形态结构玉米秸2012-Cell morphology and chemical characteristics of corn stover fractions

Industrial Crops and Products 37 (2012) 130–136Contents lists available at SciVerse ScienceDirectIndustrial Crops andProductsj o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /i n d c r opCell morphology and chemical characteristics of corn stover fractionsZhiyong Li a ,b ,Huamin Zhai a ,∗,Yan Zhang b ,Li Yu ba Jiangsu Provincial Key Lab of Pulp and Paper Science and Technology,Nanjing Forestry University,Nanjing 210037,China bUPM Asia Research &Development Center,Changshu 215536,Chinaa r t i c l ei n f oArticle history:Received 16October 2011Received in revised form 17November 2011Accepted 22November 2011Available online 11 January 2012Keywords:Corn stover fractions Cell morphologyChemical components Inorganic elements Lignin distributiona b s t r a c tThis paper investigated cell morphology,chemical components,lignin distribution and inorganic ele-ments distribution of corn stover fractions.Corn stover fractions,classified as stalk rind,stalk pith and leaf,had different tissues,cell morphology and chemical compositions.Corn stalk rind had good fiber morphological characteristics for papermaking,while stalk pith,having short fibers and high contents of parenchyma and vessel,was not suitable for papermaking.Stalk rind had the highest lignin and cellulose content but the lowest hemicellulose content among all the fractions.The major ash-forming elements in corn stover fractions were potassium,chlorine,silica,calcium,magnesium and sulfur.Potassium and chlorine took more than 86%of total inorganic elements in stalk rind while silica content was much higher in leaf and stalk pith than that in stalk rind.Perivascular sclerenchyma and subepidermal sclerenchyma of stalk rind were more lignified than the other tissues.The highest lignin concentration existed in cell middle lamella and corner.All corn stover fractions could be good biorefinery feedstock based on their main chemical compositions,though they are obviously heterogeneous in aspects of cell morphology and chemical characteristics.© 2011 Elsevier B.V. All rights reserved.1.IntroductionCurrent environmental and social pressures are limiting wood harvest volumes worldwide.Therefore wood supply is getting less and less for pulp and paper industry.Non-woody lignocellulosic biomass,such as wheat straw,rice straw and reed,has been an important fiber resource for decades in countries with a short-age of woods,like China.Meanwhile,lignocellulosic biomass is also a potential renewable feedstock for biofuels and biorefinery (Ragauskas et al.,2006).As a kind of abundant and renewable agri-cultural residue,corn (Zea mays L.)stover could be a low-cost and sustainable source for energy and chemicals in future.The term corn stover normally refers to combination of corn stalk (stem)and leaf.Annual production of corn stover in China was about 200million tons in 2008,which counted for one third of the total agricultural residues production in the country.In United States,since corn stover is the largest non-food biomass available source,it has been studied for biofuel process development at National Renewable Energy Laboratory (NREL)(Decker et al.,2007).Numerous studies have been undertaken to investigate pre-treatment and bioconver-sion of corn stover to biofuel (Akin et al.,2006;Kim and Lee,2006;van Walsum and Shi,2004;Weiss et al.,2010;Zhu et al.,2009).∗Corresponding author.Tel.:+862585428697;fax:+862585428697.E-mail address:hzhai@ (H.Zhai).Biomass is a complex heterogeneous mixture of organic com-ponents and,to a less extent,inorganic matter,containing various solid and fluid intimately associated phases or minerals with dif-ferent origins (Vassilev and Vassileva,2009).Corn stover always consists of stalks,leaves,husks and ears;and stalks are composed of stalk rind and pith.All these fractions have different tissue struc-tures,fiber properties,and chemical compositions.Vassilev et al.