The genus Populus (poplars, aspens, and cottonwoods)comprises woody plant species that can grow in subtrop-ical to boreal forests (http://www.fs.fed.us/database/feis/plants/tree/; http://www.euforgen.org/distribution-maps/and [1]); they represent important pioneer species in bore-al forests, are dominant riparian species [1], and exhibitsome of the fastest inherent growth rates for temperate
trees worldwide (17–30 dry Mg ha–1 yr–1 on 6–8 year rota-tions for certain hybrid poplars [2]). Figure 1 depicts thevariation in biomass accumulation within a P. trichocarpacommon garden trial established in the Pacific Northwestin which, after four years of growth, individuals with sub-stantial bole mass gain could be harvested [3]. Important-ly, Populus species have value for the rehabilitation of dis-turbed sites from logging or fire (i.e., land reclamation) orfor the bioremediation of soils polluted by hazardouswaste. The exact number of Populus species within thesix sections Abaso, Aigeiros, Leucoides, Populus, Taca -mahaca, Turanga is disputed and is ranging from 29 to 85 species [1]. This number can get even higher whenvarieties, cultivars and hybrids are considered due to thefrequent interspecific hybridization in nature and thecomplex morphological variation within the genus, a con-sequence of possible reticulate evolution in Populus [1],and certainly, artificial hybridizations in breeding pro-grams have also led to this considerable species expan-sion. There have been additional efforts to improve Popu-lus species delineation within and between sectionsbased on nuclear genetic markers [4, 5] and more recently
Review
Using Populus as a lignocellulosic feedstock for bioethanol
Ilga Porth and Yousry A. El-Kassaby
Forest and Conservation Sciences, University of British Columbia, Vancouver, Canada
Populus species along with species from the sister genus Salix will provide valuable feedstockresources for advanced second-generation biofuels. Their inherent fast growth characteristics canparticularly be exploited for short rotation management, a time and energy saving cultivation alter-native for lignocellulosic feedstock supply. Salicaceae possess inherent cell wall characteristicswith favorable cellulose to lignin ratios for utilization as bioethanol crop. We review economicallyimportant traits relevant for intensively managed biofuel crop plantations, genomic and pheno-typic resources available for Populus, breeding strategies for forest trees dedicated to bioenergyprovision, and bioprocesses and downstream applications related to opportunities using Sali-caceae as a renewable resource. Challenges need to be resolved for every single step of the con-version process chain, i.e., starting from tree domestication for improved performance as a bioen-ergy crop, bioconversion process, policy development for land use changes associated withadvanced biofuels, and harvest and supply logistics associated with industrial-scale biorefineryplants using Populus as feedstock. Significant hurdles towards cost and energy efficiency, envi-ronmental friendliness, and yield maximization with regards to biomass pretreatment, saccharifi-cation, and fermentation of celluloses and the sustainability of biorefineries as a whole still needto be overcome.
Correspondence: Dr. Ilga Porth, Forest and Conservation Sciences, Uni-versity of British Columbia, 2424 Main Mall, V6T1Z4 Vancouver, CanadaE-mail: [email protected]
Abbreviations: cDNA, complementary DNA; C3’H, p-coumaroylshikimate3’-hydroxylase; GS, genomic selection; GWAS, genome-wide associationstudy, studies; HCT, hydroxycinnamoyl-CoA : shikimate hydroxycinnamoyltransferase; IL, ionic liquid, liquids; MAF, minor allele frequency; P. del-toides, Populus deltoides; P. nigra, Populus nigra; P. tremuloides, Populustremuloides; P. trichocarpa, Populus trichocarpa; QTL, quantitative trait locus,loci; RNAi, RNA interference; SA, salicylic acid; SNP, single nucleotide poly-morphism; WUE, water use efficiency
Biotechnol. J. 2015, 10 DOI 10.1002/biot.201400194
www.biotechnology-journal.com
BiotechnologyJournal
Received 01 AUG 2014Revised 11 NOV 2014Accepted 30 DEC 2014
on whole-plastome re-sequencing suggesting an earlydivergence of North American species ~6–7 Myrs ago [6].
