Investigation of lactic acid production by pressurized liquid hot water from cultivated Miscanthus × giganteus.

The production of lactic acid, a polylactic acid monomer from energy crop Miscanthus × giganteus lignocellulosic biomass cultivated in Izmir was investigated. Liquid hot water (LHW) pretreatment was carried out at a temperature range of 140-200 °C, pressure 100 to 200 bar and reaction time of 15-45 min at a fixed flow rate of 2 mL/min using D-optimal experimental plan. The optimum conditions were elicited as 140 °C, 100 bar and 45 minutes, yielding the highest reducing sugar content of 77.32 mg/g, whereas 1.25 mg/mL arabinose and 1.35 mg/mL xylose as monomeric sugars. Subsequently, the enzymatic hydrolysis was applied to the solid fraction. The optimum conditions for enzymatic hydrolysis were determined as 5% (w/v) solid/liquid ratio, 20 FPU/mL enzyme loading and 72 hours, revealing the highest amount of reducing sugar as 200 mg/mL. LHW hydrolysate was used as a production medium for lactic acid manufacturing in submerged fermentation by Rhizopus oryzae. The maximum lactic acid content was found to be 6.8 g/L at 24 hours, whereas the lactic acid yield was 0.28 g/L.h. The sequential design of LHW, followed by enzymatic hydrolysis and submerged lactic acid fermentation can be utilized in industry, contributing to the bioeconomy.

Effect of Miscanthus × giganteus ash on survival, biomass, reproduction and avoidance behaviour of the endogeic earthworm Aporrectodea caliginosa.

To achieve the EU's targets for reducing energy production from fossil fuels, the use of energy crops, such as Miscanthus × giganteus, is increasing resulting in a corresponding increase in waste ash from incineration. The chemical properties of Miscanthus ash (e.g. phosphorus and potassium content) may allow this waste material (currently landfilled) to be used as a fertiliser, but no information exists on the effect of the ash on the biological properties of soil. The main aim of this study was to determine the potential impact of Miscanthus ash on earthworms by assessing the effect on survival, change in biomass, reproduction and avoidance behaviour of the geophagous, soil dwelling earthworm, Aporrectodea caliginosa. Tests utilised a range of Miscanthus ash doses from 0 to 50 t ha-1 (0, 1, 2.5, 5, 10, 25, 50). Results showed that Miscanthus ash had no significant impact on A. caliginosa survival, biomass and reproduction, but negative trends were observed for biomass from 2.5 t ha-1 and for reproduction from 10 t ha-1. In contrast, a significant avoidance response was observed in the 25 and 50 t ha-1 treatment and according to ISO guideline 17512 there is a negative impact of the Miscanthus ash on soil habitat function at 25 t ha-1 and above as more than 80% of earthworms were in the control soil. It is suggested that this negative effect on soil habitat function could be attributed to a range of factors including the presence of heavy metals in the ash and a change in substrate pH, texture and/or osmotic stress. Further laboratory-based studies conducted over extended time periods with a more refined range of ash doses and associated field-based studies are required to validate the results and determine a more precise assessment of the threshold ash value inducing a loss of soil habitat function.

Population structure of Miscanthus sacchariflorus reveals two major polyploidization events, tetraploid-mediated unidirectional introgression from diploid M. sinensis, and diversity centred around the Yellow Sea.