(2010)indicated that there are two fundamental aspects relating to biomass usage for fuels and biorefinery materials:to extend and improve basic knowledge of the composition and properties and to apply this knowledge for the most advanced and environmentally safe utilization.The chemical composition of lignocellulosic feed-stock is a key factor affecting efficiency of biofuel production during conversion processes (Hamelinck et al.,2005;Hames et al.,2003).Recently,an effective mechanical fractionation of corn stover into stalk rind,stalk pith,and leaves is realized (Sun,2003).The wider utilization of corn stover as a source of fiber,biofuel and biochemi-cals requires a comprehensive understanding of the cells (including fibers)properties,chemical characteristics,lignin distribution and inorganic elements distribution.Corn stover has been investigated as a fiber resource for pulp and papermaking since 1929(Jahan et al.,2004).Corn stalk fibers showed good performance for papermaking (Ahmed and Zhu,2006;Byrd et al.,2006;Cheng et al.,2008;Wiernik et al.,2002).However,corn stover has not yet been successfully commercial-ized for pulp production worldwide.The reason might come from two aspects:firstly,the abundant wood supply was the most0926-6690/$–see front matter © 2011 Elsevier B.V. All rights reserved.doi:10.1016/j.indcrop.2011.11.025Z.Li et al./Industrial Crops and Products37 (2012) 130–136131convenient way for pulping industry in20th century;secondly and maybe more importantly,the heterogeneity of corn stover fractions and their integrated harvest limit the application.However the sit-uation starts changing significantly because of shortage of wood supply and technology development.Recent technology develop-ment has allowed the fraction separation by either mechanical or chemical methods.Therefore,corn stover is becoming an impor-tant feedstock for integrated biorefinery process to producefibers and other valued biochemicals simultaneously.Althoughfiber properties of corn stover have been studied for decades,no systematic investigation of cell morphology andfiber quality of different corn stover fractions is done so far.In this paper,the cell morphology andfiber quality of corn stalk rind, stalk pith,leaf blade and leaf sheath will be investigated system-atically.Chemical components of corn stover fractions have been investigated in some studies.Ahmed and Zhu(2006)studied the contents of lignin,cellulose and pentosan in corn stalk rind and the sugar components of corn stalk.Hess et al.(2002)examined the chemical variations within the node,internode,and leaf sec-tions of corn stover.Tortosa Masiáet al.(2007)characterized the ash components of corn stover.However,none of the above studies provided detailed and completed information of chemical compo-nents,sugar components and inorganic elements of individual corn stover fractions,which this paper will focus on.Furthermore,lignin distribution and inorganic elements distribution in corn stalk rind will also be investigated in this paper.2.Materials and methods2.1.MaterialsThe corn stover in this study was collected from Henan,China. The air-dried corn stover was manually fractionated into leaves and stalksfirstly.Then the stalks were separated into rind and pith. Leaves included both leaf blades and leaf sheaths.Husks and cobs were not included in the study because they together just take less than1%in weight percentage.The above separated fractions were stored in plastic bags for future use.2.2.Methods2.2.1.Weight percentage of corn stover fractionsAbout5kg of air-dried corn stover was manually separated into stalk rind,stalk pith and leaves.The separated fractions were weighed individually and their dryness contents were determined according to Technical Association of Pulp and Paper Industry (TAPPI)test method T258om-02.Two batches of separation were conducted and the weight percentage of each fraction was calcu-lated on oven-dry basis.2.2.2.Cells morphology and their dimensions analysisFour fractions,stalk rind,stalk pith,leaf blade and leaf sheath, were prepared forfiber morphology and quality analysis.The frac-tions were cut into1mm wide and10mm long strips manually followed by treatment with mixture of acetic acid and30%hydro-gen peroxide(1:1,v/v)at60◦C for72h for cell dissociation.When the color of samples turned to white,the macerated cells werefil-tered and thoroughly washed with distilled water.The cells were stained with Graff C and thefiber morphological features were examined with Olympus BX51research microscopy.Thefibers were disintegrated with Metso handy disintegrator which does not introduce any mechanical damage to samples,and analyzed with Metso Kajaani-FiberLab FS-300fiber quality analyzer.Thefiber length(L),fiber diameter(D),cell wall thickness(W)andfines con-tent were length weighted values.Fines were defined asfinefibers with a length smaller than0.02mm.Three derived values,Runkel ratio,slenderness ratio andflex-ibility coefficient,were used to assess the suitability of plant raw materials for paper production(Saikia et al.,1997).2.2.3.Chemical