In addition to their commercial value for timber, ply-wood, pulp, and paper, Populus species and their hybridsare widely considered to be the premier woody perenni-als for bioenergy feedstock (second-generation biofuelcrops) owing to their abundant biomass production, rap-id growth rates, and favorable cell wall chemistry [2, 7–9].Other favored second-generation biofuel crops includeMiscanthus and switchgrass, other fast-growing hard-woods such as willows and eucalypts as well as the soft-woods pine, fir and spruce. Research efforts to establishPopulus as a bioenergy crop are primarily focused on thefollowing species/hybrids: P. trichocarpa Torr. & Gray(black cottonwood; Western poplar), P. balsamifera L., P. deltoides × P. trichocarpa (Populus × generosa), P. del-toides × P. nigra (Populus × Canadensis), P. deltoides ×P. maximowiczii, P. tomentosa Carrière (Chinese whitepoplar), P. tremula L. (European aspen), P. tremuloidesMichx (trembling aspen) and P. tremula L. × P. tremu-loides Michx (hybrid aspen), their trihybrids and evenmore complex multi-species hybrids involving thesespecies [10–12]. The cultivation of such hybrids hasincreased the variability, introduced novel gene combina-tions and has resulted in superior growth and biomassaccumulation [10]. In comparison to other woody peren-nials that can also be grown on marginal land, food crops(e.g., maize, wheat, sugar beet, and vegetable oil crops)are at present abundantly used for first-generation biofu-els. However, food crops are much more vulnerable toprice rises that follow harvest losses since their addition-al demand for biofuel production has an aggravating
effect on any production shortfall (e.g., due to pests,drought) [13]. Hence, the utilization of tree crops withfavorable lignocellulosics biomass properties for an indus-trial scaled biofuel production in lieu of food crops is envi-sioned to de-escalate the food-versus-fuel dilemma andcan therefore be considered more sustainable [13]. Also,recent studies have suggested that an integration ofgenomics has high potential in the bio-processingimprovement of wood fibre [14, 15]. Generally, wood fromtree species of the genus Populus exhibits favorable rela-tive proportions and structures of all three major cell wallcomponents – cellulose, lignin, and hemicelluloses, withglucan content ranging from 40 to 62%, xylan contentfrom 14 to 24%, and lignin content from 15 to 29% of thetotal dry weight [7–9, 16–18]. Interestingly, in dioeciouspoplar, the male P. trichocarpa individuals [18] seem toaccumulate significantly more glucan in woody stem tis-sue (J. Klápšte, Faculty of Forestry, University of BritishColumbia, personal communication). Recently, mucheffort has been dedicated towards the generation ofgenomic resources in Populus [19–28]. In addition,unprecedented phenotyping efforts with respect toimportant target traits for feedstock improvement such asbiomass related traits, wood density, polysaccharides andmonomeric sugars of the cell wall, lignin, cell wall crys-tallinity, and saccharification efficiency have been under-taken to determine the available variation for such traitsin nature. C.1,100 genotypes originating from P. tri-chocarpa natural stands within a 38.8–58.6°N latitudinalrange across the Pacific Northwest were investigated [18,29, 30]. Thus far, most effort has been focused on the “treemodel species” P. trichocarpa [19, 21, 28, 31, 32], yet this
Figure 1. Four-year-old Populus tri-chocarpa growth trial on Totem Field,UBC, British Columbia, Canada. A totalof 461 accessions with clonal replicateswere planted in a randomized blockdesign at 1.5 × 1.5 m clone spacing in a 40 × 54 m sized area. Biomass harvestand assessment took place March21–23, 2012: bole mass: 6.6 kg average(21.3 kg max); whole tree mass: 10.8 kgaverage (31.7 kg max); bole density:784.2 kg/m3 average (1516.4 kg/m3
is primarily due to the completed reference genome of the Nisqually-1 genotype [20] and its exceptionally wellmaintained genomic resources (Phytozome v3.0 athttp://www.phytozome.net/cgi-bin/gbrowse/poplar/).The poplar genome contains c.500 million base pairs (Mb)and c.40 000 genes distributed over 19 chromosomes(2n = 38) [20]. Dioecious and obligate outbreeding plantspecies such as Populus show substantial inherent natu-ral genetic variation, i.e., their genomes are highly het-erozygous [19]. Among-individual genetic diversity is alsohigh in Populus [28], with one polymorphism found atevery tenth base pair (one SNP/50 bp at minor allele fre-quency (MAF) greater 5%) and mirrors the high adaptivevariation across the wide latitudinal and longitudinal dis-tributions of these species [33].
Although Populus species exhibit considerable natu-ral variation in important cell wall traits including thecalorific value of wood as an important characteristic (P. trichocarpa: [9, 18]; P. tremuloides: [34]; P. trichocarpa ×P. deltoides: [35]), it is not clear if the required gain forhighly profitable plantation forests can be achieved with-in the naturally occurring phenotypic variation [13]. Yet,recently identified rare but genetically stable loss-of-func-tion allelic variants in wild Populus spp. [36] (similarly, rareloss-of-function alleles that are defective in certain bio -pathways were also identified in other forest tree speciesthat are prospective bioenergy crops [37, 38]) hold greatpromise for advantageously altered cell wall composition(e.g., reduced lignin) as such alleles could be easily intro-duced via breeding [39]. To which extent such null allelesthat can substantially alter cell wall properties are presentwithin the plant genome is currently unknown. Alterna-tively, efforts to design cell wall properties (such as theenhanced cell wall digestibility) that are tailored to theindustrial application of lignocellulosic feedstock inethanol production and also comply with the tree’s inher-ent cell wall biochemistry and development have recent-ly been successful [40].
Here, we review the genetic and genomic resources ofPopulus. We report the research conducted in Populus oneconomically important traits for biofuel production suchas: (i) cell wall traits; (ii) biomass traits; (iii) root develop-ment; (iv) crown architecture; (v) the genomic basis ofhybrid vigour; (vi) epigenetics/epigenomics of develop-ment; and (vii) stress resistance. Any genetic interrela-tions between stress resilience and bioenergy-relatedtraits help to develop abiotic stress-resistant phenotypesthat can thrive on marginal land otherwise unsuitable forfood crops. Breeding strategies for bioenergy Populus arealso discussed. Finally, we present the current improve-ments in terms of bioprocessing of lignocellulosic bio-mass with a specific focus on Populus.
2 Available resources for Populus
2.1 Genomic resources
The first entirely sequenced and assembled tree genomewas black cottonwood’s (genus Populus) in 2006 [20]. Thisis due to its modest genome size, less than four times larg-er than the model plant Arabidopsis thaliana (which wascompletely sequenced in 2000 [41]), but 40 times smallerthan conifers [42]. In 2014, the whole-genome sequencingof Eucalyptus grandis, another hardwood tree specieswith high potential as a bioenergy tree crop was com-pleted [43]. Both, Populus and Eucalyptus, show highdiversity, adaptability, superior wood quality and growthrates, all important characteristics for the use as a renew-able feedstock for fibre and energy [33, 43]. The availablegenomic resources for both species are also useful for theprediction of gene function based upon phylogenomics[19].