BACKGROUND AND AIMS: Miscanthus, a C4 perennial grass native to East Asia, is a promising biomass crop. Miscanthus sacchariflorus has a broad geographic range, is used to produce paper in China and is one of the parents (along with Miscanthus sinensis) of the important biomass species Miscanthus × giganteus. The largest study of M. sacchariflorus population genetics to date is reported here. METHODS: Collections included 764 individuals across East Asia. Samples were genotyped with 34 605 single nucleotide polymorphisms (SNPs) derived from restriction site-associated DNA sequencing (RAD-seq) and ten plastid microsatellites, and were subjected to ploidy analysis by flow cytometry. KEY RESULTS: Six major genetic groups within M. sacchariflorus were identified using SNP data: three diploid groups, comprising Yangtze (M. sacchariflorus ssp. lutarioriparius), N China and Korea/NE China/Russia; and three tetraploid groups, comprising N China/Korea/Russia, S Japan and N Japan. Miscanthus sacchariflorus ssp. lutarioriparius was derived from the N China group, with a substantial bottleneck. Japanese and mainland tetraploids originated from independent polyploidization events. Hybrids between diploid M. sacchariflorus and M. sinensis were identified in Korea, but without introgression into either parent species. In contrast, tetraploid M. sacchariflorus in southern Japan and Korea exhibited substantial hybridization and introgression with local diploid M. sinensis. CONCLUSIONS: Genetic data indicated that the land now under the Yellow Sea was a centre of diversity for M. sacchariflorus during the last glacial maximum, followed by a series of migrations as the climate became warmer and wetter. Overall, M. sacchariflorus has greater genetic diversity than M. sinensis, suggesting that breeding and selection within M. sacchariflorus will be important for the development of improved M. × giganteus. Ornamental M. sacchariflorus genotypes in Europe and North America represent a very narrow portion of the species' genetic diversity, and thus do not well represent the species as a whole.

Autumnal leaf senescence in Miscanthus × giganteus and leaf [N] differ by stand age.

Poor first winter survival in Miscanthus × giganteus has been anecdotally attributed to incomplete first autumn senescence, but these assessments never paired first-year with older M. × giganteus in side-by-side trials to separate the effect of weather from stand age. Here CO2 assimilation rate (A), photosystem II efficiency (ΦPSII), and leaf N concentration ([N]) were used to directly compare senescence in first, second, and third-year stands of M. × giganteus. Three M. × giganteus fields were planted with eight plots, one field each in 2009, 2010, and 2011. To quantify autumnal leaf senescence of plants within each stand age, photosynthetic and leaf [N] measurements were made twice weekly from early September until a killing frost. Following chilling events (daily temperature averages below 10 °C), photosynthetic rates in first year plants rebounded to a greater degree than those in second- and third-year plants. By the end of the growing season, first-year M. × giganteus had A and ΦPSII rates up to 4 times greater than third-year M. × giganteus, while leaf [N] was up to 2.4 times greater. The increased photosynthetic capability and leaf N status in first-year M. × giganteus suggests that the photosynthetic apparatus was not dismantled before a killing frost, thus potentially limiting nutrient translocation, and may explain why young M. × giganteus stands do not survive winter when older stands do. Because previous senescence research has primarily focused on annual or woody species, our results suggest that M. × giganteus may be an interesting herbaceous perennial system to investigate the interactive effects of plant ageing and nutrient status on senescence and may highlight management strategies that could potentially increase winter survival rates in first-year stands.

Sub-zero cold tolerance of Spartina pectinata (prairie cordgrass) and Miscanthus × giganteus: candidate bioenergy crops for cool temperate climates.

Miscanthus × giganteus grown in cool temperate regions of North America and Europe can exhibit severe mortality in the year after planting, and poor frost tolerance of leaves. Spartina pectinata (prairie cordgrass), a productive C4 perennial grass native to North America, has been suggested as an alternative biofuel feedstock for colder regions; however, its cold tolerance relative to M. × giganteus is uncertain. Here, we compare the cold tolerance thresholds for winter-dormant rhizomes and spring/summer leaves of M. × giganteus and three accessions of S. pectinata. All genotypes were planted at a field site in Ontario, Canada. In November and February, the temperatures corresponding to 50% rhizome mortality (LT(50)) were near -24°C for S. pectinata and -4°C for M. × giganteus. In late April, the LT50 of rhizomes rose to -10°C for S. pectinata but remained near -4°C for M. × giganteus. Twenty percent of the M. × giganteus rhizomes collected in late April were dead while S. pectinata rhizomes showed no signs of winter injury. Photosynthesis and electrolyte leakage measurements in spring and summer demonstrate that S. pectinata leaves have greater frost tolerance in the field. For example, S. pectinata leaves remained viable above -9°C while the mortality threshold was near -5°C for M. × giganteus. These results indicate M. × giganteus will be unsuitable for production in continental interiors of cool-temperate climate zones unless freezing and frost tolerance are improved. By contrast, S. pectinata has the freezing and frost tolerance required for a higher-latitude bioenergy crop.

