Impact of drought stress on growth and quality of miscanthus for biofuel production

Miscanthus has a high potential as a biomass feedstock for biofuel production. Drought tolerance is an important breeding goal in miscanthus as water deficit is a common abiotic stress and crop irrigation is in most cases uneconomical. Drought may not only severely reduce biomass yields, but also affect biomass quality for biofuel production as cell wall remodeling is a common plant response to abiotic stresses. The quality and plant weight of 50 diverse miscanthus genotypes were evaluated under control and drought conditions (28 days no water) in a glasshouse experiment. Overall, drought treatment decreased plant weight by 45%. Drought tolerance – as defined by maintenance of plant weight – varied extensively among the tested miscanthus genotypes and ranged from 30% to 110%. Biomass composition was drastically altered due to drought stress, with large reductions in cell wall and cellulose content and a substantial increase in hemicellulosic polysaccharides. Stress had only a small effect on lignin content. Cell wall structural rigidity was also affected by drought conditions; substantially higher cellulose conversion rates were observed upon enzymatic saccharification of drought‐treated samples with respect to controls. Both cell wall composition and the extent of cell wall plasticity under drought varied extensively among all genotypes, but only weak correlations were found with the level of drought tolerance, suggesting their independent genetic control. High drought tolerance and biomass quality can thus potentially be advanced simultaneously. The extensive genotypic variation found for most traits in the evaluated miscanthus germplasm provides ample scope for breeding of drought‐tolerant varieties that are able to produce substantial yields of high‐quality biomass under water deficit conditions. The higher degradability of drought‐treated samples makes miscanthus an interesting crop for the production of second‐generation biofuels in marginal soils.     

Enzyme hydrolysis and ethanol fermentation of dilute ammonia pretreated energy cane

This study is the first one ever to report on the use of high fiber sugarcane (a.k.a. energy cane) bagasse as feedstock for the production of cellulosic ethanol. Energy cane bagasse was pretreated with ammonium hydroxide (28% v/v solution), and water at a ratio of 1:0.5:8 at 160 °C for 1 h under 0.9-1.1 MPa. Approximately, 55% lignin, 30% hemicellulose, 9% cellulose, and 6% other (e.g., ash, proteins) were removed during the process. The maximum glucan conversion of dilute ammonia treated energy cane bagasse by cellulases was 87% with an ethanol yield (glucose only) of 23 g ethanol/100 g dry biomass. The enzymatic digestibility was related to the removal of lignin and hemicellulose, perhaps due to increased surface area and porosity resulting in the deformation and swelling of exposed fibers as shown in the SEM pictures.     

Decomposition of Phragmites australis rhizome in a shallow lake

Decomposition of Phragmites australis (Cav. Trin ex Steudel) rhizome was studied at Lake Fert?/Neusiedler See using the litter bag technique. Samples were analysed for rhizome dry mass, fibre (cellulose, hemicellulose, lignin) and nutrient content (C, N, P and S), litter-associated fungal biomass, potential microbial respiration (electron transport activity: ETS) and cellulolitic bacteria. The mass loss of decomposing rhizome was rapid in the initial period and only 13.6% of the dry mass remained at the end of the experiment during 953 days. Substantial quantities of C, N, S and P were lost during 99 days; only 18% C, 19% N, 14% S and 6.4% of the P remained after 953 days. Hemicellulose degraded more rapidly than the other fibres whilst the lignin had the slowest rate of decomposition. Bacteria were found to be the primary colonizers of plant detritus, which was followed by fungal growth. An antagonistic relationship was observed between bacteria and fungi. Fungal biomass as determined by ergosterol concentrations ranged between 4.1 and 420 μg g⁻¹ and peaked every year in September. The number of cellulolitic bacteria varied from 0 to 22 MPN g⁻¹ with higher values in summer. The ETS-activity ranged between 0.1 and 1.6 mg O₂ g⁻¹ h⁻¹. The changes in ETS-activity varied almost in parallel with the in situ temperature of the lake water.     