Several cDNA resources for Populus have been estab-lished serving different research priorities that is 19 841full-length enriched cDNA clones from abiotic stresstreated P. nigra [44]; 4664 full-length cDNA clones from P. trichocarpa × P. deltoides related to insect feeding [23];34 131 SNP genotyping array of P. trichocarpa represent-ing 3543 candidate genes relevant to wood into biofuelsconversion and environmental adaptation ([27] anddoi:10.5061/dryad.1051d), 495 000 SNPs based onsequence capture and without pre-selection according totheir MAF [26], and finally 73 013 protein-coding tran-scripts from 41 335 loci from the v3 Populus genomeassembly [20] as well as c.18 million SNPs (phytozome.jgi.doe.gov) from 544 range-wide collected and unrelatedwhole genome resequenced P. trichocarpa accessions[28].
2.2 Phenotypic resources
While whole-genome sequencing and in-depth charac-terization (including genotyping) of Populus genomes hasbecome affordable due to cost-efficient technical advancesincluding high-throughput technologies, in general, thephenotyping, i.e., the comprehensive assessment of com-plex traits, remains labour intensive, especially those ofwood quality. Therefore, the present collections of pheno-typic data from Populus trees that demonstrated consid-erable phenotypic variation in wood traits (based on thetwo high-throughput techniques, near-infrared spectra(NIRS) determination [45] and molecular beam mass spec-trometry following pyrolysis [46], as well as based on wetlab chemistry [18]) are very valuable, as these efforts allowbridging the information with the available vast genomicsresources (see above) which should ultimately cumulateinto the meta-analysis of all these resources to develop abroader understanding of phenotypic changes in biofueltraits depending on genetic variation and environmental
influences. Automated phenotyping techniques forgrowth traits have already been developed for plants andallow for phenomics-based inferences, yet such high-throughput accomplishments are mostly feasible for Ara-bidopsis due to the ease of handling this model plantspecies [47–49]. Nevertheless, non-destructive and high-throughput techniques for the assessment of cell walltraits such as density, structure (using a X-ray tomogra-phy [50]), macrochemical distribution of lignin, celluloseand hemicelluloses (using coupled differential scanningcalorimetry & thermo gravimetrical analysis, http://www.trees4future.eu/transnational-accesses/dsc-tga.html) arebeing developed. Furthermore, the tree’s physicochemi-cal profile based on near-infrared spectroscopy has beenshown to be quantitative in nature [51], hence allowingthis high-throughput phenotyping technique to be usedin genetics-based analyses. In order to establish a varietyof sustainable aromatic chemicals as additional value-added products along with biofuels, metabolomics plat-
forms that identify low molecular weight (aqueous phasesoluble, extractable) salicinoids [52] and other unusualbark metabolites from natural Salicaceae collections arebecoming more and more invaluable for high-throughputscreening.
3 Economically important traits in Populusplantations related to biofuel production
3.1 Cell wall traits
Total lignin content, syringyl-to-guaiacyl (S/G) monolig-nol ratio, hemicelluloses content and their interactionswith the remaining cell wall polymers, all are known toimpact biomass pretreatment efficiency [9, 53, 54]. Car-bohydrate saccharification efficacy and yield are influ-enced by cell wall crystallinity and density, % alpha cellu-lose, % glucose, respectively [55]. The interrelations
between these cell wall traits have been demonstrated forpoplar ([18]; Fig. 2). Thus, the variation in these cell walltraits provides opportunities for genetic improvement ofPopulus for bioenergy uses [9, 46, 53, 54].
The O-acetylation of the non-cellulosic cell wall-poly-saccharides (mostly xylan) is an important biochemicalprocess in the biosynthesis of lignocellulose in hard-woods. The established O-acetyl groups (c.4% in poplardry wood-based percentage) provide the necessary inter-actions with other cell wall polymers for mechanicalstrength. However, the freed acetic acid from xylans dur-ing the hydrolysis of hardwoods constitutes a potentinhibitor to the microorganisms that are commonly usedin the fermentation process. Although it was shown thatalkaline-pretreatment based deacetylation improvesethanol production from hardwoods [56, 57], the directmodification of the O-acetylation sites in xylans (throughtransgenic approaches) remains an important target forfeedstock improvement to enhance saccharification andmicrobial fermentation [58].
3.2 Biomass traits
Populus can be grown either single-stem or as short rota-tion coppice, the latter cultivation form (similarly impor-tant for growing bioenergy willows Salix spp.) representsan attractive alternative for the rapid production of bio-mass for fibre and fuels as the harvest cycles are readilyrepeated every few years [59]. Recently, five genomicregions on the poplar chromosomes 3, 4, 10, 14, and 19(based on the co-localization of biomass related quantita-tive trait loci (QTLs, see glossary in Supporting informa-tion, Table S1)) have been identified for yield in short rota-tion coppice bioenergy poplar [60]. Moreover, all mappedQTLs explained c.20% of the variation in the final harvestbiomass yield [60]. Such QTLs can potentially be used inmarker-assisted breeding. Shoot branching genes relatedto strigolactone plant hormone signaling have recentlybeen characterized in poplar and willow [61, 62]. TheseMAX (More AXillary growth) genes may prove to be par-ticularly important in short rotation coppice practices asthe number of re-sprouted branches defines the suitabili-ty of the trees grown for biomass production [62]. It is alsohypothesized that strigolactone represents a long dis-tance plant signal in response to the root symbiotic micro-biome and nutrient availability in the soil that regulatesplant architecture and hence also productivity [61]. Forenhanced cellulosic biomass production in bioenergytrees, the traits that need to be targeted for breeding arethose that affect cellulosic wood production via cambialactivity [63]. The increased expression of important regulators of wood development such as the auxin signalling-related Class III homeodomain-leucine-zipperpopREVOLUTA [64, 65] has the potential to induce multi-ple active cambial meristems [63].
