Sustainable bioenergy for climate mitigation: developing drought-tolerant trees and grasses.

BACKGROUND AND AIMS: Bioenergy crops are central to climate mitigation strategies that utilize biogenic carbon, such as BECCS (bioenergy with carbon capture and storage), alongside the use of biomass for heat, power, liquid fuels and, in the future, biorefining to chemicals. Several promising lignocellulosic crops are emerging that have no food role - fast-growing trees and grasses - but are well suited as bioenergy feedstocks, including Populus, Salix, Arundo, Miscanthus, Panicum and Sorghum. SCOPE: These promising crops remain largely undomesticated and, until recently, have had limited germplasm resources. In order to avoid competition with food crops for land and nature conservation, it is likely that future bioenergy crops will be grown on marginal land that is not needed for food production and is of poor quality and subject to drought stress. Thus, here we define an ideotype for drought tolerance that will enable biomass production to be maintained in the face of moderate drought stress. This includes traits that can readily be measured in wide populations of several hundred unique genotypes for genome-wide association studies, alongside traits that are informative but can only easily be assessed in limited numbers or training populations that may be more suitable for genomic selection. Phenotyping, not genotyping, is now the major bottleneck for progress, since in all lignocellulosic crops studied extensive use has been made of next-generation sequencing such that several thousand markers are now available and populations are emerging that will enable rapid progress for drought-tolerance breeding. The emergence of novel technologies for targeted genotyping by sequencing are particularly welcome. Genome editing has already been demonstrated for Populus and offers significant potential for rapid deployment of drought-tolerant crops through manipulation of ABA receptors, as demonstrated in Arabidopsis, with other gene targets yet to be tested. CONCLUSIONS: Bioenergy is predicted to be the fastest-developing renewable energy over the coming decade and significant investment over the past decade has been made in developing genomic resources and in collecting wild germplasm from within the natural ranges of several tree and grass crops. Harnessing these resources for climate-resilient crops for the future remains a challenge but one that is likely to be successful.

Expanding the biomass resource: sustainable oil production via fast pyrolysis of low input high diversity biomass and the potential integration of thermochemical and biological conversion routes.

Waste biomass is generated during the conservation management of semi-natural habitats, and represents an unused resource and potential bioenergy feedstock that does not compete with food production. Thermogravimetric analysis was used to characterise a representative range of biomass generated during conservation management in Wales. Of the biomass types assessed, those dominated by rush (Juncus effuses) and bracken (Pteridium aquilinum) exhibited the highest and lowest volatile compositions respectively and were selected for bench scale conversion via fast pyrolysis. Each biomass type was ensiled and a sub-sample of silage was washed and pressed. Demineralization of conservation biomass through washing and pressing was associated with higher oil yields following fast pyrolysis. The oil yields were within the published range established for the dedicated energy crops miscanthus and willow. In order to examine the potential a multiple output energy system was developed with gross power production estimates following valorisation of the press fluid, char and oil. If used in multi fuel industrial burners the char and oil alone would displace 3.9 × 105 tonnes per year of No. 2 light oil using Welsh biomass from conservation management. Bioenergy and product development using these feedstocks could simultaneously support biodiversity management and displace fossil fuels, thereby reducing GHG emissions. Gross power generation predictions show good potential.

Impact of Miscanthus x giganteus senescence times on fast pyrolysis bio-oil quality.

In this study the impact of senescence and harvest time in Miscanthus on the quality of fast pyrolysis liquid (bio-oil) was investigated. Bio-oil was produced using a 1 kg h(-1) fast pyrolysis reactor to obtain a quantity of bio-oil comparable with existing industrial reactors. Bio-oil stability was measured using viscosity, water content, pH and heating value changes under specific conditions. Plant developmental characteristics were significantly different (P≤0.05) between all harvest points. The stage of crop senescence was correlated with nutrient remobilisation (N, P, K; r2=0.9043, r2=0.9920, r2=0.9977 respectively) and affected bio-oil quality. Harvest time and senescence impacted bio-oil quality and stability. For fast pyrolysis processing of Miscanthus, the harvest time of Miscanthus can be extended to cover a wider harvest window whilst still maintaining bio-oil quality but this may impact mineral depletion in, and long term sustainability of, the crop unless these minerals can be recycled.

Seasonal variation in Laminaria digitata and its impact on biochemical conversion routes to biofuels.