Applications of Anammox based processes to treat anaerobic digester supernatant at room temperature

The supernatant of an anaerobic digester was treated at 20 °C in two systems. The first one is a two units configuration, conformed by two sequencing batch reactors (SBR), carrying out partial nitrification and Anammox processes, respectively. Partial nitrification was achieved by granular biomass with a mean diameter of 3 mm, operating at a dissolved oxygen concentration of 2.7 mg/L. The combined system allowed the removal of nitrogen loading rates around 0.08 g N/(L d).     

Effect of thermal pretreatment on equilibrium moisture content of lignocellulosic biomass

The equilibrium moisture content (EMC) of raw lignocellulosic biomass, along with four samples subjected to thermal pretreatment, was measured at relative humidities ranging from 11% to 97% at a constant temperature of 30 °C. Three samples were prepared by treatment in hot compressed water by a process known as wet torrefaction, at temperatures of 200, 230, and 260 °C. An additional sample was prepared by dry torrefaction at 300 °C. Pretreated biomass shows EMC below that of raw biomass. This indicates that pretreated biomass, both dry and wet torrefied, is more hydrophobic than raw biomass. The EMC results were correlated with a recent model that takes into account additional non-adsorption interactions of water, such as mixing and swelling. The model offers physical insight into the water activity in lignocellulosic biomass.     

Phenol and phenolics from lignocellulosic biomass by catalytic microwave pyrolysis

Catalytic microwave pyrolysis of biomass using activated carbon was investigated to determine the effects of pyrolytic conditions on the yields of phenol and phenolics. The high concentrations of phenol (38.9%) and phenolics (66.9%) were obtained at the temperature of 589 K, catalyst-to-biomass ratio of 3:1 and retention time of 8 min. The increase of phenol and its derivatives compared to pyrolysis without catalysts has a close relationship with the decomposition of lignin under the performance of activated carbon. The concentration of esters was also increased using activated carbon as a catalyst. The high content of phenols obtained in this study can be used either directly as fuel after upgrading or as feedstock of bio-based phenols for chemical industry.     

Pretreatment of switchgrass for sugar production with the combination of sodium hydroxide and lime

Sodium hydroxide (NaOH) and lime (Ca(OH)₂) were innovatively used together in this study to improve the cost-effectiveness of alkaline pretreatment of switchgrass at ambient temperature. Based on the sugar production in enzymatic hydrolysis, the best pretreatment conditions were determined as: residence time of 6 h, NaOH loading of 0.10 g/g raw biomass, NaOH addition at the beginning, Ca(OH)₂ loading of 0.02 g/g raw biomass, and biomass wash intensity of 100 ml water/g raw biomass, at which the glucose and xylose yields were respectively 59.4% and 57.3% of the theoretical yields. The sugar yield of the biomass pretreated using the combination of 0.10 g NaOH/g raw biomass and 0.02 g Ca(OH)₂/g raw biomass was found comparable with that of the biomass pretreated using 0.20 g NaOH/g raw biomass at the same conditions, while the chemical expense was remarkably reduced due to the low cost of lime and the reduced loading of NaOH.     

Influence of environmental factors on Vallisneria americana seed germination

Over the course of a growing season (April–October) water quality (water temperature, light, salinity, dissolved oxygen) and reproductive phenology (biomass, production of flowering shoots and seed pods, seed bank densities) were quantified in three Vallisneria americana beds in Nanjemoy Creek, MD, a tributary to the Chesapeake Bay. Clonal production of V. americana biomass increased at all sites when water temperatures rose above 25 °C. Flowering occurred during peak biomass (August–September) and resulted in the production of up to 16,000 seeds m⁻² at the end of the growing season. However, observed seed bank densities represented <1% of seed production. Laboratory experiments quantified the effects of dissolved oxygen (0.29–8.00 mg l⁻¹), light (0–160 μmol m² s⁻¹), temperature (13–29 °C), salinity (0.1–17.4 psu), sediment composition (3–86% sand; 0.9–8.3% sediment organic content), and burial depth (0.2–10 cm) on V. americana seed germination. Germination of V. americana seeds was enhanced (greater overall germination and shorter time to germination) under oxygenated conditions (8.00 mg l⁻¹), temperatures >22 °C, salinities of <1 psu, and in sediments composed of ≤3% organic content and >40% sand. Light (<160 μmol m⁻² s⁻¹) and burial depth (0.2–10 cm) had no significant effects on germination. Temperatures most favorable for seed germination (>22 °C) occurred in June, 2 months in the growing season just prior to development of peak vegetative standing stock. Seedlings were therefore at a distinct disadvantage to plants developed from over wintering buds. A lack of viable seed retention and inadequate environmental conditions at critical times in the growing season may be limiting seed germination success and subsequent seedling establishment within V. americana beds in the Chesapeake Bay. However, ungerminated seeds were found to maintain high viability, especially at salinities of 10 psu that can have significant negative effects of shoot growth survival. This suggests that seeds may serve as a source of reproductive material for bed recovery after periods of drought or other stressful conditions in estuarine systems.     