There are many ways to improve wood biomass yieldand quality in Populus. Opportunities include: (i) the sus-tainable use of forest biomass resource by harnessing soilmicrobial diversity [66]; (ii) the development of special-ized bioenergy poplar cultivars carrying alleles with highbreeding values for wood biomass yield and quality. Thelatter can be achieved by: (i) chemical mutagenesis-induced point mutations and small deletions that subse-quently create novel germplasm with unique genotypes[67]; or (ii) irradiation-induced unique combinations ofchromosomal regions in polyploid/aneuploid hybridpoplar individuals [68, 69]. Populus with superior feed-stock potential can also be generated based on genomics-and proteomics-informed breeding for enhanced biomassaccumulation and facilitated bioethanol production [70, 71]. The recently gained knowledge in P. trichocarparegarding the metabolic flux through the monolignolpathways should facilitate this undertaking [72].
3.3 Root development and water use efficiency(WUE)
The root architecture is essential for nitrogen and wateruse enabling robust biomass productivity even undermarginal conditions for a sustainable lignocellulosic bio-mass production [73]. Root architecture also becomesincreasingly important for plantation stability againstwind, specifically if lignin is selected against [74]. Thus,understanding root development regulation for the bioen-ergy crop Populus is important. Understanding how rootendophytic bacteria can alter plant growth and produc-tivity provides opportunities for the functional manipula-tion of root endophyte populations for feedstock improve-ment [75]. The recent findings that growth traits are large-ly decoupled from WUE provides opportunities for highyielding bioenergy dedicated forest trees with high WUEin short-rotation practices [76, 77].
3.4 Crown and whole plant architecture
Full canopy photosynthesis is tightly linked to crop yield[78]. The phytochrome-mediated elongation response toneighbors represents an adaptive and plastic trait inplants which is tightly linked to plant fitness, and isdependent on stand density [79]. In Populus, understand-ing the phytochrome-mediated responses to competitionin dense plantings is important to maximize carbon cap-ture per unit of land area for increased biomass produc-tion. Furthermore, whole plant architecture can bemanipulated based on phytohormones such as gib-berellins or cytokinins, promoting height growth and sec-ondary branching, respectively, to increase biomass perunit planting area [80, 81]. The repression of gibberellinbiosynthesis to produce semi-dwarf trees in dense standsis anticipated to effectively provide higher levels of bio-mass production and carbon sequestration under short-
rotation practices [82, 83]. The positive aspects of semi-dwarf plant stature include the improved wind-firmnessbased on larger root systems (see also above), less reac-tion wood, easier harvest, and greater number of stemsand greater fibre yields per unit area [84].
3.5 Epigenetic/epigenomic control of bioenergy traits in poplar
Much of the yield improvement in domesticated crops isdue to capturing hybrid vigour (i.e., heterosis). Poplarbreeding has also extensively relied on breeding forhybrids [85]. Recent studies were able to link heterosis tochromatin modification (epigenetics) associated withdistinct changes in the DNA methylation patterns [86].Hence, epigenetics affects breeding efficiency. Moreover,epigenetic plasticity impacts growth and development(e.g., bud dormancy induction), but also stress resilience[86] and, consequently, epigenetics affects productivity.
4 Breeding strategies for bioenergy Populus
With the discovery of rare loss-of-function allelic variantsin wild Populus spp. germplasm as beneficial variations inplant metabolism and their introduction into poplar indi-viduals of diverse genetic background, Populus can besubstantially improved as a lignocellulosic feedstock (seebelow) [39]. Genome-wide association studies (GWAS,see glossary in Supporting information, Table S1),designed to associate genetic markers with functionalgenes, typically identify only the common allelic variantsunderlying the quantitative trait under study. This is pri-marily due to the convention that the lower threshold forthe SNP MAF needs to be above 10% or even greater than15% in the analysis [49]. The MAF restriction to at least10% in GWAS was proposed to avoid the detection of spu-rious associations, as p-value distributions tend to bebiased for MAF with an enrichment of statistically signif-icant polymorphisms at low MAF in the linear mixed mod-el of GWAS [49]. Nevertheless, rare alleles (defined by anMAF below 1%) determine strong phenotypic variation[87]. The importance of rare allelic variants became par-ticularly evident in the genetics of complex diseases inhumans. The access to such rare mutations and their car-riers are facilitated by a genotype screening methodtermed tilling by sequencing (or ecotilling) that utilizesnext-generation sequencing (NGS) platforms [88–94].This approach has recently been proposed for plants [36,87, 95]. In a proof-of-concept case study for breeding withrare defective alleles (BRDA) in P. nigra, Vanholme et al.[39] backcrossed the previously identified non-functionalallele from a phenylpropanoid pathway gene (containinga premature stop codon [36]) in different P. nigra het-erozygous carrier genomes (aAxAA) to generate a collec-tion of F1 homozygous offspring. P. nigra homozygots of