characterizationFor chemical analysis,the40–60mesh fractions of stalk rind, stalk pith and leaves were prepared in accordance with TAPPI test method T257cm-02.All chemical composition results were calcu-lated based on oven-dried corn stover fractions.The extractives and ash were analyzed according to TAPPI test method T204cm-97and T211om-02respectively.The holocel-lulose was obtained by treating the extractive-free samples with NaClO2and acetic acid to remove lignin,and then the␣-cellulose content was determined by further treating holocellulose with 17.5%NaOH to remove the hemicellulose(Han and Rowell,1996). Klason lignin content was determined according to TAPPI test method T222om-02.Acid-soluble lignin content was determined by a spectrophotometric method based on absorption of ultravio-let radiation(Schoening and Johansson,1965).Acetyl and formyl contents were determined by hydrolysis with NaOH and IC analysis (Anttila et al.,2007).Nitrogen content was determined according to the Kjeldahl method.The crude protein content was calculated from nitrogen content by multiplying a coefficient of6.25.2.2.4.Sugar analysis of hemicellulose and pectinsThe sugar components of hemicellulose and pectins were determined by acid methanolysis and gas chromatography(GC) (Sundberg et al.,1996).About10–20mg of freeze dried sample was weighted in a conical bottle with cap.Acid methanolysis was performed with2ml of2M anhydrous HCl/MeOH at100◦C for 3h.After cooling in room temperature for15min,the sample was neutralized with300␮l of pyridine.Additionally,4ml of sorbitol standard(0.10mg/ml)was added as internal standard.Accurate 1ml of the clear upper phase was transferred to another conical bottle and evaporated to dryness with nitrogen.Derivation was conducted by adding200␮l of pyridine,300␮l of hexamethylsi-lazane(HMDS)and200␮l of trimethylchlorosilane(TMCS).The derived sugars were analyzed by Varian3900GC with FactorFour capillary column VF-1ms.2.2.5.Inorganic components analysisThe silica content was determined according to related TAPPI test method T244cm-99for acid-insoluble ash determination.To determine the contents of sodium,potassium,calcium,magnesium, manganese,iron,copper,phosphorus,sulfur,aluminum and zinc, the40–60mesh fractions were digested in microwave digester by adding7ml of HNO3and1ml of H2O2.The digested samples were then washed into a50-ml volumetricflask and diluted to the mark with18-M ultrapure water.The elements were determined by Thermo Scientific iCAP6000ICP-OES.For chloride content determination,samples were boiled in dis-tilled water for1.5h and thenfiltered.Thefiltrate was analyzed for chloride by ICS-1000with IonPac AS23anion-exchange column.2.2.6.Inorganic elements distribution in stalk rindThe stalk rind was cut into3mm wide and10mm long strips manually.The strips were extracted with benzene–ethanol(2:1, v/v)for24h and then dehydrated with graded ethanol solutions. Then the samples were embedded in epoxy resin and polished with Struers LaboPol-5polisher.The polished samples,coated with car-bon by JEC-560auto carbon coater,were examined with a JEOL 6009analytical scanning electron microscopy(SEM)with energy dispersive X-ray analysis(EDXA).132Z.Li et al./Industrial Crops and Products37 (2012) 130–136Table1Fiber dimensional parameters of corn stover fractions compared with China white poplar and reed.Fraction Stalk rind Stalk pith Leaf blade Leaf sheath China white poplar a Reed(internode)bFiber length,mm 1.11±0.010.38±0.010.86±0.030.81±0.04 1.18 1.22Fiber diameter,␮m19.7±0.118.4±0.519.3±0.418.8±0.421.017.3Cell wall thickness,␮m 5.3±0.1 4.3±0.1 5.3±0.1 5.5±0.1 2.4 4.4Lumen diameter,␮m9.19.88.77.816.28.5Runkel ratio 1.160.88 1.22 1.410.37 1.04Slenderness ratio562145435671Flexibility coefficient465345417749Fines content,% 5.529.518.717.1––Vessels,pcs/1000181299136196––Lumen diameter=fiber diameter−(2×cell wall thickness);Runkel ratio=(2×cell wall thickness)/lumen diameter;slenderness ratio=fiber length/fiber diameter;flexibility coefficient=(fiber lumen diameter/fiber diameter)×100.a Xu et al.(2006).b Ververis et al.