Can the exceptional chilling tolerance of C4 photosynthesis found in Miscanthus × giganteus be exceeded? Screening of a novel Miscanthus Japanese germplasm collection.

BACKGROUND AND AIMS: A clone of the hybrid perennial C4 grass Miscanthus × giganteus (Mxg) is known for achieving exceptionally high rates of leaf CO2 uptake during chilling. This is a requisite of success in the early spring, as is the ability of the leaves to survive occasional frosts. The aim of this study was to search for genotypes with greater potential than Mxg for photosynthesis and frost survival under these conditions. METHODS: A total of 864 accessions representing 164 local populations of M. sacchariflorus (Msa), M. sinensis (Msi) and M. tinctorius (Mti) collected across Japan were studied. Accessions whose leaves survived a natural late frost in the field were screened for high maximum photosystem II efficiency (Fv/Fm) following chilling weather, as an indicator of their capacity for light-limited photosynthesis. Those showing the highest Fv/Fm were transferred to a high-light-controlled environment and maintained at chilling temperatures, where they were further screened for their capacities for high-light-limited and light-saturated leaf uptake of CO2 (ΦCO2,max and Asat, respectively). KEY RESULTS: For the first time, relatives of Mxg with significantly superior capacities for photosynthesis at chilling temperatures were identified. Msa accession '73/2' developed leaves in the spring that survived night-time frost, and during growth under chilling maintained a statistically significant 79 % higher ΦCO2,max, as a measure of light-limited photosynthesis, and a 70 % higher Asat, as a measure of light-saturated photosynthesis. A second Msa accession, '73/3' also showed significantly higher rates of leaf uptake of CO2. CONCLUSIONS: As remarkable as Mxg has proved in its chilling tolerance of C4 photosynthesis, this study shows that there is still value and potential in searching for yet more superior tolerance. Msa accession '73/2' shows rates of light-limited and light-saturated photosynthesis at chilling temperatures that are comparable with those of the most cold-tolerant C3 species. This adds further proof to the thesis that C4 photosynthesis is not inherently limited to warm climates.

Microbially inoculated chars strongly reduce the mobility of alachlor and pentachlorobenzene in an alluvial sediment.

The objective of this study was to investigate the transport behavior of two organic and persistent contaminants (alachlor and pentachlorobenzene) on Danube alluvial sediment in the absence and in the presence of microbially inoculated biochar produced at 400 °C and three hydrochars produced at 180, 200, and 220 °C. Stainless steel columns were used for the sorption experiments in nonequilibrium conditions. Obtained results were modeled using the advective-dispersive equation under nonequilibrium conditions. Transport of these compounds through the alluvial sediment column showed that the retention time increased with increasing molecular hydrophobicity. Inoculated biochar increases the retardation of both compounds: twofold for pentachlorobenzene compared with alachlor as a consequence of a higher hydrophobicity. Obtained results indicate that the highest biodegradation coefficient was observed for pentachlorobenzene (λ = 10) in alluvial sediment with addition of an inoculated hydrochar, which is assumed to be a consequence of biosorption. Moreover, all experiments on the columns indicate that the addition of inoculated chars yields a significantly higher Rd coefficient for pentachlorobenzene than for alachlor. Bacterial counts increased in all of the column experiments, which indicates the successful adaptation of microorganisms to experimental conditions and their potential for the removal of a large number of organic pollutants. Thus, addition of inoculated chars to contaminated sediments has the potential as a remediation technique to inhibit the leaching of pollutants to groundwaters. Integr Environ Assess Manag 2023;19:933-942. © 2022 SETAC.