the recessive allele – with otherwise different geneticbackground – consistently showed the altered lignin composition where plants incorporated a dramaticallyhigher amount (10–18 times) of p-hydroxyphenyl units(“H-lignin”) accompanied by an increased S:G monolignolratio [39]. These resulting cell wall alterations are highlyadvantageous to an efficient downstream lignocellulosicbiomass processing for future use of poplar wood in anindustrially scaled biofuel production. A major advantageof utilizing such naturally occurring null (i.e., loss-of-func-tion) alleles in breeding programs is their already provengenetic stability within the natural populations (in con-trast to problems with introduced transgenes that eitherget potentially silenced or induce major pleiotropiceffects, [13]). As outlined previously [39], so far, only veryfew, i.e., three, cases of natural loss-of-function alleles arecurrently known for trees. All three genes (cinnamyl alco-hol dehydrogenase CAD, cinnamoyl-CoA reductase CCR,and hydroxycinnamoyl-CoA: shikimate hydroxycin-namoyl transferase HCT) function within the phenyl-propanoid pathway. Their non-functional variants wereshown to impact lignification (as in the case of CAD andHCT) or fibre properties (microfibril angle as in the case ofCCR) [36, 38]. Yet, since both the frequency as well as thelocation of additional null alleles within the poplar genomewith potential effects on enzymes of the biopathways iscurrently unknown, further investigations are warranted.However, by merging the now available data from high-throughput phenotyping for wood quality traits [30, 96]and the high quality reads from in-depth genomesequencing (using NGS) performed for hundreds of natu-ral range-wide accessions [28], their identification andcorrelation with considerable wood trait variation shouldbecome feasible. Prior to their potential wider introduc-tion by tree breeding, these rare allelic variants will needto be validated by conventional Sanger sequencing andtested in segregation studies. As an additional asset,once such studies validated null alleles with major phe-notypic effects in poplar, the underlying genes becomeunambiguously annotated for their function. Thisapproach has two advantages over transgenic approach-es, as natural loss-of-function variants have proven to begenetically stable compared to the potential silencing ofan introduced transgene, and the avoidance of majorpleiotropic effects that obscure the study of normal genefunction as commonly observed in several RNAi genesilencing experiments. BRDA [39] is also considered high-ly complementary to GWAS and the genomic selection(GS, see glossary in Supporting information, Table S1)efforts that aim at accelerating conventional treeimprovement programs.
GWAS studies the individual SNP effects on the phe-notype and the uncovered effects typically explain only asmall portion of the heritable phenotypic variance (that isbetween 3–7% of the variation in 16 studied wood prop-erties in a P. trichocarpa GWAS study [96], while 1–4% for
variation in S:G, C6 sugars and lignin in a P. trichocarpacandidate gene study [46], and 5% for variation in ligninand S:G in P. nigra [45]; 2–8% for variation in 11 biomasstraits likewise assessed through GWAS in P. trichocarpa[97]). The obtained results are also highly context-dependent (i.e., varying for different genetic backgroundsin different environments) which represents a majorobstacle for implementing GWAS results into operationaltree breeding. Similarly, GS may show limitations as phe-notypic predictions are also population dependent andmay not be broadly transferable (however, the estimatedpredictive accuracies are mostly valid within the estimat-ed breeding zones, i.e., for similar genetic background)[98]. For GS to be universally applicable, phenotypes inthe training population need to be assessed across differ-ent environments involving multiple independent obser-vations and using the same genetic material to measureenvironmental influences (GxE effects) [99]. In addition,the actual functional variants (i.e., the QTLs that explainthe phenotypic variance) that should represent the stableentities associated with the phenotypic trait in questionare not necessarily required for the predictive model inGS. Thus, to conclusively elucidate the importance offunctional variants for tree breeding with respect toimproved wood characteristics (“if causative polymor-phism are found, known, and constant,” B. Stanton,GreenWood Resources, OR, USA, personal communica-tion), the results from GS, GWAS, and QTL mappingefforts that study SNPs genome-wide and use the identi-cal populations within a designated breeding zone needto be evaluated. It is also important that herein the genefunctions and the potential gene–gene interactions (epis-tasis) are elucidated, such that the implementation of theuncovered markers leads to consistent results [99, 100].Currently, no such comparative analysis is known. GS,GWAS, and QTL mapping studies all make use of the nat-ural variation for the trait.
For example, GS yielded 50% predictive ability for thepercentage of galactose content in dry wood (a pectin-derived cell wall-associated carbohydrate) by investigat-ing natural P. trichocarpa accessions from a geneticallyunrelated cohort. The same analysis also suggested thatgalactose content represents a wood trait that is geneti-cally much less complex than, for example, the percent-age of alpha cellulose content in dry wood (J. Klápšte, Faculty of Forestry, UBC, personal communication). How-ever, our knowledge regarding the mode of genetic con-trol of the individual wood traits and the genetic relation-ships among the traits is essential for efficient programsin tree breeding [18]. Galactose content, for example,shows high heritability (h2 = 0.64) and negative geneticcorrelations exist with microfibril angle (rg = –0.79) andtotal lignin (rg = –0.30) [18]. From this we can concludethat galacturonans (and their structure) influence otherwood properties [101, 102]. The aim of breeding programs(that exploit the natural variation in a specific trait) is to
increase the allele frequency of the target allele to attaingain while maintaining the genetic variability in otherattributes for future selection and adaptation (the evolu-tion component). For example, the finding that lignin andhemicellulose content are negatively correlated with den-sity and alpha cellulose content appears to be highlyadvantageous for optimizing poplar as a lignocellulosicfeedstock for bioethanol production (Fig. 2). Consequent-ly, breeding for lower lignin or hemicellulose content couldsimultaneously select for higher density and alpha cellu-lose content, and vice versa [18].
