Analysis of Wheat Straw Biodiversity for Use as a Feedstock for Biofuel Production
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Wheat straw is a potential source of feedstock for biofuel production that does not compete with food. We have screened 48 wheat lines from a collection representing a broad range of the biodiversity of wild and domestic wheat. Wheat straw was fractionated into water-soluble and nonsoluble fractions. In the water-soluble fraction (WSF), we found a broad variation in the concentration of free soluble sugars (FSS) and a narrow variation in starch. The FSS fraction could reach levels of reducing sugars as high as 130 g sugar/kg of straw. The analysis of the FSS by ion chromatography indicated that fructose and glucose were the major sugar monomers in this fraction. The composition of the nonsoluble cell wall fraction was determined by both pyrolysis and direct chemical analysis. These analyses showed a limited variation in the lignin or the cellulose fraction. There was a significant degree of variation among wheat lines in the enzymatic saccharification of the straw, following acid pretreatment. Interestingly, the straw from wild wheat had the highest degree of saccharification compared to domestic lines. These findings are of interest for the biofuel industry because they mean that wheat lines can be developed in which a significant amount of free soluble sugars can be easily extracted from straw without the need for costly pretreatment and enzymatic deconstruction. Moreover, the high FSS trait might be combined with the high enzymatic saccharification trait suggesting that wheat lines can be developed with a straw composition better adapted for biofuel production.
KeywordsLignocellulosic biomass Bioethanol Wheat straw Free soluble sugars (FSS) Water-soluble fraction (WSF)
Wheat straw is a by-product left after the grain harvest, and thus has a great economic potential to generate bioethanol fuels worldwide. The annual global production of wheat grain in 2012 was estimated to be 670 million tons , and it keeps increasing each year. Assuming that the ratio between residues (straw) to grains is 1.3 , the residue weighs 887 million tons. Considering other applications of the wheat straw such as plowing back to the soil to prevent soil erosion or for tillage practices, burning, bedding, and forage for ruminants and more, it is estimated that half of this amount, i.e., ~400 million tons, will remain unused and could be an available biomass for ethanol production . According to biofuels-OECD-FAO Agricultural Outlook 2013–2022 , ethanol production is expected to increase by 67 % over the next 10 years, and the forecast is to reach up to 168 billion liters by 2022, most of it from sugarcane and grains. The prospect to produce bioethanol from these starch/sugar-rich crops exacerbates the food versus fuel conflict . The feedstock for second generation biofuels produced from the lignocellulosic biomass of crops has the advantage of being cheap and abundant, and its production is not competing on agricultural lands or food unlike many other first generation biofuel feedstocks. Bioethanol produced from lignocellulosic biomass could satisfy a proportion of the increasing demand for liquid fuels , it can be used as sole fuel in cars with dedicated engines or as an additive (up to 30 %) with no engine modification requirements . Still, technological progress is essential in order to increase the implementation of second generation biofuels and to reduce the processing cost to make it cost-competitive [5, 6]. The lignocellulosic biomass inside the plant cell wall is composed of three main components: lignin, hemicellulose, and cellulose. The conversion of lignocellulose biomass into ethanol requires three main steps: (i) pretreatment, which disrupts the connection between the carbohydrates and the lignin, depolymerizes and solubilizes the hemicellulose polymers, and exposes the cellulose and the hemicellulose for enzymatic degradation; (ii) saccharification, the enzymatic hydrolysis of the cell wall carbohydrates to their sugar monomers; (iii) microbial fermentation of the six carbon sugar monomers into ethanol . During the development of cool season cereals (C3 plants) (e.g., wheat, barley, oats), water-soluble carbohydrates can accumulate in the stem and leaf sheath. They are mainly composed of fructans, sucrose, glucose, and fructose [8, 9]. The phenomenon of nonstructural carbohydrate accumulation has been well documented in rice, in which the stem behaves as a tentative source of sugar translocation to the grains. Rice stem carbohydrates consist of starch, sucrose, and soluble reducing sugars (without fructans) . Carbohydrates temporarily accumulate in wheat stems during the early reproductive growth phase, predominantly as water-soluble carbohydrates, and are subsequently remobilized during grain filling. This carbon accumulation becomes more important for grain yield in cereal crops under abiotic stress. In wheat plants undergoing drought stress, the carbon supply from photosynthesis is reduced due to both stomatal closure in the leaves and downregulation of genes involved in the Calvin Cycle . Under these conditions, the stem water-soluble carbohydrates can contribute up to 50 % of grain yield . Wheat straw was used as a feedstock for bioethanol production [11, 12, 13, 14], but successful engineering or breeding of straw that is well adapted to bioethanol production are still needed. Traits to improve include both the efficiency of enzymatic saccharification and the contents of sugars in the water-soluble fraction. Genetic engineering of wheat remains problematic due to the technical difficulty at performing transformation and because regulations for genetically modified wheat will probably be more draconian than for other crops not directly consumed by humans. On the other hand, there is a great biodiversity that has not been harnessed for improving straw as a feedstock for biofuel, and that could be used by breeders without regulatory hurdles. The wheat group (Aegilops and Triticum species) includes several species whose chromosomes can recombine with their homologs or homeologs in bread or durum wheat . This enables the transfer of genes from these species into modern cultivars.