Evaluation of Miscanthus × giganteus Tolerance to Trace Element Stress: Field Experiment with Soils Possessing Gradient Cd, Pb, and Zn Concentrations.

Miscanthus × giganteus demonstrated good phytostabilization potentials by decreasing the trace elements (T.E.s) mobility and enhancing the degraded soil quality. Nevertheless, most of the published work was performed under controlled conditions in ex situ pot experiments and/or with soils being spiked. Hence, data about the plant's tolerance to increased T.E. concentrations in real conditions is still scarce and requires further investigation. For this sake, a field experiment was established by cultivating miscanthus plants in three different agricultural plots representing gradient trace element (Cd, Pb and Zn) concentrations. Another uncontaminated plot was also introduced. Results showed that T.E. concentrations in the leaves were tolerable to the plant. In addition, no variations were detected between the miscanthus cultivated in the contaminated and uncontaminated soils at the level of antioxidant enzymatic activities (ascorbate peroxidase and superoxide dismutase), photosynthetic pigments (chlorophyll a and b and carotenoids), and secondary metabolites (phenolic compounds, flavonoids, anthocyanins, and tannins). These outcomes validate the high capacity of miscanthus to resist and tolerate contaminated conditions. Such results may contribute to further understanding of the miscanthus tolerance mechanisms.

Remediation of soils on municipal rendering plant territories using Miscanthus × giganteus.

Phytoremediation, as a cost-effective, highly efficient, environmentally friendly, and green approach, gained attention to the removal of metals, including heavy metals, from contaminated soils. The toxic nature of heavy metals can have an adverse effect on human health and the ecosystem, and their removal remains a worldwide problem. Therefore, in this study, a field experiment was carried out to evaluate the potential of Miscanthus × giganteus for the removal of ten microelements and heavy metals (Al, Zn, Fe, Pb, Cd, Co, Cr, Cu, Mn, Ni) from contaminated soil in the territory of a Municipal Waste Rendering Plant. Moreover, the effect of the incorporation of soil improver obtained upon composting biodegradable waste as well as the addition of highly contaminated post-industrial soil on the efficiency of phytoremediation and plant growth was described. The soil improver (SK-8) was applied to the soil at a rate of 200 Mg ha-1 and 400 Mg‧ha-1. Meanwhile, in the last object, 100 Mg‧ha-1 of highly contaminated post-industrial soil was added. Herein, the research was aimed at assessing the possibility of phytoextraction of heavy metals from soils with different physicochemical properties. The results showed that plants cultivated in soil with 400 Mg‧ha-1 of soil improver exhibited the highest yield (approximately 85% mass increase compared to the soil without additives). Furthermore, the application of a single dose of SK-8 (200 Mg ha-1) increased the uptake of Al, Fe, Co, Pb, Mn, Ni, and Cd by Miscanthus × giganteus compared to the soil without additives. Additionally, the performed biotests demonstrated no or low toxicity of the investigated soils affecting the test organisms. However, in all experiments, the phytorecovery of the elements did not exceed 1% of the amount introduced to the soil, which may result from a short cultivation period and large doses of SK-8 or highly contaminated post-industrial soil.

PGPR-driven phytoremediation and physiobiochemical response of Miscanthus × giganteus to stress induced by the trace elements.

The effect of inoculation of Miscanthus × giganteus Greef et Deu by the plant growth promoting rhizobacteria (PGPRs) to the phytoremediation process and physio-biochemical plant's parameters was investigated in soil contaminated with the trace elements (TEs) from the Tekeli mining complex, Kazakhstan. Yeast Trichosporon sp. CA1, strains Rhizobium sp. Zn1-1, Shinella sp. Zn5-6, and Pseudomonas sp. CHA1-4, resistant to Zn and Pb, were isolated from the rhizosphere of M × g when the plant was cultivated in the same contaminated soil. Results illustrated that inoculation improved M × g adaptability to TEs toxicity by increasing the tolerance index to 2.9. The treatment enhanced the aboveground biomass yield by up to 163%, root biomass by up to 240%, chlorophyll content by up to 30%, and Chla/b ratio by up to 21%. Through M × g active growth and development, the peak activity of antioxidant enzymes was observed: activity of superoxide dismutase and glutathione reductase was induced, while the activity of catalase and ascorbate peroxidase was inhibited. Based on bioconcentration and translocation factors it was revealed that PGPRs selectively increased the uptake of TEs or stabilised them in the M × g rhizosphere. Inoculation with PGPRs increased the stabilization of Pb, V, Cr, Co, Ni, Cu, Cd, As, and Ba in the soil and plant tissues. Further research should focus on ex situ experiments using isolated PGPRs.