Production of 5-hydroxymethylfurfural in ionic liquids under high fructose concentration conditions

Acid-promoted, selective production of 5-hydroxymethylfurfural (HMF) under high fructose concentration conditions was achieved in ionic liquids (ILs) at 80 °C. A HMF yield up to 97% was obtained in 8 min using 1-butyl-3-methylimidazolium chloride ([C₄mim]Cl) catalyzed with 9 mol % hydrochloric acid. More significantly, an HMF yield of 51% was observed when fructose was loaded at a high concentration of 67 wt % in [C₄mim]Cl. Water content below 15.4% in the system had little effect on HMF yield, whereas a higher water content was detrimental to both reaction rate and HMF yield. In situ NMR analysis suggested that the transformation of fructose to HMF was a highly selective reaction that proceeded through the cyclic fructofuranosyl intermediate pathway. This work increased our capacity to produce HMF, and should be valuable to facilitate cost-efficient conversion of biomass into biofuels and bio-based products.     

Comparative LCA studies of simulated HMF biorefineries from maize and miscanthus as an example of first- and second-generation biomass as a tool for process development

5-Hydroxymethylfurfural (HMF) is a versatile platform chemical for a fossil free, bio-based chemical industry. HMF can be produced by using fructose as a feedstock. Using edible, first-generation biomass to produce chemicals has been questioned in terms of potential competition with food supply. Second-generation biomass like miscanthus could be an alternative. However, there is a lack of information if second-generation lignocellulosic biomass is a more sustainable feedstock to produce HMF. Therefore, a life cycle assessment was performed in this study to determine the environmental impacts of HMF production from miscanthus and to compare it with HMF from high-fructose corn syrup (HFCS). HFCS from either Hungary or Baden-Württemberg (Germany) was considered. Compared to the HFCS biorefineries the miscanthus concept is producing less emissions in all impact categories studied, except land occupation. Overall, the production and usage of second-generation biomass could be especially beneficial in areas where the use of N fertilizers is restricted. Besides, conclusions for the further development of the on-farm biorefinery concept were elaborated. For this purpose, process simulations from a previous study were used. Results of the previous study in terms of TEA and the current LCA study in terms of environmental sustainability indicate that the lignin depolymerization unit in the miscanthus biorefinery has to be improved. The scenario without lignin depolymerization performs better in all impact categories. The authors recommend to not further convert the lignin to products like phenol and other aromatic compounds. The results of the contribution analyses show that the major impact in the HMF production is caused by the auxiliary materials in the separation units and the required heat. Further technical development should focus on efficient heat as well as solvent use and solvent recovery. At this point further optimizations will lead to reduced emissions and costs at the same time.     