Forest trees show two important characteristics: (i) complex polygenic trait architecture; and (ii) long-termexposure to a wide array of environmental contingencies.Both have important consequences for tree breeding, as:(i) a large number of allelic combinations needs to be cap-tured for a significant heritable portion of the observedphenotypic variance; and (ii) realized QTL effects maychange over time as they are context-dependent. Thus,an investigation into the hierarchies of gene action with-in the genetic architecture of wood traits is warranted,such that the most important genes and allelic variantscontrolling the trait in question within the complexity ofinteracting traits can be identified and selected for in asimplification of the breeding activities and maximizationof response to selection [100]. However, despite the pres-ence of substantial variation for important cell wall traitsin natural populations, it is unclear if the required gain forhighly profitable plantation forests can be achieved with-in this naturally occurring phenotypic variation [13].Specifically, the energy and/or chemical input require-ments for feedstock pretreatment need to be drasticallyreduced to make lignocellulosics derived biofuels cost-efficient and sustainable [103]. Interspecific Populushybrids represent highly productive trees for biomass pro-duction [10, 104]. Recently, strategies have been exploredto understand the genotypic and karyotypic underpin-nings of the superior hybrid poplar performance. Con-comitantly, new genomic combinations leading to uniquephenotypes of considerable commercial importance havebeen identified by pollen treatment with ionizing radia-tion during interspecific hybridization (http://comailab.genomecenter.ucdavis.edu/index.php/Poplar). Individu-als carrying such unique phenotypes related to superiorgrowth or feedstock properties (based on the induced“gene dosage effect”) can then be clonally propagated ina time efficient manner.
Another attractive strategy is the generation ofpoplars that have altered cell wall properties that arealready “designed for deconstruction” by using geneticengineering (GE) strategies [40]. However, such changescan also unfavorably affect plant growth and developmentwhen the extreme phenotype is selected for and/or trans-genes are constitutively or ectopically expressed usingthe common cauliflower mosaic virus 35S promoter [101,105, 106]. Carefully conducted GE strategies that only tar-
get mature woody tissue (via an inducible promoter sys-tem) and also comply with the tree’s development andinherent cell wall biochemistry should circumvent suchnegative effects on tree physiology. The lignin biosyn-thetic pathway, for example, shows considerable meta-bolic plasticity in nature; this concerns the ligninmonomer composition (H/S/G) as well as the monolignolside chain variation (leading in the extreme cases to theformation of linear homopolymers). Even unconventionalmonomers can be artificially introduced without nega-tively affecting lignification per se, such that benefits forbioprocessing can be achieved by: (i) reducing the cross-linking to other biopolymers in the cell wall [17]; and (ii) allowing for an easier cleavage of the monolignolinterunit bonds by introducing ester bonds into the back-bone structure [40]. The latter improvement was intro-duced by Wilkerson et al. [40] who overexpressed mono-lignol ferulate monotransferase under the cellulose syn-thase A catalytic subunit 8 promoter (pCesA8::FMT) inhybrid poplar (P. alba × grandidentata) to alter ligninstructure in said way and the authors attained an almostdoubled saccharification yield compared to the unim-proved reference poplars. Glucose yield per dry weightamounted to 26% in the most extreme case compared toonly 13% yield from non-transgenics [40]. Besides alteredlignin structure and reduced cross-linking of lignins tocarbohydrate cell wall polymers in secondary cell walls,the GE targeted degradation of components of the pri-mary cell wall and the middle lamella has also attractedattention in research to improve saccharification yield inPopulus (i.e., pectate lyase to target homogalacturonan(pectin) [101]; xyloglucanase to target xyloglucan (hemi-celluloses) [107]). However, such GE trees still necessitateextensive testing in field settings [108]. Moreover, asthese transgenic lines exhibited different transgeneexpression due to different transfer-DNA insertion loca-tion or/and divergent number of incorporated copies intothe host genome, they also showed individually differenttrait expression (an unfavorable feature when the biocon-version process should be streamlined). Thus, a more sta-ble (controlled) overall phenotype is required, but this may
be easily accomplished by employing techniques thatpromote the precise integration of recombinant DNA intothe tree genome (as already shown for Populus [109]) orother alternative “DNA-free” genome-editing approaches[110, 111]. This may in fact accelerate the anticipatedderegulation procedures for such poplar transgenics andreduce the costs associated with deregulation [13, 108].
Plantation forests of substantially improved feedstockin terms of wood quality and high yields for industrialdemands need to be intensively managed to guaranteehigh productivity (Fig. 3). However, Populus species areknown to be strong isoprene emitters, with negative con-sequences on the environment [112]. Recently, researchefforts have been directed towards generation of lowemitting poplars by means of targeted RNA interferencethat suppressed isoprene biosynthesis. However, as atrade-off, stress responses in those trees were negativelyaffected with unfavorable consequences for host defens-es against herbivores [112]. Interestingly, wood from non-isoprene emitting RNAi lines tended to have increasedalpha cellulose content and concomitantly, lowered lignincontent, yet these values were not significantly differentfrom the control trees. However, it was noticed that over-all, wood from such transgenics had a distinct chemicalprofile [112].