Simultaneous Production of Cellulose Nitrates and Bacterial Cellulose from Lignocellulose of Energy Crop.

This study is focused on exploring the feasibility of simultaneously producing the two products, cellulose nitrates (CNs) and bacterial cellulose (BC), from Miscanthus × giganteus. The starting cellulose for them was isolated by successive treatments of the feedstock with HNO3 and NaOH solutions. The cellulose was subjected to enzymatic hydrolysis for 2, 8, and 24 h. The cellulose samples after the hydrolysis were distinct in structure from the starting sample (degree of polymerization (DP) 1770, degree of crystallinity (DC) 64%) and between each other (DP 1510-1760, DC 72-75%). The nitration showed that these samples and the starting cellulose could successfully be nitrated to furnish acetone-soluble CNs. Extending the hydrolysis time from 2 h to 24 h led to an enhanced yield of CNs from 116 to 131%, with the nitrogen content and the viscosity of the CN samples increasing from 11.35 to 11.83% and from 94 to 119 mPa·s, respectively. The SEM analysis demonstrated that CNs retained the fiber shape. The IR spectroscopy confirmed that the synthesized material was specifically CNs, as evidenced by the characteristic frequencies of 1657-1659, 1277, 832-833, 747, and 688-690 cm-1. Nutrient media derived from the hydrolyzates obtained in 8 h and 24 h were of good quality for the synthesis of BC, with yields of 11.1% and 9.6%, respectively. The BC samples had a reticulate structure made of interlaced microfibrils with 65 and 81 nm widths and DPs of 2100 and 2300, respectively. It is for the first time that such an approach for the simultaneous production of CNs and BC has been employed.

Species selection determines carbon allocation and turnover in Miscanthus crops: Implications for biomass production and C sequestration.

Growing Miscanthus species and hybrids has received strong scientific and commercial support, with the majority of the carbon (C) modelling predictions having focused on the high-yield, sterile and noninvasive hybrid Miscanthus × giganteus. However, the potential of other species with contrasting phenotypic and physiological traits has been seldom explored. To better understand the mechanisms underlying C allocation dynamics in these bioenergy crops, we pulse-labelled (13CO2) intact plant-soil systems of Miscanthus × giganteus (GIG), Miscanthus sinensis (SIN) and Miscanthus lutarioriparius (LUT) and regularly analysed soil respiration, leaves, stems, rhizomes, roots and soils for up to 190 days until leaf senescence. A rapid isotopic enrichment of all three species was observed after 4 h, with the amount of 13C fixed into plant biomass being inversely related to their respective standing biomass prior to pulse-labelling (i.e., GIG < SIN < LUT). However, both GIG and LUT allocated more photoassimilates in the aboveground biomass (leaves+stems = 78 % and 74 %, respectively) than SIN, which transferred 30% of fixed 13C in its belowground biomass (rhizomes+roots). Although less fixed 13C was recovered from the soils (<1 %), both rhizospheric and bulk soils were signficantly more enriched under SIN and LUT than under GIG. Importantly, the soils under SIN emitted less CO2, which suggests it could be the best choice for reaching C neutrality. These results from this unique large-scale study indicate that careful species selection may hold the success for reaching net GHG mitigation.

Effect of Heavy-Metal-Resistant PGPR Inoculants on Growth, Rhizosphere Microbiome and Remediation Potential of Miscanthus × giganteus in Zinc-Contaminated Soil.