Enhancing alfalfa conversion efficiencies for sugar recovery and ethanol production by altering lignin composition

Alfalfa (Medicago sativa L.) biomass was evaluated for biochemical conversion into ethanol using dilute-acid and ammonia pretreatments. The two alfalfa lines compared were a reduced S-lignin transgenic cultivar generated through down regulation of the caffeic acid O-methyltransferase gene and a wild-type control. Both were harvested at two maturities. All the samples had similar carbohydrate contents including a mean composition of 316 g glucan and 497 g total neutral carbohydrates per kg dry biomass, which corresponds to a theoretic ethanol yield of 382 l/ton. Ethanol yields for alfalfa stems pretreated with dilute-acid were significantly impacted by harvest maturity and lignin composition, whereas when pretreated with dilute-ammonia, yield was solely affected by lignin composition. Use of a recombinant xylose-fermenting Saccharomyces strain, for converting the ammonia pretreated alfalfa samples, further increased ethanol yields. Ethanol yields for the xylose-fermenting yeast were 232-278 l/ton and were significantly enhanced for the reduced S lignin cultivars.     

Selective extraction of hemicelluloses from spruce using switchable ionic liquids

Switchable ionic liquids (SILs) made from alcohols, either hexanol or butanol, and CO₂ together with an amidine (1,8-diazabicyclo-[5.4.0]-undec-7-ene (DBU)) were investigated as dissolution/fractionation solvents for wood material. Both native spruce (Picea abies), and pre-extracted spruce were treated with either butanol SIL (SIL1) or hexanol SIL (SIL2) for 5 days at 55 °C under normal pressure. The SILs were formed by bubbling CO₂ through an equimolar mixture of either 1-hexanol or 1-butanol and DBU. The viscosity of the mixture increased from 7.1 mPa s to 2980 mPa s for SIL2 and 5.1 to 1600 mPa s for SIL1. Melting points of the SILs 1 and 2 were at 8 and 14 °C, respectively. After the treatment time (5 days), the undissolved fraction contained 38 wt.% less hemicelluloses compared to native spruce. There was an increase in the glucose content of the milled spruce treated with both SILs, since the milling step reduced the cellulose crystallinity of the wood and facilitated an easier SIL access into the wood. The solvents were very neutral in terms of lignin removal. Consequently, only about 2% of the lignin was removed from native wood. Moreover, a priori removal of the wood extractives did not influence the lignin removal.     

Responses of a fringing Cyperus papyrus L. swamp to changes in water level

Over a 9-year period (1993–2001), the land-water width of a papyrus fringe on the southern shore of Lake Naivasha, Kenya, varied between 40 and 60 m. Increases in width via rhizome spreading into open water followed the 1997/1998 El Niño flood when water depths rose by about 2 m. Germination of papyrus seeds also responded to water depth with a mean ± S.E. rate in experiments of 23 ± 6% after 21 days when water level was 5 cm below the sediment surface. No germination occurred when sediment was flooded or allowed to desiccate. Rhizome spreading from floating mats appeared to be favoured by deep water with seedling spread favoured on newly inundated, low-gradient slopes in shallow water. Although natural regenerative capacity was influenced by water depth, the height, density, biomass and chemical content of papyrus were not. Total average biomass along a land-water transect was 6950 ± 860 g m⁻² which was large in relation to nutrient and mineral contents. Culms contained 0.47 ± 0.14% N and 0.06 ± 0.05% P and rhizomes 0.71 ± 0.21% and 0.10 ± 0.06%. Sediment underlying the swamp was aerobic and there were small land-water gradients in the BOD of swamp water and sediment. However, chemical gradients were weak compared with wider papyrus swamps elsewhere. Lake and swamp water mixed in the narrow fringe studied and residence times for organic matter may not have been long enough for organic material to mineralise before entering lake water.     

How can miscanthus fields be reintegrated into a crop rotation?