In this work, we have screened wheat biodiversity to identify lines whose straw is well adapted as a feedstock for bioethanol production. We screened a core collection of 48 wheat lines representing a broad spectrum of genetic variation, including wild and domestic lines, tetraploid and hexaploid, as well as traditional lines or modern varieties. The straw composition was analyzed, including the main components of the water-soluble fraction, such as starch, fructans, or free soluble sugars and also the cell wall components, such as cellulose, hemicellulose, and lignin . We identified wheat lines with a high concentration of free soluble sugars (FSS) in the straw. Fructose and glucose were the two major sugars in this fraction. Such lines produce significant yield of fermentable sugars without the need for harsh pretreatment or for enzymatic deconstruction. In addition, we identified variation among wheat lines in the efficiency of enzymatic saccharification of the straw, following acid pretreatment.
Materials and Methods
Plant Material and Growth Conditions
The 48 wheat lines were grown in three replicates, each in a different block. Plants were grown in 5-l pots, each pot corresponding to a different replica. All the 48 lines and their replicates were grown under the same conditions, in winter 2012, in a net house at the Weizmann Institute, Rehovot, Israel. The 48 lines of wheat were from six different genetic groups (Online Resource 1): Tetraploid wild emmer wheat (Triticum turgidum var. diccocoides, TTD), tetraploid domesticated emmer wheat (Triticum turgidum var. diccocum, TTC), tetraploid durum wheat (Triticum turgidum var. durum, TTR) both landrace (traditional) and modern and hexaploid bread wheat (Triticum aestivum var. aestivum, TAA) both landrace and modern . Harvested stems and leaves from three upper nodes of the wheat straw were ground and dried at 60 °C.
A volume of 1.5 ml DW was added to 25 mg of dried and ground wheat straw in tube 1. The sample was boiled for 1 h and centrifuged at 10,000 rpm for 5 min. A volume of 1.3 ml of the supernatant was transferred into tube 2, was freeze-dried, and weighed.
DNS (3,5-Dinitrosalicylic Acid) Assay for Reducing Sugar Measurements
The concentration of the reducing sugars [mM] was determined using the 3,5-dinitrosalicylic acid (DNS) assay using glucose as standard for the calibration curve. Each sample was diluted 20× with DDW and vortexed. Calibration was made by preparing glucose standards. A 20-mM glucose solution was diluted in DDW to obtain final concentrations of glucose from 0 to 5 mM. Then, 150-μl DNS  reagent was added to 100 μl of the diluted hydrolyzate and to the glucose standards. The standards and samples were boiled for 5 min. The absorption (OD) was read at 540 nm. A calibration curve was plotted from the glucose standards and used to find the concentration of reducing sugars [mM] in the experimental samples.
Determination of FSS and Starch Quantities in the WSF
To quantify the free reducing sugars in the WSF, the DNS assay was carried out for half of the WSF, without any pretreatment. The second half was treated with α amylase and pullulanase enzymes (see cell wall isolation). The amount of reducing sugars in the untreated WSF was subtracted from this treatment to obtain the net amount of glucose obtained by starch enzymatic hydrolysis.
Determination of Fructan Quantities in the WSF
The amount of fructans was estimated by measuring the total amount of reducing sugars using the DNS assay before and after hydrolysis with 1 % H2SO4 for 1 h at 121 °C. Reducing sugars generated due to the H2SO4 treatment would originate from fructan hydrolysis.