5 Bioenergy Populus – improvements for downstream processing
Bioethanol production from lignocellulosic plant materialinvolves four major steps: (i) the pretreatment in whichlignin, the phenolic polymer-based “glue” to the complexpolysaccharide matrix, is broken up for access to the cellwall carbohydrates; (ii) the enzymatic hydrolysis/saccha-rification to generate the free monomeric sugars; (iii) themicrobial fermentation to convert these free monomersugars into alcohol; and finally (iv) the distillation to puri-fy ethanol from residue [113]. The utilization of the cellu-losic biomass from forest woody feedstocks still requiresconsiderable optimization in terms of performance and
cost effectiveness of the conversion process [113]. This isthe case, because the process is currently optimized forfirst generation biofuel crops such as corn (starch-basedbioethanol) and sugarcane (sucrose-based bioethanol)which are much less recalcitrant to the release of the fer-mentable carbohydrates and therefore still have the costadvantage over cellulosic biomass [114]. By modeling theoverall sustainability of a poplar-derived bioethanol industry, the authors Guo et al. and Littlewood et al.demonstrated that optimized poplar feedstock (throughadvanced breeding or GE) is crucial for maximizedethanol yields, an improved environmental profile (with50% less environmental impacts than from unimprovedpoplar feedstock) as well as higher price competitivenesswith conventional fuels (41% improvement) [115, 116].Thus, in line with this finding with respect to the use ofPopulus as a lignocellulosic feedstock for bioethanol pro-duction, our review has put substantial emphasis on thecurrent research progress regarding the opportunities forimproved feedstock quality of poplars.
The major obstacles associated with lignocellulosicbiomass utilization for bioethanol involve, for example,the heterogeneity of the cell wall components, the protec-tion of cellulose microfibrils by lignin and hemicelluloses,the inaccessibility of enzymes to the cellulose moieties,and cellulose that is 50% crystalline [53, 117]. Increasedethanol yield from lignocellulosic biomass generallyrequires that the feedstock has lowered lignin and xylancontent, fewer or modified hemicellulose–lignin interac-tions and above average syringyl lignin composition(syringyl-to-guaiacyl (S/G) ≥ 2.0) [9, 54]. This has alreadybeen demonstrated for other biofuel crops (e.g., alfalfa[118]; switchgrass [119]). However, if poplar wood ismeant to provide benefits as a renewable feedstock in itsentirety, xylan and lignin (both combined contribute~50% to the woody biomass in poplar, [18]) will also needto be efficiently valorized as resources for variousadvanced biomaterials [8, 17, 120, 121]. To preserve theirintact structures, lignin and xylans are recovered imme-diately after the pretreatment of the feedstock in lignocel-lulosic biorefineries. The fractionation of lignin, a pheno-lics-based polymer and therefore an ideal precursor forcarbon fibres, plant-based plastics and composites, needsto occur prior to the enzymatic breakdown of cellulose andideally uses a countercurrent flow-through [17]. Certainly,efforts to introduce lignin as feedstock for the value-addedbiomaterial market are facilitated by in planta geneticengineering of more H-lignin (which offers lignin with lessinherent structural diversity), uniform molecular-weightlignins and lignins with chemically labile backbone link-ages for an easier fractionation of lignin from cellulosicbiomass [17].
Lignin has negative side effects on enzymatic hydrol-ysis by generating phenolic-derived cellulase inhibitors.However, due to the substantial environmental benefitscellulosic crops provide to the agricultural landscape
[114], active research in improved cell wall deconstructionand increased saccharification efficiency for the conver-sion into liquid biofuels is ongoing. For example, whenlow-lignin engineered biomass is used, pretreatmentmight become obsolete [122]. While plants with down-regulated lignin suffer from stunted growth that is oftenstress-induced via salicylic acid (SA), yet by depleting SAaccumulation [123], growth can be restored in certain cas-es (for HCT-RNAi plants, e.g., [124]). C3’H (p-coumaroyl-shikimate 3′-hydroxylase) RNAi poplars with significant-ly reduced Klason lignin content were impaired in variousaspects of plant growth, leaf mass per area, WUE, andhydraulic conductivity [74, 126]. To optimize yield inplants with modified lignin (missense mutation withinthe C3’H enzyme, e.g., causing an interruption of themetabolic flux to G-monolignol), where dwarfism is notassociated with SA (e.g., c3’h plants), gene stacking thatincludes disruption of the (lignin-modification-induceddwarfism) Mediator REF4/RFR (reduced epidermal fluo-rescence 4/REF4-related) is proposed. This was found topossibly represent an attractive strategy to restore plantgrowth and also yield plants with advantageously high H-lignin [125]. Recently, tension wood (a wood type char-acterized by altered cell wall composition and structure:gelatinous (G-) layer formed in the fibre cell walls withc.89% cellulose that is highly crystalline [127, 128]) wasexamined for optimized bioethanol production [129].Here, tension wood was induced by enforced stem incli-nation [129]. Significantly enhanced enzymatic sacchari-fication yield (i.e., a 38% increase in released glucose/gglucan) was noticed due to increased glucan accessibili-ty to the cell wall degrading enzymes. Overall, the tensionwood induction did not compromise total biomass yield,yet would make harsh pretreatment unnecessary [129]. Inthis respect, interestingly, cell wall crystallinity (from ten-sion wood) and overall lignin levels in non-pretreated bio-mass did not influence inherent glucan accessibility [129].With respect to low-lignin phenotypes, the study ofextreme variant phenotypes present within the naturalmetabolic plasticity of lignin synthesis, promise a lucra-tive research opportunity as these variants do not haveimpaired plant integrity and therefore are suitable forcommercial applications [39, 100, 129]. Low-recalcitrantbiomass variants can be screened for by using a high-throughput pretreatment and streamlined co-hydrolysissystem [130].