(2004).2.2.7.Lignin distribution in stalk rindStalk rind was cut into1mm wide and10mm long strips.The strips were extracted with benzene–ethanol(2:1,v/v)mixture for 24h and then dehydrated with graded ethanol solutions.After embedding with epoxy resin,the transverse section with5-␮m thickness was cut with Leica RM2265microtome.The section sam-ples were stained with1%toluidine blue,and were viewed with Olympus BX51research microscopy.Another section sample was stained with1%KMnO4solution and coated with carbon for SEM-EDXA analysis.3.Results and discussion3.1.Cell morphologyAfter harvest of corn,corn stover mainly consists of stalk and leaves,while ears and husks take only tiny portions.Corn stalk was separated into two distinct fractions:rind and pith,and leaf fraction was divided into leaf blade and leaf sheath.The cell mor-phologies of four fractions are shown in Fig.1.Obvious differences can be observed in the light microscopy pictures where several cell types can be distinguished.Plant parenchyma is unspecialized plant tissue consisting of simple thin-walled cells with interven-ing air spaces.These parenchyma cells often have thin cell walls, which,in small quantities,can aid infiber bonding.However,in larger percentages,thin walled parenchyma cells can plug paper sheet so as to reduce paper machine drainage and increase paper density.Parenchyma cells of stalk rind dispersed throughout the vascular bundles.Generally,four kinds of parenchyma cells could be distinguished in Fig.1:claviform,round,pillow-like and spongy.Claviform parenchyma cells,mainly existing in stalk rind,had relatively higher wall thickness than the other kinds of parenchyma,so they can almost keep their original shapes after defiberation. Parenchyma cells of stalk pith were almost spongy tissues that occupy most of the pith volume.This feature makes pith unsuit-able for papermaking.The leaf blade contained more pillow-like and round parenchyma cells than leaf sheath.Fibers in pith fraction were much shorter and thinner than that in other fractions.The vessel elements are usually short,thin-walled,with pitting and open ends.Vessels of stalk rind,leaf blade, and leaf sheath were much thinner than those of stalk pith.Epi-dermal cells in stalk rind,leaf blade and leaf sheath had serrated borders,and epidermal cells of stalk rind were longer than that of leaf blade and leaf sheath.There was no epidermis in stalk pith.Thefiber dimensional parameters compared to China white poplar and reed are shown in Table1.It is well known thatfiber morphology with long length,high slenderness ratio(>33)and/or low Runkel ratio(<1)is essential for pulping and papermaking(Xu et al.,2006).Corn stalk rind had longfiber length(1.11mm),which Table2Weight percentage of corn stover fractions on dry basis.Stalk rind Stalk pith Leaf Weight percentage,%52.7±1.57.0±0.540.3±1.2 Reference a,%53.915.830.3a Byrd et al.(2006).was similar to China white poplar,whereas cell walls were signif-icantly thicker than those in the white poplar.Slenderness ratio of stalk rindfibers was also similar to China white poplarfibers but lower than reedfibers.Runkel ratio andflexibility of stalk rindfibers were worse than China white popularfibers but sim-ilar to reedfibers.In principle,poor Runkel ratio means that stalk rindfibers are difficult to be collapsed in papermaking,and low flexibility could have some negative effect on tensile and bursting strengths as well as on folding endurance(Ogbonnaya et al.,1997). However,the reality of reedfibers have shown proved good prop-erties for papermaking(Shatalov and Pereira,2006).Therefore we could expect that stalk rindfibers have similar good performance for papermaking.Pithfibers were short(0.38mm)with poor slenderness ratio, which in return might reduce tearing resistance dramatically.This is partly because short and thickfibers do not produce good surface contact andfiber-to-fiber bonding(Ogbonnaya et al.,1997).High vessel andfines contents in pith were expected to lead to poor drainage and dense paper.Therefore there might be runnability issues especially for high speed paper.Similar performance could be expected due to similarfiber dimension andfine content in the leaf blade and leaf sheath to pithfiber.3.2.Chemical characteristicsThe corn stover was separated into stalk rind,stalk pith and leaf. The percentage of each fraction was determined based on the total dry weight of the sample.As shown in Table2,stalk rind took about half of the whole stover,which is similar to reference.However, pith and leaf content differed a lot.Leaf content variation may come from the harvest process.Mechanical harvest,especially baling,can lead to more leaf loss than manual harvest.The storage method can also influence the weight loss of corn stover fractions,since volatile substances will disappear in outdoor circumstance.The different fractions of corn stover have different cell mor-phologies and play different functions;hence their chemical