Thermogravimetric analysis (TGA)  of the samples was performed using the SDT Q600 V8.3 Build 101 thermal analyzer. Samples of about 7–10 mg were taken from the dried and ground straw for analysis at a temperature ranging between 23° and 800 °C with a heating rate of 10 °C/min and a flow of nitrogen at 100 ml/min. The crucible used was of alumina (Al2O3), which also served as a reference for the sample. From this, we obtained the data to determine the percent of weight loss for each sample and first derivative of the thermogram (DTG) which indicates the temperature of decomposition for the components in the wheat straw.
Ion Chromatography Analysis for WSF
The WSF was analyzed by high-performance anion-exchange chromatography  coupled to pulsed amperometric detection (HPAEC-PAD) using an Dionex ICS3000 system equipped with a CarboPac PA1 column coupled to a CarboPac PA1 guard column (both from Dionex). The amperometric detector was equipped with a disposable gold electrode (Dionex), and the standard carbohydrate waveform recommended by the manufacturer was applied. Injection volume was set at 10 μl for all samples. Flow was set at 1 ml/min. Sugars were eluted using two methods: gradient elution for the assignment of the differential compounds and isocratic elution for the absolute quantification of glucose, fructose, and sorbitol in WSF. Eluent A was DDW, eluent B was 0.1 M NaOH. Gradient elution method was adopted from Couturier et al. . Gradient program started from isocratic elution at 20 % B for 20 min; then, a linear gradient to 100 % B was applied for 0.2 min, isocratic elution at 100 % B for 14.8 min to flush the column, linear gradient to 20 % B for 0.2 min and reconditioning of the column at 20 % B for 14.8 min (total run time was 50 min). Peak assignment was based on the analysis of individual standards: glucose, fructose, sorbitol, mannose, arabinose, galactose, ribose (Sigma). Isocratic elution method: 50 % A-50 % B for 20 min. External calibration curves were prepared for fructose in the range of 1–100 μg/ml, for glucose 1–25 μg/ml, for sorbitol 1–15 μg/ml.
Enzymatic Hydrolysis of Acid-Pretreated Samples by Commercial Cellulase Preparation
An acid pretreatment protocol  was carried out using 1-ml 1 % H2SO4 on 50-mg ground straw in 2-ml tubes. Each tube was autoclaved at 121 °C for 20 min (overall cycle length 90 min). After 4 washes with 1 ml DDW and one wash with 1-ml acetate buffer (50 mM, pH = 4.9), enzymatic hydrolysis of cellulose was performed. We added 1 ml of acetate buffer (50 mM, pH = 4.9) in the presence of 32 μl Celluclast and 8 μl β-glucosidase (final enzyme loading is 25 mg cellulase proteins per 1 g of dry sample). For this purpose, a commercial cellulase preparation (Sigma) supplemented with β-glucosidase (Sigma) was used. The tubes were placed in a shaker incubator at 50 °C and were agitated at 150 rpm for 24 h. The concentration of the reducing sugars [mM] generated by the enzymatic hydrolysis after 24 h was determined using the DNS assay.
Cell Wall Component Analysis
Cell Wall Isolation
Dried wheat straw was ground, weighed, and washed in 70 % ethanol, vortexed and centrifuged at 10,000 rpm for 10 min, and then the supernatant was aspirated. The pellet was resuspended in chloroform/methanol (1:1 v/v solution), centrifuged at 10,000 rpm, decanted, and resuspended in acetone. The solvent was evaporated, and the pellet was resuspended in sodium acetate buffer (pH 5.0), α-amylase (Sigma), and pullulanase (Sigma) in order to remove starch from the pellet. The pellet was washed three times with DDW. Acetone was added, and the pellet was air-dried .
Crystalline Cellulose Content
The dried cell wall was weighed into a tube. Updegraff  reagent (acetic acid/nitric acid/water, 8:1:2 v/v) was added, vortexed, and heated at 100 °C for 30 min, when only crystalline cellulose remains insoluble in the pellet. Samples were cooled, centrifuged, washed with DDW and acetone, and air-dried. The supernatant was discarded. The crystalline cellulose content was quantified after being completely hydrolyzed into glucose with the colorimetric Antherone assay . For that purpose, 72 % sulfuric acid was added and incubated at room temperature for 30 min. The assay was performed in a 96-well plate. A glucose standard curve was prepared using 1-mg/ml glucose stock in order to create standard solutions of 0, 2, 4, 6, 8, and 10 μg glucose in 100 μl DDW. From the samples, 2 μl was added to 98 μl DDW. To this, 200 μl of freshly prepared Anthrone reagent was added. By heating the plate at 80 °C, glucose containing samples turn from yellow into blue green. Absorption (OD) was read at 625 nm. Glucose (and hence crystalline cellulose content) was calculated based on the absorbance compared to the standard curve established on the same plate.
