Establishment and Early Growth of Willow at Different Levels of Weed Competition and Nitrogen Fertilization

To evaluate the effects of weed competition and nitrogen fertilization on the early growth performance of willow, cuttings of the clone Tora (Salix schwerinii x S. viminalis) were planted in buckets together with model weeds (spring barley or white mustard) sown 15, 26, and 30 days after willow planting. The buckets were fertilized with 30 or 90 kg N ha^?1. Willow with weeds sown after 15 days produced less biomass and smaller leaf area and had a lower maximum shoot height compared to willow planted without weeds and willow with weeds sown after 26 or 30 days. Fertilization with 90 kg N ha^?1 gave higher willow biomass production in willow with weeds sown after 26 or 30 days. Type of model weed had no effect on willow performance. Weed biomass and maximum shoot height were higher in weeds planted without willows compared to the willow-weed mixtures. A high nitrogen level gave more weed biomass when planted without willows and in the willow-weed mixture with weeds sown after 15 days. We conclude that for the given high density of willow, competition from weeds emerging soon after willow planting had strong effect on early production. Furthermore, if there is a risk of weed infestation, fertilization should be delayed.     

Simultaneous Saccharification and Fermentation of Hydrothermal Pretreated Lignocellulosic Biomass: Evaluation of Process Performance Under Multiple Stress Conditions

Industrial lignocellulosic bioethanol processes are exposed to different environmental stresses (such as inhibitor compounds, high temperature, and high solid loadings). In this study, a systematic approach was followed where the liquid and solid fractions were mixed to evaluate the influence of varied solid loadings, and different percentages of liquor were used as liquid fraction to determine inhibitor effect. Ethanol production by simultaneous saccharification and fermentation (SSF) of hydrothermally pretreated Eucalyptus globulus wood (EGW) was studied under combined diverse stress operating conditions (30–38 °C, 60–80 g of liquor from hydrothermal treatment or autohydrolysis (containing inhibitor compounds)/100 g of liquid and liquid to solid ratio between 4 and 6.4 g liquid in SSF/g unwashed pretreated EGW) using an industrial Saccharomyces cerevisiae strain supplemented with low-cost byproducts derived from agro-food industry. Evaluation of these variables revealed that the combination of temperature and higher solid loadings was the most significant variable affecting final ethanol concentration and cellulose to ethanol conversion, whereas solid and autohydrolysis liquor loadings had the most significant impact on ethanol productivity. After optimization, an ethanol concentration of 54 g/L (corresponding to 85 % of conversion and 0.51 g/Lh of productivity at 96 h) was obtained at 37 °C using 60 % of autohydrolysis liquor and 16 % solid loading (liquid to solid ratio of 6.4 g/g). The selection of a suitable strain along with nutritional supplementation enabled to produce noticeable ethanol titers in quite restrictive SSF operating conditions, which can reduce operating cost and boost the economic feasibility of lignocellulose-to-ethanol processes.     

Impact of Different Lignin Fractions on Saccharification Efficiency in Diverse Species of the Bioenergy Crop Miscanthus

Lignin is a key factor limiting saccharification of lignocellulosic feedstocks. In this comparative study, various lignin methods—including acetyl bromide lignin (ABL), acid detergent lignin (ADL), Klason lignin (KL), and modified ADL and KL determination methods—were evaluated for their potential to assess saccharification efficiency. Six diverse accessions of the bioenergy crop miscanthus were used for this analysis, which included accessions of Miscanthus sinensis, Miscanthus sacchariflorus, and hybrid species. Accessions showed large variation in lignin content. Lignin estimates were different between methods, but (highly) correlated to each other (0.54?≤?r?≤?0.94). The strength of negative correlations to saccharification efficiency following either alkaline or dilute acid pretreatment differed between lignin estimates. The strongest and most consistent correlations (?0.48?≤?r?≤??0.85) were obtained with a modified Klason lignin method. This method is suitable for high throughput analysis and was the most effective in detecting differences in lignin content (p?<?0.001) between accessions.     

Sequential Thermochemical Hydrolysis of Corncobs and Enzymatic Saccharification of the Whole Slurry Followed by Fermentation of Solubilized Sugars to Ethanol with the Ethanologenic Strain <Emphasis Type="Italic">Escherichia coli</Emphasis> MS04

Interest in the use of corncobs as feedstock for bioethanol production is growing. This study assesses the feasibility of sequential thermochemical diluted sulfuric acid pretreatment of corncobs at moderate temperature to hydrolyze the hemicellulosic fraction, followed by enzymatic hydrolysis of the whole slurry, and fermentation of the obtained syrup. The total sugar concentration after enzymatic hydrolysis was 85.21 g/l, i.e., 86 % of the sugars were liberated from the polymeric fractions, together with a low amount of furfural (0.26 g/l) and 4.01 g/l of acetic acid. The syrups, which contained 36.3, 40.9, 4.47, and 1.84 g/l of xylose, glucose, arabinose, and mannose, respectively, were fermented (pH 7, 37 °C, 150 rpm) to ethanol with the metabolically engineered acetate-tolerant Escherichia coli strain MS04 under non-aerated conditions, producing 35 g/l of ethanol in 18 h (1.94 g_EtOH/l/h), i.e., a conversion yield greater than 80 % of the theoretical value based on total sugars was obtained. Hence, using the procedures developed in this study, 288 l of ethanol can be produced per metric ton of dry corncobs. Strain MS04 can ferment sugars in the presence of acetate, and the amount of furans generated during the sequential thermochemical and enzymatic hydrolysis was low; hence, the detoxification step was avoided. The residual salts, acetic acid, and solubilized lignin present in the syrup did not interfere with the production of ethanol by E. coli MS04 and the results show that this strain can metabolize mixtures of glucose and xylose simultaneously.     