Microbial-assisted phytoremediation is considered a more effective approach to soil rehabilitation than the sole use of plants. Mycolicibacterium sp. Pb113 and Chitinophaga sp. Zn19, heavy-metal-resistant PGPR strains originally isolated from the rhizosphere of Miscanthus × giganteus, were used as inoculants of the host plant grown in control and zinc-contaminated (1650 mg/kg) soil in a 4-month pot experiment. The diversity and taxonomic structure of the rhizosphere microbiomes, assessed with metagenomic analysis of rhizosphere samples for the 16S rRNA gene, were studied. Principal coordinate analysis showed differences in the formation of the microbiomes, which was affected by zinc rather than by the inoculants. Bacterial taxa affected by zinc and the inoculants, and the taxa potentially involved in the promotion of plant growth as well as in assisted phytoremediation, were identified. Both inoculants promoted miscanthus growth, but only Chitinophaga sp. Zn19 contributed to significant Zn accumulation in the aboveground part of the plant. In this study, the positive effect of miscanthus inoculation with Mycolicibacterium spp. and Chitinophaga spp. was demonstrated for the first time. On the basis of our data, the bacterial strains studied may be recommended to improve the efficiency of M. × giganteus phytoremediation of zinc-contaminated soil.

Response of Three Miscanthus × giganteus Cultivars to Toxic Elements Stress: Part 2, Comparison between Two Growing Seasons.

The positive impact on restoring soil functionality, decreasing toxic elements (TE) bioaccessibility, and enhancing soil physicochemical and biological parameters established a consensus on considering a Miscanthus × giganteus convenient species for phytomanaging wide TE contaminated areas. Nevertheless, information about the plant's mode of reaction to elevated soil multi-TE concentrations is still scarce. For the sake of investigating the miscanthus response to stressful TE concentrations, an ex-situ pot experiment was initiated for 18 months, with three miscanthus cultivars referred to as B, U, and A planted in soils with gradient Cd, Pb, and Zn concentrations. A non-contaminated control soil was introduced as well, and plants were cultivated within. Results revealed that the long exposure to increasing soil TE concentrations caused the number of tillers per plant to decline and the TE concentrations in the leaves to boost progressively with the soil contamination. The photosynthetic pigments (chlorophyll a, b, and carotenoids) were negatively affected as well. However, the phenolic compounds, flavonoids, tannins, and anthocyanins, along with the antioxidant enzymatic activities of superoxide dismutase, ascorbate peroxidase, and glutathione reductase elevated progressively with the TE concentration and exposure duration. Conclusively, miscanthus plants demonstrated an intensified and synchronized antioxidative activity against the TE concentration.

Response of Total (DNA) and Metabolically Active (RNA) Microbial Communities in Miscanthus × Giganteus Cultivated Soil to Different Nitrogen Fertilization Rates.

Miscanthus × giganteus is a promising high-yielding perennial plant to meet growing bioenergy demands; however, the degree to which the soil microbiome affects its nitrogen cycling and subsequently, biomass yield remains unclear. In this study, we hypothesize that contributions of metabolically active soil microbial membership may be underestimated with DNA-based approaches. We assessed the response of the soil microbiome to nitrogen availability in terms of both DNA and RNA soil microbial communities from the Long-term Assessment of Miscanthus Productivity and Sustainability (LAMPS) field trial. DNA and RNA were extracted from 271 samples, and 16S small subunit (SSU) rRNA amplicon sequencing was performed to characterize microbial community structure. Significant differences were observed in the resulting soil microbiomes and were best explained by the sequencing library of origin, either DNA or RNA. Similar numbers of membership were detected in DNA and RNA microbial communities, with more than 90% of membership shared. However, the profile of dominant membership within DNA and RNA differed, with varying proportions of Actinobacteria and Proteobacteria and Firmicutes and Proteobacteria. Only RNA microbial communities showed seasonal responses to nitrogen fertilization, and these differences were associated with nitrogen-cycling bacteria. The relative abundance of bacteria associated with nitrogen cycling was 7-fold higher in RNA than in DNA, and genes associated with denitrifying bacteria were significantly enriched in RNA, suggesting that these bacteria may be underestimated with DNA-only approaches. Our findings indicate that RNA-based SSU characterization can be a significant and complementing resource for understanding the role of soil microbiomes in bioenergy crop production. IMPORTANCEMiscanthus × giganteus is a promising candidate for bioeconomy cropping systems; however, it remains unclear how the soil microbiome supplies nitrogen to this low-input crop. DNA-based techniques are used to provide community characterization, but may miss important metabolically active taxa. By analyzing both DNA- and actively transcribed RNA-based microbial communities, we found that nitrogen cycling taxa in the soil microbiome may be underestimated using only DNA-based approaches. Accurately understanding the role of microbes and how they cycle nutrients is important for the development of sustainable bioenergy crops, and RNA-based approaches are recommended as a complement to DNA approaches to better understand the microbial, plant, and management interactions.