Laminaria digitata is a highly prevalent kelp growing off the coast of the UK but has rarely been considered as a source of biomass to date. This study shows it can be used as a feedstock in both ethanol fermentation and anaerobic digestion for methane production. The study optimised several parameters in the fermentation of L. digitata and investigated the suitability of the macroalgae through the year using samples harvested every month. For both methane and ethanol production, minimum yields were seen in material harvested in March when the carbohydrates laminarin and mannitol were lowest. July material contained the highest combined laminarin and mannitol content and maximum yields of 167 mL ethanol and 0.219 m(3) kg(-1)L. digitata.

Expression of bacterial levanase in yeast enables simultaneous saccharification and fermentation of grass juice to bioethanol.

This study demonstrates use of recombinant yeast to simultaneously saccharify and ferment grass juice (GJ) to bioethanol. A modified Bacillus subtilis levanase gene (sacC) in which the native bacterial signal sequence was replaced with a yeast α-factor domain, was synthesised with yeast codon preferences and transformed into Saccharomyces cerevisiae (strain AH22) using the expression vector pMA91. AH22:psacC transformants secreted sacCp as an active, hyper-glycosylated (>180 kDa) protein allowing them to utilise inulin (ß[2-1] linked fructose) and levan (ß[2-6] linkages) as growth substrates. The control (AH22:pMA91) strain, transformed with empty plasmid DNA was not able to utilise inulin or levan. When cultured on untreated GJ levels of growth and bioethanol production were significantly higher in experiments with AH22:psacC than with AH22:pMA91. Bioethanol yields from AH22:psacC grown on GJ (32.7[±4] mg mL(-1)) compared closely to those recently achieved (Martel et al., 2010) using enzymatically pre-hydrolysed GJ (36.8[±4] mg mL(-1)).

Variation in Miscanthus chemical composition and implications for conversion by pyrolysis and thermo-chemical bio-refining for fuels and chemicals.

Different species and genotypes of Miscanthus were analysed to determine the influence of genotypic variation and harvest time on cell wall composition and the products which may be refined via pyrolysis. Wet chemical, thermo-gravimetric (TGA) and pyrolysis-gas chromatography-mass spectrometry (Py-GC-MS) methods were used to identify the main pyrolysis products and determine the extent to which genotypic differences in cell wall composition influence the range and yield of pyrolysis products. Significant genotypic variation in composition was identified between species and genotypes, and a clear relationship was observed between the biomass composition, yields of pyrolysis products, and the composition of the volatile fraction. Results indicated that genotypes other than the commercially cultivated Miscanthus x giganteus may have greater potential for use in bio-refining of fuels and chemicals and several genotypes were identified as excellent candidates for the generation of genetic mapping families and the breeding of new genotypes with improved conversion quality characteristics.

A flexible quantitative methodology for the analysis of gene-flow between conventionally bred maize populations using microsatellite markers.

Previous studies of gene-flow in agriculture have used a range of physical and biochemical markers, including transgenes. However, physical and biochemical markers are not available for all commercial varieties, and transgenes are difficult to use when trying to estimate gene flow in the field where the use of transgenes is often restricted. Here, we demonstrate the use of simple sequence repeat microsatellite markers (SSRs) to study gene flow in maize. Developing the first quantitative analysis of pooled SSR samples resulted in a high sampling efficiency which minimised the use of resources and greatly enhanced the possibility of hybrid detection. We were able to quantitatively distinguish hybrids in pools of ten samples from non-hybrid parental lines in all of the 24 pair-wise combinations of commercial varieties tested. The technique was used to determine gene flow in field studies, from which a simple model describing gene flow in maize was developed.

Seasonal variation in the chemical composition of the bioenergy feedstock Laminaria digitata for thermochemical conversion.

To avoid negative impacts on food production, novel non-food biofuel feedstocks need to be identified and utilised. One option is to utilise marine biomass, notably fast-growing, large marine 'plants' such as the macroalgal kelps. This paper reports on the changing composition of Laminaria digitata throughout it growth cycle as determined by new technologies. The potential of Laminaria sp. as a feedstock for biofuel production and future biorefining possibilities was assessed through proximate and ultimate analysis, initial pyrolysis rates using thermo-gravimetric analysis (TGA), metals content and pyrolysis gas chromatography-mass spectrometry. Samples harvested in March contained the lowest proportion of carbohydrate and the highest ash and alkali metal content, whereas samples harvested in July contained the highest proportions of carbohydrate, lowest alkali metals and ash content. July was therefore considered the most suitable month for harvesting kelp biomass for thermochemical conversion to biofuels.

Miscanthus as a feedstock for fast-pyrolysis: does agronomic treatment affect quality?