Ecosystem Services of Woody Crop Production Systems

Short-rotation woody crops are an integral component of regional and national energy portfolios, as well as providing essential ecosystem services such as biomass supplies, carbon sinks, clean water, and healthy soils. We review recent USDA Forest Service Research and Development efforts from the USDA Biomass Research Centers on the provisioning of these ecosystem services from woody crop production systems. For biomass, we highlight productivity and yield potential, pest susceptibility, and bioenergy siting applications. We describe carbon storage in aboveground woody biomass and studies assessing the provision of clean and plentiful water. Soil protection and wildlife habitat are also mentioned, in the context of converting lands from traditional row-crop agriculture to woody production systems.     

Effective and Low-Cost Saccharification of Pineapple Peel by <Emphasis Type="Italic">Trichoderma viride</Emphasis> Crude Extract with Enhanced β-Glucosidase Activity

Efficient, low-cost enzymatic hydrolysis of lignocellulosic biomass is essential for cost-effective production of bioethanol. The aim of this study was to establish a fungal fermentation-based strategy for the economic enzymatic conversion of pineapple peel into fermentable sugars. Trichoderma viride was grown on passion fruit peel in order to improve its β-glucosidase production, and a crude extract was then used to hydrolyze pineapple peel. The effects of medium pH, cultivation time, and passion fruit peel concentration on β-glucosidase production were evaluated using a central composite rotational design (CCRD) combined with response surface methodology (RSM). Optimal β-glucosidase activity of 2.40 U mL^?1 was found after 6.5 days of cultivation in medium at pH 6.0, containing 2.0 % passion fruit peel. Saccharification of pineapple peel was also optimized by RSM and CCRD with respect to pH, temperature, β-glucosidase concentration, and reaction time and proceeded optimally at pH 4.0, 55 °C, with a β-glucosidase loading of 31.25 U g^?1 dry feedstock and 75 h of reaction. Under these conditions, T. viride crude extract hydrolyzed pineapple peel with a glucose yield of 65.3 %. This study therefore presents passion fruit peel as an attractive raw material for the production of β-glucosidases. In addition, it describes an improved, effective, and low-cost enzymatic method for the production of fermentable sugars from pineapple peel, an abundant and inexpensive agro-industrial waste.     

Understanding the Physicochemical Characteristics and the Improved Enzymatic Saccharification of Corn Stover Pretreated with Aqueous and Gaseous Ammonia

Physicochemical characteristics of corn stover pretreated by soaking in aqueous ammonia (SAA) and low-moisture anhydrous ammonia (LMAA) were compared and investigated. The glucan digestibility of the treated biomass reached 90 % (SAA) and 84 % (LMAA). The LMAA pretreatment enhanced the digestibility by cleaving cross-linkages between cell wall components, whereas the SAA pretreatment additionally improved the digestibility by efficiently removing a major portion of the lignin under mild reaction conditions without significant loss of carbohydrates. Fourier transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR), and gel permeation chromatography (GPC) revealed the structural and chemical transformations of lignin during the pretreatments. Both pretreatments effectively cleaved ferulate cell wall cross-linking that is associated with the recalcitrance of grass lignocellulosics toward enzymatic saccharification. Extracted lignin from SAA pretreatment was extensively depolymerized but retained “native” character, as evidenced by the retention of β-ether linkages.     

Imaging Membrane Potential with Two Types of Genetically Encoded Fluorescent Voltage Sensors

Genetically encoded voltage indicators (GEVIs) have improved to the point where they are beginning to be useful for in vivo recordings. While the ultimate goal is to image neuronal activity in vivo, one must be able to image activity of a single cell to ensure successful in vivo preparations. This procedure will describe how to image membrane potential in a single cell to provide a foundation to eventually image in vivo. Here we describe methods for imaging GEVIs consisting of a voltage-sensing domain fused to either a single fluorescent protein (FP) or two fluorescent proteins capable of Förster resonance energy transfer (FRET) in vitro. Using an image splitter enables the projection of images created by two different wavelengths onto the same charge-coupled device (CCD) camera simultaneously. The image splitter positions a second filter cube in the light path. This second filter cube consists of a dichroic and two emission filters to separate the donor and acceptor fluorescent wavelengths depending on the FPs of the GEVI. This setup enables the simultaneous recording of both the acceptor and donor fluorescent partners while the membrane potential is manipulated via whole cell patch clamp configuration. When using a GEVI consisting of a single FP, the second filter cube can be removed allowing the mirrors in the image splitter to project a single image onto the CCD camera.