composition should vary very much.Table3shows the chemical composition of corn stover fractions.The category of extractives in biomass includes all plant materials that are extracellular or not part of the three-dimensional cell wall structure(Hames, 2009).Total water-soluble and/or benzene–ethanol soluble mate-rials are typically quantified gravimetrically and identified only asZ.Li et al./Industrial Crops and Products37 (2012) 130–136133Table3Chemical composition of corn stover fractions.Stalk rind Stalk pith LeafBenzene–ethanol extractives,% 2.5±0.1 6.6±0.2 4.5±0.2 Hot water extractives,%17.8±0.223.1±0.322.7±0.1 Total lignin,%20.1±0.417.0±0.217.4±0.3 Klason lignin,%18.1±0.314.4±0.114.9±0.2 Acid-soluble lignin,% 2.0±0.1 2.6±0.1 2.5±0.1␣-Cellulose,%39.4±0.131.6±0.131.3±0.3 Acetyl content,%7.7±0.19.1±0.5 4.3±0.3 Formyl content,ppm348±3665±33548±33 Nitrogen as N,%0.95±0.02 1.04±0.02 1.21±0.03 Crude protein,% 5.9±0.1 6.5±0.17.6±0.2 Ash,525◦C,%9.9±0.110.0±0.111.3±0.1 Hemicellulose and pectins a,%25.2±0.727.3±0.621.2±0.5a Hemicellulose and pectins content was the sum of sugar components listed in Table4.extractives(Thanmmasouk et al.,1997).Stalk pith had the highest benzene–ethanol extractives content,while stalk rind had the low-est one.Stalk pith and leaf had similar hot water extractives content which was higher than stalk rind.Monomeric sugars(primarily glu-cose and fructose)were found to be the predominant water-soluble components of corn stover,accounting for30–46%of the dry weight of extractives.Other water-soluble extractives may include various alditols,aliphatic acids,inorganic ions,oligomeric sugars and a dis-tribution of oligomers derived from phenolic glycosides(Chen et al., 2007).The highest lignin content existed in stalk rind,while pith and leaf had about3–4%lower lignin content.High lignin content can help corn plant to resist mechanical as well as fungal injury.How-ever,high lignin content is not desired for pulping,because lignin must be removed during chemical pulping.The␣-cellulose,which is a unique molecule differing only in degree of polymerization and crystallinity,content can be regarded Table4Sugar components of hemicellulose and pectins.Stalk rind Stalk pith LeafArabinose,% 1.4±0.1 2.7±0.1 2.9±0.2 Rhamnose,%0.04±0.010.15±0.010.13±0.01 Xylose,%14.6±0.215.9±0.212.4±0.1 Mannose,%0.19±0.010.31±0.010.33±0.01 Glucose,% 1.43±0.07 2.65±0.07 1.99±0.05 Galactose,%0.27±0.010.53±0.010.73±0.02 Glucuronic acid,%0.96±0.03 1.08±0.020.71±0.02 Galacturonic acid,% 6.3±0.3 3.9±0.2 2.0±0.1 Total,%25.2±0.727.3±0.621.2±0.5as an indication of pulping yield.Certainly,higher␣-cellulose con-tent also means more ethanol output in bioethanol process.The stalk rind had much higher␣-cellulose content(39.4%)than that of stalk pith(31.6%)and leaf(31.3%).Unlike cellulose,hemicelluloses are noncrystalline het-eropolysaccharides and classically defined as the alkali soluble material after removal of the pectic substances.The term of pectins or pectic substances is associated with acidic polysaccharides con-sisting of a backbone of mainly(1→4)-␣-bound d-galacturonic acid residues interrupted by the insertion of(1→2)-␣-linked l-rhamnose residues(Xu,2010).Sugar components of hemicel-lulose and pectins in corn stover fractions are shown in Table4. Xylose was the predominant sugar in hemicellulose of corn stover fractions.Stalk pith had the highest xylose content while leaf had the lowest one.Arabinose and glucose appeared as notice-able amounts.Ramnose,mannose,galactose and glucuronic acid were observed as minor constituents.The galacturonic acid in corn stover fractions was very high.This meant that the pectic substances content was very high in corn stover fractions.The hemicelluloses of leaf and stalk have been shown to be arabino-glucuronoxylans of similar structure.Each has a xylanbackboneFig.1.Cell morphology of corn stover fractions by light microscopy:(a):stalk rind;(b):stalk pith;(c):leaf blade;(d)leaf sheath.F,fiber;E,epidermis cells;V,vessels;P, parenchyma.Scale bar=200␮m.134Z.Li et al./Industrial Crops and Products37 (2012) 130–136Table5Inorganic components of corn stover fractions.Stalk rind Stalk pith LeafSilica as Si,%0.38±0.010.37±0.01 2.68±0.01 Chloride as Cl,% 2.35±0.01 2.52±0.020.95±0.01 Potassium as K,% 4.03±0.02 4.17±0.03 2.13±0.08 Calcium as Ca,%0.25±0.020.22±0.020.49±0.03 Magnesium as Mg,%0.26±0.010.14±0.010.34±0.02 Sulfur as S,%0.10±0.010.11±0.010.27±0.01 Phosphorus as P,ppm394±23485±31757±35 Sodium as Na,ppm326±13242±11396±14 Iron as Fe,ppm108±852±4339±15 