The objectives of the experiment were to assess the impact of nitrogen (N) and potassium (K) fertiliser application on the cell wall composition and fast-pyrolysis conversion quality of the commercially cultivated hybrid Miscanthus x giganteus. Five different fertiliser treatments were applied to mature Miscanthus plants which were sampled at five intervals over a growing season. The different fertiliser treatments produced significant variation in concentrations of cell wall components and ash within the biomass and affected the composition and quality of the resulting fast-pyrolysis liquids. The results indicated that application of high rates of N fertiliser had a negative effect on feedstock quality for this conversion pathway: reducing the proportion of cell wall components and increasing accumulation of ash in the harvested biomass. No exclusive effect of potassium fertiliser was observed. The low-N fertiliser treatment produced high quality, low ash-high lignin biomass most suitable as a feedstock for thermo-chemical conversion.

Expression, purification and use of the soluble domain of Lactobacillus paracasei beta-fructosidase to optimise production of bioethanol from grass fructans.

Microbial inulinases find application in food, pharmaceutical and biofuel industries. Here, a novel Lactobacillus paracasei beta-fructosidase was overexpressed as truncated cytosolic protein ((t)fosEp) in Escherichia coli. Purified (t)fosEp was thermostable (10-50 degrees C) with a pH optimum of 5; it showed highest affinity for bacterial levan (beta[2-6] linked fructose) followed by nystose, chicory inulin, 1-kestose (beta[2-1] linkages) and sucrose (K(m) values of 0.5, 15, 15.6, 49 and 398 mM, respectively). Hydrolysis of polyfructose moieties in agriculturally-sourced grass juice (GJ) with (t)fosEp resulted in the release of >13 mg/ml more bioavailable fructose than was measured in untreated GJ. Bioethanol yields from fermentation experiments with Brewer's yeast and GJ+(t)fosEp were >25% higher than those achieved using untreated GJ feedstock (36.5[+/-4.3] and 28.2[+/-2.7]mg ethanol/ml, respectively). This constitutes the first specific study of the potential to ferment ethanol from grass juice and the utility of a novel core domain of beta-fructosidase from L. paracasei.

Direct fermentation of fodder maize, chicory fructans and perennial ryegrass to hydrogen using mixed microflora.

This study examined the feasibility of producing hydrogen by direct fermentation of fodder maize, chicory fructooligosaccharides and perennial ryegrass (Lolium perenne) in batch culture (pH 5.2-5.3, 35 degrees C, heat-treated anaerobically digested sludge inoculum). Gas was produced from each substrate and contained up to 50-80% hydrogen during the peak periods of gas production with the remainder carbon dioxide. Hydrogen yields obtained were 62.4+/-6.1mL/g dry matter added for fodder maize, 218+/-28mL/g chicory fructooligosaccharides added, 75.6+/-8.8mL H(2)/g dry matter added for wilted perennial ryegrass and 21.8+/-8mL H(2)/g dry matter added for fresh perennial ryegrass. Butyrate, acetate and ethanol were the main soluble fermentation products. Hydrogen yields of 392-501m(3)/hectare of perennial ryegrass per year and 1060-1309m(3)/hectare of fodder maize per year can be obtained based on the UK annual yield per hectare of these crops. These results significantly extend the range of substrates that can be used for hydrogen production without pre-treatment.

Identification of perennial ryegrass (Lolium perenne (L.)) and meadow fescue (Festuca pratensis (Huds.)) candidate orthologous sequences to the rice Hd1(Se1) and barley HvCO1 CONSTANS-like genes through comparative mapping and microsynteny.

Microsynteny with rice and comparative genetic mapping were used to identify candidate orthologous sequences to the rice Hd1(Se1) gene in Lolium perenne and Festuca pratensis. A F. pratensis bacterial artificial chromosome (BAC) library was screened with a marker (S2539) physically close to Hd1 in rice to identify the equivalent genomic region in F. pratensis. The BAC sequence was used to identify and map the same region in L. perenne. Predicted protein sequences for L. perenne and F. pratensis Hd1 candidates (LpHd1 and FpHd1) indicated they were CONSTANS-like zinc finger proteins with 61-62% sequence identity with rice Hd1 and 72% identity with barley HvCO1. LpHd1 and FpHd1 were physically linked in their respective genomes (< 4 kb) to marker S2539, which was mapped to L. perenne chromosome 7. The identified candidate orthologues of rice Hd1 and barley HvCO1 in L. perenne and F. pratensis map to chromosome 7, a region of the L. perenne genome which has a degree of conserved genetic synteny both with rice chromosome 6, which contains Hd1, and barley chromosome 7H, which contains HvCO1.

Molecular tagging of a senescence gene by introgression mapping of a stay-green mutation from Festuca pratensis.