Cell Wall Properties Determine Genotype-Specific Response to Cold in Miscanthus × giganteus Plants.

The cell wall plays a crucial role in plant growth and development, including in response to environmental factors, mainly through significant biochemical and biomechanical plasticity. The involvement of the cell wall in C4 plants' response to cold is, however, still poorly understood. Miscanthus × giganteus, a perennial grass, is generally considered cold tolerant and, in contrast to other thermophilic species such as maize or sorgo, can maintain a relatively high level of photosynthesis efficiency at low ambient temperatures. This unusual response to chilling among C4 plants makes Miscanthus an interesting study object in cold acclimation mechanism research. Using the results obtained from employing a diverse range of techniques, including analysis of plasmodesmata ultrastructure by means of transmission electron microscopy (TEM), infrared spectroscopy (FTIR), and biomechanical tests coupled with photosynthetic parameters measurements, we present evidence for the implication of the cell wall in genotype-specific responses to cold in this species. The observed reduction in the assimilation rate and disturbance of chlorophyll fluorescence parameters in the susceptible M3 genotype under cold conditions were associated with changes in the ultrastructure of the plasmodesmata, i.e., a constriction of the cytoplasmic sleeve in the central region of the microchannel at the mesophyll-bundle sheath interface. Moreover, this cold susceptible genotype was characterized by enhanced tensile stiffness, strength of leaf wall material, and a less altered biochemical profile of the cell wall, revealed by FTIR spectroscopy, compared to cold tolerant genotypes. These changes indicate that a decline in photosynthetic activity may result from a decrease in leaf CO2 conductance due to the formation of more compact and thicker cell walls and that an enhanced tolerance to cold requires biochemical wall remodelling. Thus, the well-established trade-off between photosynthetic capacity and leaf biomechanics found across multiple species in ecological research may also be a relevant factor in Miscanthus' tolerance to cold. In this paper, we demonstrate that M. giganteus genotypes showing a high degree of genetic similarity may respond differently to cold stress if exposed at earlier growing seasons to various temperature regimes, which has implications for the cell wall modifications patterns.

Response of Three Miscanthus × giganteus Cultivars to Toxic Elements Stress: Part 1, Plant Defence Mechanisms.

Miscanthus × giganteus demonstrated good phytostabilization potentials in toxic element (TE) contaminated soils. However, information about its tolerance to elevated concentrations is still scarce. Therefore, an ex-situ pot experiment was launched using three cultivars (termed B, U, and A) grown in soils with a gradient Cd, Pb and Zn concentrations. Control plants were also cultivated in non-contaminated soil. Results show that the number of tillers per plant, stem diameter as well as leaf photosynthetic pigments (chlorophyll a, b and carotenoids) were negatively impacted by soil contamination. On the other hand, phenolic compounds, flavonoids, tannins, and anthocyanins levels along with the antioxidant enzymatic activities of superoxide dismutase, ascorbate peroxidase and glutathione reductase increased in the plants grown on contaminated soils. Altogether, these data demonstrate that miscanthus is impacted by concentrations of toxic elements yet is able to tolerate high levels of soil contamination. These results may contribute to clarifying the miscanthus tolerance strategy against high contamination levels and its efficiency in phytoremediation.

