrates, lignin,
and potassium were lower than those of the other mineral elements, as observed
by comparing cpH factors (Figures 7 and 8); cpH values of xylose and arabinose
were low. As pH constituted merely a contribution to the severity of
pretreatment by opening up the cell wall, it had no direct implications on
solubilization of xylose and arabinose. For elements with high cpH (phosphorus,
magnesium, calcium, zinc, manganese), low pH could, in addition to opening up
the cell wall, increase the solubility of the elements. The solubility of
calcium phytate, for example, increases significantly below pH 4 [29]. As seen
in Figure 7, contents of glucose and lignin increased with higher
pretreatment-severity, because the relative proportion of glucose and lignin
increased when xylose and arabinose contents decreased. Variations in glucose
and lignin recoveries in the fiber fraction did not depend on the severity of
pretreatment, so any change in content must have been governed by removal of
other main constituents of the fiber fraction, namely hemicellulose. This
explains why the cpH of glucose and lignin were in
the range of those of xylose and
arabinose.
Conclusion
By optimizing a factor, cpH,
indicating pH dependency for each constituent of the biomass, it was possible
to model the composition of the wheat straw fiber fraction after hydrothermal pretreatment
with respect to xylose, arabinose, glucose, lignin, and mineral elements at
varying pretreatment-severities. Solubilization of phosphorus and the mineral
elements magnesium, calcium, zinc, and manganese showed high pH dependency. At
low pH, these elements were solubilized so that less than 20% by weight compared
with the initial amounts present in the untreated wheat straw were recovered in
the fiber fraction. At high pH, recovery of these elements was temperature-dependent,
presumably due to a combined effect of opening of the cell walls by
solubilizing cell wall constituents (mainly hemicellulose) and increased
solubility of some elements at acidic pH. The levels of other elements in the fiber fraction, that is,
iron, copper, aluminum and silicon, did not depend on pretreatment conditions,
and hence could not be modeled.
Materials
and methods
Wheat straw material Wheat
(Triticum aestivum L.) straw was grown and harvested in Denmark in 2012, and
cut into pieces approximately 10 cm long prior to hydrothermal pretreatment (see
below). The chemical composition of the untreated wheat straw (determined
according to National Renewable Energy Laboratory (NREL) procedures [30,31] and
multi-elemental analyses respectively (the latter method is described further
below) was: 337 g/kg dry matter (DM) glucose, 225 g/kg DM xylose, 30 g/kg DM
arabinose, 182 g/kg DM lignin, 57 g/kg DM extractives (fats and proteins), 92
g/kg DM ash, 13.4 g/kg DM potassium, 12.4 g/kg DM silicon, 4.0 g/kg DM calcium,
1.7 g/kg DM phosphorus, 1.1 g/kg DM iron, 1.1 g/kg DM aluminum, 0.9 g/kg DM magnesium,
0.1 g/kg DM sodium, 0.1 g/kg DM manganese,0.1 g/kg DM zinc, and 0.01 g/kg DM
copper. Throughout this study, contents of monosaccharides are presented as
dehydrated values.
Hydrothermal
pretreatment
Hydrothermal pretreatments were
performed in controlled batch runs using Mini-IBUS equipment (Technical University
of Denmark, Risø Campus, Roskilde, Denmark). Wheat straw (1 kg DM) was soaked
at pH 2, 6 or 10 for 30 minutes, and thereafter treated at 170°C, 183°C or
196°C for 14, 18, or 22 minutes according to a
Box-Behnken statistical design
with duplicate runs of the center point. pH was adjusted with sulfuric acid and
ammonium hydroxide; the concentrations needed to reach the desired pH values of
the soaking straw was determined on a small scale prior to the pretreatment
campaign. After hydrothermal pretreatment, the pressure was relieved in the
reactor, and the biomass was immediately pressed to 30 ± 4% DM. Afterwards, the
fiber fraction was washed in Milli-Q-grade deionized water (1:8 solid: liquid ratio)
for 30 minutes at 50°C and 150 rpm, and pressed to 34 ± 5% DM. Liquid fractions
were discarded, while all fiber fractions were weighed, frozen ,and then stored
at −24°C until further analysis.