* Intergeneric hybrids between Lolium multiflorum and Festuca pratensis (Lm/Fp) and their derivatives exhibit a unique combination of genetic and cytogenetic characteristics: chromosomes undergo a high frequency of homoeologous recombination at meiosis; the chromosomes of the two species can easily be discriminated by genomic in situ hybridization (GISH); recombination occurs along the entire length of homoeologous bivalents; a high frequency of marker polymorphism is observed between the two species. * This combination of characters has been used to transfer and isolate a F. pratensis chromosome segment carrying a mutant 'stay-green' gene conferring a disrupted leaf senescence phenotype into L. multiflorum. * The genetic location within the introgressed F. pratensis segment of the senescence gene has been mapped using amplified fragment length polymorphisms (AFLPs), and F. pratensis-specific AFLP markers closely flanking the green gene have been cloned. * The use of these cloned sequences as markers for the stay-green locus in marker-assisted selection programmes has been tested. The potential application of Lm/Fp introgressions as a tool for the map-based cloning of introgressed Fp genes is discussed.

Modification of senescence in ryegrass transformed with IPT under the control of a monocot senescence-enhanced promoter.

We report here the genetic modification of ryegrass senescence. Embryogenic cell suspensions of Lolium multiflorum were transformed by microprojectile bombardment with plasmid constructs containing 1.98 kb of the 5' flanking sequence of SEE1 (a maize cysteine protease gene showing enhanced expression during senescence) fused either to the Agrobacterium tumefaciens cytokinin biosynthesis gene IPT (designated PSEE1::IPT) or to the beta-glucuronidase reporter gene UIDA (PSEE1::UIDA). Plants were regenerated under selection for the HPH hygromycin resistance gene in the vector. PSEE1::UIDA transformants confirmed that the SEE1 flanking sequence functioned as a senescence-enhanced promoter in ryegrass. The IPT transgene was detected in 28 regenerants (PSEE1::IPT) from five independent transformation events. PSEE1::IPT leaves displayed a stay-green phenotype. Some PSEE1::IPT lines developed spontaneous lesions.

Synteny between a major heading-date QTL in perennial ryegrass (Lolium perenne L.) and the Hd3 heading-date locus in rice.

The genetic control of induction to flowering has been studied extensively in both model and crop species because of its fundamental biological and economic significance. An ultimate aim of many of these studies has been the application of the understanding of control of flowering that can be gained from the study of model species, to the improvement of crop species. The present study identifies a region of genetic synteny between rice and Lolium perenne, which contains the Hd3 heading-date QTL in rice and a major QTL, accounting for up to 70% of the variance associated with heading date in L. perenne. The identification of synteny between rice and L. perenne in this region demonstrates the direct applicability of the rice genome to the understanding of biological processes in other species. Specifically, this syntenic relationship will greatly facilitate the genetic dissection of aspects of heading-date induction by enabling the magnitude of the genetic component of the heading-date QTL in L. perenne to be combined with the sequencing and annotation information from the rice genome.

Physical and genetic mapping in the grasses Lolium perenne and Festuca pratensis.

A single chromosome of the grass species Festuca pratensis has been introgressed into Lolium perenne to produce a diploid monosomic substitution line 2n = 2x = 14. In this line recombination occurs throughout the length of the F. pratensis/L. perenne bivalent. The F. pratensis chromosome and recombinants between it and its L. perenne homeologue can be visualized using genomic in situ hybridization (GISH). GISH junctions represent the physical locations of sites of recombination, enabling a range of recombinant chromosomes to be used for physical mapping of the introgressed F. pratensis chromosome. The physical map, in conjunction with a genetic map composed of 104 F. pratensis-specific amplified fragment length polymorphisms (AFLPs), demonstrated: (1) the first large-scale analysis of the physical distribution of AFLPs; (2) variation in the relationship between genetic and physical distance from one part of the F. pratensis chromosome to another (e.g., variation was observed between and within chromosome arms); (3) that nucleolar organizer regions (NORs) and centromeres greatly reduce recombination; (4) that coding sequences are present close to the centromere and NORs in areas of low recombination in plant species with large genomes; and (5) apparent complete synteny between the F. pratensis chromosome and rice chromosome 1.

Chlorophyll breakdown in Chlorella protothecoides: characterization of degreening and cloning of degreening-related genes.