Biorefining Potential of Wild-Grown Arundo donax, Cortaderia selloana and Phragmites australis and the Feasibility of White-Rot Fungi-Mediated Pretreatments.

Arundo donax, Cortaderia selloana and Phragmites australis are high-biomass-producing perennial Poalean species that grow abundantly and spontaneously in warm temperate regions, such as in Mediterranean-type climates, like those of Southern Europe, Western United States coastal areas, or in regions of South America, South Africa and Australia. Given their vigorous and spontaneous growth, biomass from the studied grasses often accumulates excessively in unmanaged agro-forestry areas. Nonetheless, this also creates the demand and opportunity for the valorisation of these biomass sources, particularly their cell wall polymers, for biorefining applications. By contrast, a related crop, Miscanthus × giganteus, is a perennial grass that has been extensively studied for lignocellulosic biomass production, as it can grow on low-input agricultural systems in colder climates. In this study Fourier transform mid-infrared spectroscopy (FTIR), high-performance anion-exchange chromatography (HPAEC) and lignin content determinations were used for a comparative compositional characterisation of A. donax, C. selloana and P. australis harvested from the wild, in relation to a trial field-grown M. × giganteus high-yielding genotype. A high-throughput saccharification assay showed relatively high sugar release values from the wild-grown grasses, even with a 0.1M NaOH mild alkali pretreatment. In addition to this alkaline pretreatment, biomass was treated with white-rot fungi (WRF), which preferentially degrade lignin more readily than holocellulose. Three fungal species were used: Ganoderma lucidum, Pleurotus ostreatus and Trametes versicolor. Our results showed that neutral sugar contents are not significantly altered, while some lignin is lost during the pretreatments. Furthermore, sugar release upon enzymatic saccharification was enhanced, and this was dependent on the plant biomass and fungal species used in the treatment. To maximise the potential for lignocellulose valorisation, the liquid fractions from the pretreatments were analysed by high performance liquid chromatography - photodiode array detection - electrospray ionisation tandem mass spectrometry (HPLC-PDA-ESI-MS n ). This study is one of the first to report on the composition of WRF-treated grass biomass, while assessing the potential relevance of breakdown products released during the treatments, beyond more traditional sugar-for-energy applications. Ultimately, we expect that our data will help promote the valorisation of unused biomass resources, create economic value, while contributing to the implementation of sustainable biorefining systems.

Bioprospecting of a Novel Plant Growth-Promoting Bacterium Bacillus Altitudinis KP-14 for Enhancing Miscanthus × giganteus Growth in Metals Contaminated Soil.

Use of plant growth-promoting bacteria (PGPB) for cultivation of the biofuel crop Miscanthus × giganteus (Mxg) in post-military and post-mining sites is a promising approach for the bioremediation of soils contaminated by metals. In the present study, PGPB were isolated from contaminated soil and screened for tolerance against abiotic stresses caused by salinity, pH, temperature, and lead (Pb). Selected strains were further assessed and screened for plant growth-promoting attributes. The isolate showing the most potential, Bacillus altitudinis KP-14, was tested for enhancement of Mxg growth in contaminated soil under greenhouse conditions. It was found to be highly tolerant to diverse abiotic stresses, exhibiting tolerance to salinity (0-15%), pH (4-8), temperature (4-50 °C), and Pb (up to 1200 ppm). The association of B. altitudinis KP-14 with Mxg resulted in a significant (p ≤ 0.001) impact on biomass enhancement: the total shoot and dry root weights were significantly enhanced by 77.7% and 55.5%, respectively. The significant enhancement of Mxg biomass parameters by application of B. altitudinis KP-14 strongly supports the use of this strain as a biofertilizer for the improvement of plant growth in metal-contaminated soils.