Results and Discussion
WSF Composition in 48 Wheat Lines
Free Sugar Monomers Comprising the FSS of Wheat Straw
Wheat Straw Main Components by Pyrolysis
A clear difference was obtained between high and low FSS lines (Fig. 4a). The high FSS lines (in green) show a peak at 200 °C that is almost absent in the low FSS lines (in red). The WSF was extracted from the samples, and the dried nonsoluble fraction was analyzed by TGA. As expected, the peak at 200 °C disappeared in all of the samples after the WSF extraction (Fig. 4b). The other peaks, corresponding to hemicellulose, cellulose, and lignin, did not show significant difference between the lines.
Differences Among 48 Wheat Lines in Enzymatic Hydrolysis After Acid Pretreatment
Analysis of the Straw Cell Wall Main Components
In order to test if there is a correlation between the sugar release (following acid pretreatment and enzymatic digestion) and the cell wall components, we measured lignin and crystalline cellulose in six high-sugar release lines (TAA 131,TAA 1, TAA Zahir, TAA Gedera, TTR 2, TAA55) and in six low-sugar release lines (TAA Bernir, TTD31, TTD 68, TTD 5, TTC 8, TAA 222). Lignin content in the cell wall of wheat straw ranged between 18 and 23 %, with no correlation to the high- and low-sugar release groups (Online Resource 7a). Crystalline cellulose content in the cell wall ranged between 27 and 37 % (Online Resource 7b), with no correlation to the high- and low-sugar release lines. FSS amount in these lines also did not show any correlation to sugar release (Online Resource 7c).
We discovered much variation in the content of FSS in the straw among the wheat lines analyzed. Free sugars consist mostly of fructose and glucose and to a lesser extent of sorbitol, three monosaccharaides that are easy to ferment into ethanol using standard industrial yeast strains . Extracting these sugars from the straw requires only grinding and boiling, with no need for costly pretreatment or enzymatic treatment for deconstruction. In quantitative terms, the potential availability of such resource for bioethanol production is very significant. Despite fluctuations with the methods (DNS, ion chromatography and GC-MS) and the lines, we consider for sake of simplicity that the FSS in the WSF of wheat straw could represent up to 10 % of the biomass ~100 g/kg. Considering the 400 million tons of straw (see “Introduction”) that are available worldwide as a feedstock for biofuel, this means that 40 million tons of sugars could be extracted at low cost and converted to ~20 billion liters of ethanol. This value could be further increased, using glucose from the starch in the WSF, which is also easy to extract, requiring a simple amylase treatment, or alternatively amylase activity can be engineered in yeast . Further studies should help determine if the high free sugar trait in the straw is desirable or is associated negatively to agronomic performances, and genetic and genomic analysis should be helpful to determine the molecular basis for this phenotype. In Sorghum, high sugar in the stems and high grain yield can coexist ; therefore, it might also be possible in wheat. The ease of extraction of the WSF opens the prospect for simple scenarios, such as processing of the wheat straw on the farms where wheat is harvested, providing farmers with a means of producing ethanol on-site. Finally, the enzymatic saccharification of the nonsoluble fraction, i.e., the cell wall, after pretreatment, showed limited but significant differences (~30 %) between lines (Fig. 5). Increasing both the WSF sugars and the efficiency of enzymatic saccharification in wheat straw would contribute to its improvement as a feedstock for bioethanol production.
We would like to thank Prof. Moshe Feldman (Weizmann Institute of Sciences) for providing seeds of the wheat collection and for advice and Prof. Asaph Aharoni (Weizmann Institute of Sciences) for advice with the sugar composition analysis. We thank Dr. Eli Morag and Dr. Michael Loelovich (Designer Energy) for help with the cellulose hydrolysis. We thank Dr. Miron Abramson and Tali Eylat (Futuragene) for help with the cell wall component analysis. We also thank Ilan Bessudo for help with preparation of straw samples for analysis and Dr. Zvi Tamari for help with sugar composition analysis. This work was supported by a grant from the ICORE foundation, solar fuel project and by the Leona and Harry Helmsley charitable trust to A.A.L. YT was supported by the Ministry of Science and Technology, Israel.
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