Bioethanol production from lignocellulosic biomassstill faces three major challenges in downstream biopro-cessing that have led to yield restrictions due to fermen-tation inhibitors: (i) limited availability of ethanol-tolerantbacteria that can also process xylose sugar; (ii) presenceof microbial contamination from the cellulosic materialthat competes with fermenting yeast and produces toxicend-products; and (iii) formation of toxic components bypretreatment processes [113]. One great challenge is toestablish an environmentally friendly pretreatment
process [131, 132] that allows for efficient hydrolysis andcan replace the conventional physicochemical processes[133]. When new methods are tested on lignocellulosicfeedstock, it is particularly important to monitor the spe-cific structural changes caused by these pretreatmentprocedures (Fig. 4) as such macromolecular alterationscould affect downstream processes and applications [132,134–136].
Cellulose bioconversion rates following the biologicalpretreatment with the white rot fungus Trametes velutinahave recently been studied for poplar (P. tomentosa Carr.)[132]. The simultaneous saccharification and fermenta-tion of fungal pretreated poplar achieved up to 2.5 timesthe conversion rate of cellulose into ethanol compared tountreated poplar [132] (Fig. 4). As no changes in cellulose
crystallinity were observed, the modification of ligninstructure during biodegradation was supposedly key toimproved cellulose conversion rates [132]. By comparison,the oxidative enzymatic pretreatment of E. globulus woodusing recombinant Myceliophthora thermophila laccase(and the methyl syringate as a natural phenolic mediatorin delignification) resulted in 55% glucose yield at com-paratively low cellulase loading following the removal of~50% Klason lignin [134]. Lignin degradation via laccasewas found to follow a different mechanism than the onereported for white rot fungi.
Ionic liquids (IL) define as salts that are molten atambient temperatures, and are commonly derived frompetroleum sources, have now been tested extensively forbiomass pretreatment. Imidazolium-based salts are potent
agents that effectively reduce the recalcitrance of ligno-cellulosic biomass towards enzymatic hydrolysis (basedon their high delignification efficiency [137]). They pro-vide very high yields of c.90% glucose release from theoriginal biomass [138, 139]. In the procedure, the biomassis dissolved by application of IL followed by cellulose pre-cipitation by an anti-solvent. IL pretreatment also allowsfor the recovery of a separate lignin fraction that can beconverted into aromatic chemicals [138]. However, due tohigh costs associated with IL (contributing almost half ofthe total raw material costs in biofuel production compar-ing to one-fifth of the costs for conventional pretreatmentmethods, [140]), technologies that allow for the continu-ous recovery and recycling of high purity IL during theprocess are required [140, 141]. When different hardwoodspecies were tested, it was shown that the reactivity of ILvaried for anatomical differences among wood fibres (i.e.,early wood vs. late wood) and between species (differ-ences in chemical composition and microfibril angle)[135]. Thus, research on specific IL for the pretreatment of Populus lignocellulosic biomass to obtain increasedsaccharification efficiency is now warranted (tested[C4C1im][HSO4] for hybrid aspen pretreatment [142], e.g.).In a forward looking work, ILs were directly derived frombiomass (based on the reductive amination of aldehydesderived from switchgrass lignin and hemicelluloses,[143]). This provides a first outlook into a potential closedcircuit process for lignocellulosic biorefineries.
6 Conclusions
Populus species along with species from the sister genusSalix will provide valuable feedstock for second-genera-tion biofuels. Synthesis of poplar related research is pro-vided in Supporting information, Table S2. The ecologicalbenefits of growing woody plant species instead of annu-al food crops are apparent: perennials contribute to soiland ground water conservation, nutrient recycling, andsoil carbon accumulation. The inherent fast growth char-acteristics of Populus can also be exploited economicallyin short rotation management, a time and energy savingcultivation alternative for lignocellulosic crops. Theirinherent cell wall characteristics with advantageouslyhigher cellulose to lignin ratios (compared to conifer trees)provide indeed profitable opportunities for their utiliza-tion in bioenergy production. However, significant hur-dles towards cost and energy efficiency, environmentalfriendliness, and yield maximization with respect to thepretreatment of the lignocellulosic biomass, the sacchar-ification and fermentation of the recovered celluloses andthe sustainability of biorefineries as a whole still need tobe overcome. Challenges need to be overcome for eachstep of the conversion process chain should biorefineriesproduce on an industrial scale, i.e., starting from thedevelopment of improved lignocellulosic biofuel crops,
the actual bioconversion steps, the policy developmentfor land use changes (related to the anticipated large-scale land conversion) associated with advanced biofuels,and the harvest logistics associated with large-scalebiorefinery plants [14, 113, 144, 145]. In this respect, theInternational Poplar Symposium, held every four years,represents an important venue for researchers, practi-tioners, regulatory and policy experts involved in thedomestication of Salicaceae as agricultural productionsystems for wood fibre [146–148].
Funds from the Johnson’s Family Forest BiotechnologyEndowment, The British Columbia Forest InvestmentAccount Forest Genetics Conservation and ManagementProgram, the National Science and Engineering ResearchCouncil of Canada Discovery and Industrial ResearchChair (YAE.) are greatly appreciated. The authors apolo-gize to all researchers whose work could not be includedin this review due to the limited size of the manuscript.
The authors declare no financial or commercial conflict ofinterest.
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