Chemical
analysis
Fiber fractions after
hydrothermal pretreatment were analyzed for chemical composition by methods
based on the standard NREL analytical procedures [30,31]. The analysis of all
samples was performed in duplicate with a coefficient of variation (CV) of less
5%, and included DM and ash content determination and strong sulfuric acid
hydrolysis for structural carbohydrates and lignin. Untreated wheat straw was
subjected to ethanol extraction for 24 hours prior to strong acid hydrolysis
because of its high content of extractives (fats and proteins).
Multi-element
analysis
Multi-element analyses of the
untreated wheat straw and fiber fractions were conducted by inductively coupled
plasma-optical emission spectroscopy (Optima 5300 DV, PerkinElmer, Waltham, MA,
USA). To enable silicon analysis with low background values, the sample
introduction system was mounted with a hydrogen fluoride (HF)-resistant,
silicon-free kit comprising a Dura Mist nebulizer, a Tracey TFE spray chamber,
and a Sapphire injector. Prior to analysis, samples (100 mg) were digested at
2,300°C for 25 minutes in a medium consisting of a mixture (v/v) of 47.3% HNO3,
4% H2O2, and 2.65% HF in Teflon tubes in a pressurized microwave oven
(UltraWave, Milestone Inc., Sorisole, Italy). The addition of HF ensured that
silicon was solubilized and remained in solution during the analysis. Before
analysis, samples were diluted to 3.5% HNO3 with Milli-Q element water (Merck
Millipore). Data quality was evaluated using a certified reference material
(spinach; NCS ZC73013, National Analysis Center for Iron and Steel, China),
internal standard additions of silicon, and true blanks. Data were processed
using WinLab32 software (v3.1.0.0107; PerkinElmer, Waltham, MA, USA). For each
element, more than one wavelength was used for analysis to decrease the possibility
of matrix interference.
Statistical
analysis
R statistical software (v3.0.2)
was used for statistical data analysis [32]. Response surface modeling was
performed on the recoveries of constituents in the fiber fraction and presented
as perspective plots of the response surfaces. PCA was performed to study and
visualize correlations between the constituents of the fiber fractions. Score
plots were used to deduce which PCs were governed by which pretreatment
factors, while loading plots were used to show the correlation between the
different constituents. Cluster analyses were performed by ascendant hierarchical
clustering using the ClustOfVar package [33]. To allow prediction of fiber
fraction composition after hydrothermal pretreatment, an empirical factor, denoted
cpH, in an extended pretreatment-severity equation, Eq. (1), was optimized in
the interval 0 to 1 to obtain the best linear or exponential fit to the data. log
Re ð Þ¼Log t⋅eT−100 14:75 _ _ −cpH⋅pHinitial
ð1Þ where Re is the extended pretreatment-sevxerity factor, tis the treatment
time in minutes, T is the treatment temperature (°C), and 100 is the reference
temperature (°C). 14.75 is a fitted value of an arbitrary activation energy
constant (ω) when assuming pseudo-first-order kinetics [26,34]. All models were
validated by QQ plot of the residuals (data not shown). Abbreviations HF:
Hydrofluoric acid; NREL: National Renewable Energy Laboratory; PC: Principal
component; PCA: Principal component analysis. Competing interests The authors declare
that they have no competing interests. Authors’ contributions DL carried out
and participated in the design of the experiments, conducted the statistical
analysis and modeling of the data, and drafted the manuscript; HS and NK
participated in the design of the experiments, and in discussion and
interpretation of the results; JS was responsible for the multi-element analysis,
and the interpretation and discussion of these results; and AM contributed to
conceiving the study, designing the experiments, analyzing the data, and
writing the manuscript. All authors read and approved the final manuscript. Acknowledgement
We thank Ingelis Larsen and Tomas Fernqvist (Technical University of Denmark,
Risø campus) for their assistance in the laboratory and execution of the
hydrothermal pretreatment campaign. This work was supported by
the Danish National Advanced
Technology Foundation via the Technology Platform ‘Biomass for the 21st
century—B21st’. Author details 1DONG Energy, Kraftværksvej 53, DK-7000
Fredericia, Denmark. 2Center for BioProcess Engineering, Department of Chemical
and Biochemical Engineering, Technical University of Denmark, DK-2800 Lyngby,
Denmark. 3Plant and Soil Science Section, Department of Plant and Environmental
Sciences, Faculty of Science, University of Copenhagen, DK-1871 Frederiksberg
C, Copenhagen, Denmark. Received: 10 April 2014 Accepted: 16 September 2014
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