Manganese as Mn,ppm 6.9±0.1 6.5±0.153.3±0.2 Copper as Cu,ppm8.6±0.18.1±0.110.3±0.1 Aluminum as Al,ppm86±365±2292±11 Zinc as Zn,ppm19±132±136±1 with units joined1→4in the␤-d-configuration.The degree of polymerization is about55for leaf xylan and70for stalk xylan (Dutton and Kabir,1972).Acetyl contents in corn stover fractions were more than100 times higher than formyl content,while pith fraction had the highest acetyl content and leaf fraction had the lowest one.Dur-ing organic acids based cooking,acetyl and formyl groups can be released from feedstocks to compensate part of the acids con-sumption.However,the released acids will consume more alkaline during alkaline and kraft cooking.Leaf fraction had the highest nitrogen content,which meant the highest crude protein content,while the stalk rind fraction had the lowest one.The highest protein content and less lignified tissues may make leaf fraction as good animal feedstock.The crude pro-tein contents in all fractions were considerably high,which were much higher than that of wheat straw(1.9%)but lower than that of rice straw(8.1%)(Pan and Sano,2005).Protein tends to con-dense with lignin under acidic conditions(Whitmore,1982)and retard subsequent delignification.Like most agricultural lignocel-lulosic materials,corn stover fractions had much higher ash content (about10%)than most of woody biomass(Cuiping et al.,2004).Table5shows the detailed inorganic components of corn stover fractions.For both stalk rind fraction and stalk pith frac-tion,the inorganic elements contents ranged from high to low as K>Cl>Si>Ca,Mg>S>P,Na>Fe,Al>Mn,Cu,Zn.For leaf fraction, the sequence was Si>K>Cl>Ca>Mg,S>P>Na,Fe,Al>Mn,Cu,Zn. Obviously,K,Si,Cl,Ca,Mg and S were the main inorganic compo-nents in corn stover fractions.Chloride and potassium contents in leaf fraction were much lower than that of stalk fractions,while the silica content in leaf fraction was almost7times higher than that of stalk fractions.The high chloride and potassium contents in corn stover fractions might be due to the purposive fertilization with KCl,since KCl has been approved to increase introduced lignin content and improve the resistance of corn to stalk rot(Liu et al.,2007).The silica content in stalk rind fraction was only0.81%(as SiO2)which was extremely lower than that of wheat straw(7.3%as SiO2)and rice straw(11.7% as SiO2)(Pan and Sano,2005).The benefits of silica nutrition in plants include the enhancement of growth and yield,exposure of leaves to light,resistance to lodging,reduction of transpiration and resistance to drought stress,resistance to salinity,alleviation of metal toxicities,improvement of nutrient imbalance,resistance to radiation,effects on enzyme activities and increased resistance to pathogens and insect pests(Ma and Yamaji,2006).Manganese,copper and zinc could be considered as trace ele-ments,whose contents were lower than100ppm.The ash-forming element,viewed as non-processing elements in pulp mill,in black liquor can affect the liquor properties and,sometimes,hazard the continuous operation of the industrial black liquor recovery plant(Cardoso et al.,2009).Szemmelveisz et al.(2009)found Fig.2.Lignin distribution in cross section of corn stalk rind by SEM-EDXA.E,epi-dermis;P,parenchyma;F,fibers.Scale bar=250␮m.that operational problems may occur when co-firing biomass with coal directly in boilers for energy production because of the high ash compositions.It was also found that the common cations of ash(K+,Mg2+,Ca2+,Al3+,Mn2+,Fe3+,Cu2+,and Zn2+)showed inhibitive effects on cellulase at different levels,except for the stimulative effects of Ca2+and Mg2+on␤-glucosidase(Bin and Hongzhang,2010).In respect of high potassium and chloride con-tents in corn stover fractions and their negative effects on lots of biomass-processing processes,it is quite necessary to separate them economically and use them as chemicals.3.3.Lignin distribution in stalk rindMany methods have been developed for investigating lignin dis-tribution in plan cell wall(Abdul Khalil et al.,2010;Donaldson, 1992;Fromm et al.,2003;Xu et al.,2006).The stained ultra-thin sections with potassium permanganate(KMnO4)can be ana-lyzed by transmission electron microscopy coupled with energy dispersive X-ray analysis(TEM-EDXA)or SEM-EDXA for lignin dis-tribution.In this case,the lignin molecule is oxidized by KMnO4.The permanganate anion is reduced to MnO2,which thenprecipitates,Fig.3.Lignin distribution in cross section of corn stalk rind by toluidine blue stain-ing.Scale bar=50␮m.。