Chlorella protothecoides cultures grown in a nitrogen-free bleaching medium (BM-N) in the dark rapidly degraded chlorophyll (Chl) to red catabolites. This degreening process was investigated under different growth conditions. Supply of nitrogen to the culture medium (BM+N) inhibited bleaching and the synthesis of catabolites as did the addition to BM-N of cycloheximide or a chelator, 2,2'-bipyridyl. In contrast, chloramphenicol or the protease inhibitor E64 had no effect. During bleaching, Chl breakdown was accompanied by the degradation of cellular proteins such as light-harvesting complex II, cytochrome f and protochlorophyllide oxido-reductase. During growth in BM-N, protease activity increased and proteins immunologically detectable with an antibody against a senescence-enhanced cysteine protease accumulated. cDNAs from BM-N and BM+N cells were used for differential and subtractive screening to isolate cDNAs representing genes with degreening-enhanced expression (dee) in C. protothecoides. Several different dees were identified with different patterns of expression during Chlorella growth but which were all expressed at higher levels during bleaching. Among these, dee4 was most abundant and its expression was exclusive in BM-N cultures. Analysis of the dee sequences showed that they encode different proteins including a novel amino acid carrier (dee4), ferritin, ATP-dependent citrate lyase, a Ca2+-binding protein, MO25, ubiquinone-cytochrome c-reductase and several new proteins.

Molecular cloning, functional expression and characterisation of RCC reductase involved in chlorophyll catabolism.

Red chlorophyll catabolite (RCC) reductase (RCCR) and pheophorbide (Pheide) a oxygenase (PaO) catalyse the key reaction of chlorophyll catabolism, porphyrin macrocycle cleavage of Pheide a to a primary fluorescent catabolite (pFCC). RCCR was purified from barley and a partial gene sequence was cloned (pHvRCCR). The gene was expressed at all stages of leaf development and in roots. By comparison with different databases, genomic sequences and expressed sequence tags similar to RCCR were found in phylogenetically diverse species, and activity of RCCR was demonstrated in two of them, Arabidopsis thaliana and Marchantia polymorpha. The gene of A. thaliana (AtRCCR) was employed for molecular cloning, heterologous expression and the production of polyclonal antibodies. With recombinant RCCR, the major product of RCC reduction was pFCC-1, but small quantities of its C1 epimer, pFCC-2, also accumulated. The reaction required reduced ferredoxin and was sensitive to oxygen. AtRCCR encoded a 35 kDa protein which was used for chloroplast import experiments. Upon transport, it was processed to a mature form of 31 kDa. The significance of cloning of RCCR is discussed in respect to the evolution of chlorophyll catabolism and to the cloning of PaO.

Technical advance: single pollen typing combined with laser-mediated manipulation.

We combined single pollen typing with laser-mediated manipulation. After drilling a hole in the wall of a pollen grain from a dioecious plant (Silene latifolia) with a UV-laser microbeam, the single pollen grain was recovered directly in the cap of a PCR tube, using a non-contact method called laser pressure catapulting. The entire genome of the single pollen grain was then amplified with improved primer-extension-preamplification PCR (I-PEP PCR). Nested PCR with sequence tagged site (STS)-specific primers was used to analyze several loci in the haploid genome. The single copy gene MROS1 was detected in most of the single pollen grains analyzed. Bgl10, which is localized on the Y chromosome, was detected in approximately half of the pollen grains. MROS3 is reported to be localized on the X chromosome. Using inverse PCR, we isolated two genomic clones of MROS3: MROS3A and MROS3B. The single pollen analysis using nested PCR showed that MROS3A and MROS3B are derived from different loci that are not located on the X chromosome. Single pollen typing not only reveals sex chromosome-linkage within the haploid genome, but can also discriminate between alleles and different loci. This method should also be useful for measuring recombination frequencies without genetic crossover analysis.

Isolation of Y chromosome-specific sequences from Silene latifolia and mapping of male sex-determining genes using representational difference analysis.

The genomic subtraction method representational difference analysis (RDA) was used to identify male-specific restriction fragments in the dioecious plant Silene latifolia. Male-specific restriction fragments are linked to the male sex chromosome (the Y chromosome). Four RDA-derived male-specific restriction fragments were used to identify polymorphisms in a collection of X-ray-generated mutant plants with either hermaphroditic or asexual flowers. Some of the mutants have cytologically detectable deletions in the Y chromosome that were correlated with loss of male-specific restriction fragments. One RDA-derived probe detected a restriction fragment present in all mutants, indicating that each has retained Y chromosomal DNA. The other three probes detected genomic fragments that were linked in a region deleted in some hermaphroditic and some asexual mutants. Based on the mutant phenotypes and the correlation of cytologically visible deletions with loss of male-specific restriction fragments, these markers were assigned to positions on the Y chromosome close to the carpel suppression locus. This RDA mapping also revealed a Y-linked locus, not previously described, which is responsible for early stamen development.