The bioeconomy, with its aim of replacing fossil by biobased resources, is increasingly focusing on biomass production from perennial crops, such as miscanthus. To date, research on miscanthus has explored a number of cultivation aspects; however, one major issue has not yet been addressed: How can former miscanthus fields be reintegrated into a crop rotation? This encompasses the questions of which following crop most efficiently suppresses resprouting miscanthus and what happens to the soil nitrogen content after a miscanthus removal. This study aimed to answer both questions. For this purpose, four spring crops (ryegrass, rapeseed, barley, maize) and fallow as control were cultivated after a Miscanthus sinensis removal. To test the effect of the removal on soil nitrogen content, each spring crop (excluding fallow) was divided into fertilized and unfertilized plots. After the spring crop harvest, winter wheat was cultivated to clarify which spring crop had most efficiently suppressed the resprouting miscanthus. The results indicate that fertilized crops had 35% less miscanthus biomass per hectare than unfertilized crops, probably due to the higher plant density and/or better development of the fertilized crops during the growing season. The soil mineral nitrogen (N_min) content was found to increase during the vegetation period following the miscanthus removal (average +14.85 kg/ha), but was generally on a low level. We conclude that nitrogen from miscanthus residues is partly fixed in organic matter and is thus not plant‐available in the first cropping season. As some nitrogen is supplied by the decomposition of miscanthus residues, our results suggest that the crop cultivated after a miscanthus removal requires less fertilization. Of all the follow‐on spring crops tested, maize coped with the prevailing soil conditions and resprouting miscanthus most efficiently, resulting in satisfactory yields, and thus seems to be a suitable crop for cultivation after miscanthus.     

Effect of sludge age on simultaneous nitrification and denitrification in membrane bioreactor

This study evaluated the effect of sludge age on simultaneous nitrification and denitrification in a membrane bioreactor treating black water. A membrane bioreactor with no separate anoxic volume was operated at a sludge age of 20 days under low dissolved oxygen concentration of 0.1-0.2 mg/L. Its performance was compared with the period when the sludge age was adjusted to 60 days. Floc size distribution, apparent viscosity, and nitrogen removal differed significantly, together with different biomass concentrations: nitrification was reduced to 40% while denitrification was almost complete. Modelling indicated that both nitrification and denitrification kinetics varied as a function of the sludge age. Calibrated values of half saturation coefficients were reduced when the sludge age was lowered to 20 days. Model simulation confirmed the validity of variable process kinetics for nitrogen removal, specifically set by the selected sludge age.     

Characterization of lignin-rich residues remaining after continuous super-critical water hydrolysis of poplar wood (Populus albaglandulosa) for conversion to fermentable sugars

Poplar wood flour (Populous albaglandulosa) was treated with sub- and super-critical water (subcritical: 325, 350 °C; super-critical: 380, 400, 425 °C) for 60 s at 220 ± 10 atm. Hydrochloric acid (0.05% v/v) was added to samples as acidic catalyst. The final products were separated into water soluble fraction and undegraded solids. The yields of undegraded solids were thoroughly dependent on temperature severity and mainly composed of lignin fragments. Average molecular weights of the lignins were between 1500 and 4400 Da, which was only 1/3-1/8-fold of poplar milled wood lignin (13,250 Da). DFRC (Derivatization Followed by Reductive Cleavage) analysis revealed that C6C3 phenols (coniferyl and sinapyl alcohol) were rarely detected in the lignins, indicating occurrence of two probable lignin reactions during SCW hydrolysis: lignin fragmentation via splitting of β-O-4 linkage and loss of propane side chains. These results were also confirmed by ¹H and ¹³C NMR spectroscopic analysis.     

Production of bioethanol, methane and heat from sugarcane bagasse in a biorefinery concept

The potential of biogas production from the residues of second generation bioethanol production was investigated taking into consideration two types of pretreatment: lime or alkaline hydrogen peroxide. Bagasse was pretreated, enzymatically hydrolyzed and the wastes from pretreatment and hydrolysis were used to produce biogas. Results have shown that if pretreatment is carried out at a bagasse concentration of 4% DM, the highest global methane production is obtained with the peroxide pretreatment: 72.1 L methane/kg bagasse. The recovery of lignin from the peroxide pretreatment liquor was also the highest, 112.7 ± 0.01 g/kg of bagasse. Evaluation of four different biofuel production scenarios has shown that 63-65% of the energy that would be produced by bagasse incineration can be recovered by combining ethanol production with the combustion of lignin and hydrolysis residues, along with the anaerobic digestion of pretreatment liquors, while only 32-33% of the energy is recovered by bioethanol production alone.     

Characterization of compost produced from separated pig manure and a variety of bulking agents at low initial C/N ratios

The aim of this study was to investigate the composting of separated pig manure solids with or without a variety of bulking agents at a low initial C/N ratio (12.5-23.3). Compost stability was investigated using an oxygen uptake rate (OUR) test and compost maturity was investigated using a germination index test. All treatments showed typical patterns of compost temperature. Temperatures above 60 °C were achieved by Day 2, followed by a thermophilic phase (50-60 °C), which lasted for 1 to 2 weeks followed by a cooling phase. The stability of one of treatments which did not contain any bulking agent - OUR of 25 mmol O₂ kg⁻¹ OM hour⁻¹ - was negatively affected by its initial high water content (69%). The addition of a bulking agent and initial water content below 60% were necessary to compost the separated solid fraction of pig manure at a low initial C/N ratio.     

Miscanthus for biogas production: Influence of harvest date and ensiling on digestibility and methane hectare yield

The 8,000 biogas plants currently in operation in Germany are mainly fed with biomass from annual crops. However, feedstock from perennial crops such as miscanthus is expected to be more environmentally benign. If miscanthus is to be used in greater amounts as a substrate for anaerobic digestion, storage will become a relevant topic, as a continuous supply of biomass throughout the year is necessary. The objective of this study was to identify the miscanthus harvest time that best balances the simultaneous achievement of high silage quality, high digestibility and high methane hectare yields. For this purpose, biomass from four miscanthus genotypes with varying senescence characteristics was harvested on three different dates in autumn 2017. Part of the biomass was ensiled, and the methane yield of both ensiled and non‐ensiled biomass was analysed in a biogas batch test to assess the effect of ensiling on the methane hectare yield and digestion velocity. The ensiled biomass was found to have an up to 7% higher substrate‐specific methane yield and also showed a higher digestion velocity than the non‐ensiled biomass. The silage quality was best when miscanthus was harvested in mid‐October, due to highest lactic acid content (average: 3.0% of DM) and lowest pH (average: 4.39) compared to the harvests in mid‐September and beginning of October. Mass losses during ensiling (as high as 7.6% of fresh matter for the M. sinensis genotype Sin55) were compensated for by a higher substrate‐specific methane yield (up to 353 Nml CH₄ (g oDM)^?1) in ensiled miscanthus. This resulted in non‐significantly different methane hectare yields for non‐ensiled (average: 4.635 Nm³ CH₄/ha) and ensiled miscanthus biomass (4.803 Nm³ CH₄/ha). A comparison of the four genotypes suggests that Miscanthus x giganteus is the most suitable genotype for ensiling as it had the best silage quality.     

Evapotranspiration,crop coefficient and water use efficiency of giant reed (Arundo donax L.) and miscanthus (Miscanthus × giganteus Greef et Deu.) in a Mediterranean environment.

Giant reed (Arundo donax L.) and miscanthus (Miscanthus × giganteus Greef et Deu.) are two perennial rhizomatous grasses (PRGs), considered as promising sources of lignocellulosic biomass for renewable energy production. Although the agronomic performance of these species has been addressed by several studies, the literature dedicated to the crop water use of giant reed and miscanthus is still limited. Our objective was thus to investigate giant reed and miscanthus water use by assessing crop evapotranspiration (ET_c), crop coefficients (K_c) and water use efficiency (WUE). The study was carried out in central Italy and specifically designed water-balance lysimeters were used to investigate the water use of these PRGs during the 2010 and 2011 growing seasons. Giant reed showed the highest cumulative evapotranspiration, with an average consumption of approximately 1100 mm, nearly 20% higher than miscanthus (900 mm). Crop evapotranspiration rates differed significantly between the species, particularly during the midseason (from June to September), when average daily ET_c was 7.4 and 6.2 mm in giant reed and miscanthus respectively. The K_c values determined in our study varied from 0.4 to 1.9 for giant reed and 0.3 to 1.6 for miscanthus. Finally, WUE was higher in miscanthus than in giant reed, with average values of 4.2 and 3.1 g L⁻¹ respectively. Further studies concerning water use under nonoptimal water conditions should be carried out and an assessment of the response to water stress of both crops is necessary to integrate the findings from this study.