Improved Sugar Conversion and Ethanol Yield for Forage Sorghum (Sorghum bicolor L. Moench) Lines with Reduced Lignin Contents

Lignin is known to impede conversion of lignocellulose into ethanol. In this study, forage sorghum plants carrying brown midrib (bmr) mutations, which reduce lignin contents, were evaluated as bioenergy feedstocks. The near-isogenic lines evaluated were: wild type, bmr-6, bmr-12, and bmr-6 bmr-12 double mutant. The bmr-6 and bmr-12 mutations were equally efficient at reducing lignin contents (by 13% and 15%, respectively), and the effects were additive (27%) for the double mutant. Reducing lignin content was highly beneficial for improving biomass conversion yields. Sorghum biomass samples were pretreated with dilute acid and recovered solids washed and hydrolyzed with cellulase to liberate glucose. Glucose yields for the sorghum biomass were improved by 27%, 23%, and 34% for bmr-6, bmr-12, and the double mutant, respectively, compared to wild type. Sorghum biomass was also pretreated with dilute acid followed by co-treatment with cellulases and Saccharomyces cerevisiae for simultaneous saccharification and fermentation (SSF) into ethanol. Conversion of cellulose to ethanol for dilute-acid pretreated sorghum biomass was improved by 22%, 21%, and 43% for bmr-6, bmr-12, and the double mutant compared to wild type, respectively. Electron microscopy of dilute-acid treated samples showed an increased number of lignin globules in double-mutant tissues as compared to the wild-type, suggesting the lignin had become more pliable. The mutations were also effective for improving ethanol yields when the (degrained) sorghum was pretreated with dilute alkali instead of dilute acid. Following pretreatment with dilute ammonium hydroxide and SSF, ethanol conversion yields were 116 and 130 mg ethanol/g dry biomass for the double-mutant samples and 98 and 113 mg/g for the wild-type samples.     

Inter-Annual Precipitation Variability Decreases Switchgrass Productivity from Arid to Mesic Environments

Cellulosic biofuels are an important source of renewable biomass within the alternative energy portfolio. Switchgrass (Panicum virgatum L.), a perennial C₄ grass native to North America, is widely studied as a biofuel feedstock for its consistently high yields and minimal input requirements. The influences of precipitation amount and temporal variability on the fertilizer response of switchgrass productivity are not fully understood. Moreover, global climate models predict changes in rainfall patterns towards lower and increasingly variable soil water availability in several productive areas worldwide, which may impact net primary production of biofuel crops. We conducted a meta-analysis of aboveground net primary production of switchgrass from 48 publications encompassing 82 different locations, 11 soil types, 52 switchgrass cultivars, fertilizer inputs between 0 to 896 kg N ha^?1 year^?1, and 1 to 6 years of annual productivity measures repeated on the same stand. Productivity of the lowland ecotype doubled with N rates >?131 kg N ha^?1 year^?1, but upland ecotype productivity increased only by 50%. Results showed an optimum N rate of 30 to 60 kg N ha^?1 year^?1 for both ecotypes, after which biomass gain per unit of N added decreased. Growing season precipitation (GSPPT) and inter-annual precipitation variability (inter-PPTvar) affected both ecotypes similarly. Long-term mean annual precipitation (MAP) differentially affected lowland and upland productivity, depending on the N level. Productivity responses to MAP and GSPPT were similar for both upland and lowland ecotypes at none or low N rates. When N increased beyond 60 kg N ha^?1 year^?1, lowland cultivars had a greater growth response to MAP than uplands. Productivity increased with increasing GSPPT and MAP and had a positive linear response to MAP ranging from 600 to 1200 mm year^?1. One third of the variability in switchgrass production was accounted for by inter-PPTvar. After accounting for MAP, sites with higher inter-PPTvar had lower switchgrass productivity than sites with lower inter-PPTvar. Increased inter-annual variation in precipitation reduced production of both ecotypes. Predicted changes in the amount and timing of precipitation thus likely will exert greater influence on production of upland than lowland ecotypes of switchgrass.     

The Feasibility of Producing Adequate Feedstock for Year-Round Cellulosic Ethanol Production in an Intensive Agricultural Fuelshed

To date, cellulosic ethanol production has not been commercialized in the United States. However, government mandates aimed at increasing second-generation biofuel production could spur exploratory development in the cellulosic ethanol industry. We conducted an in-depth analysis of the fuelshed surrounding a starch-based ethanol plant near York, Nebraska that has the potential for cellulosic ethanol production. To assess the feasibility of supplying adequate biomass for year-round cellulosic ethanol production from residual maize (Zea mays) stover and bioenergy switchgrass (Panicum virgatum) within a 40-km road network service area of the existing ethanol plant, we identified ～14,000 ha of marginally productive cropland within the service area suitable for conversion from annual rowcrops to switchgrass and ～132,000 ha of maize-enrolled cropland from which maize stover could be collected. Annual maize stover and switchgrass biomass supplies within the 40-km service area could range between 429,000 and 752,000 metric tons (mT). Approximately 140–250 million liters (l) of cellulosic ethanol could be produced, rivaling the current 208 million l annual starch-based ethanol production capacity of the plant. We conclude that sufficient quantities of biomass could be produced from maize stover and switchgrass near the plant to support year-round cellulosic ethanol production at current feedstock yields, sustainable removal rates and bioconversion efficiencies. Modifying existing starch-based ethanol plants in intensive agricultural fuelsheds could increase ethanol output, return marginally productive cropland to perennial vegetation, and remove maize stover from productive cropland to meet feedstock demand.     

Euglena sp. as a suitable source of lipids for potential use as biofuel and sustainable wastewater treatment

Prolific algal growth in sewage ponds with high organic loads in the tropical regions can provide cost-effective and efficient wastewater treatment and biofuel production. This work examines the ability of Euglena sp. growing in wastewater ponds for biofuel production and treatment of wastewater. The algae were isolated from the sewage treatment plants and were tested for their nutrient removal capability. Compared to other algae, Euglena sp. showed faster growth rates with high biomass density at elevated concentrations of ammonium nitrogen (NH₄-N) and organic carbon (C). Profuse growth of these species was observed in untreated wastewaters with a mean specific growth rate (μ) of 0.28 day^?1 and biomass productivities of 132 mg ?L^?1? day^?1. The algae cultured within a short period of 8 days resulted in the 98 % removal of NH₄-N, 93 % of total nitrogen 85 % of ortho-phosphate, 66 % of total phosphate and 92 % total organic carbon. Euglenoids achieved a maximum lipid content of 24.6 % (w/w) with a biomass density of 1.24 g ?L^?1 (dry wt.). Fourier transform infrared spectra showed clear transitions in biochemical compositions with increased lipid/protein ratio at the end of the culture. Gas chromatography and mass spectrometry indicated the presence of high contents of palmitic, linolenic and linoleic acids (46, 23 and 22 %, respectively), adding to the biodiesel quality. Good lipid content (comprised quality fatty acids), efficient nutrient uptake and profuse biomass productivity make the Euglena sp. as a viable source for biofuel production in wastewaters.     

Switchgrass Biomass and Nitrogen Yield with Over-Seeded Cool-season Forages in the Southern Great Plains

In dry climates with long, hot summers and freezing winters, such as that of the southern Great Plains of North America, switchgrass (Panicum virgatum L.) has proven potential as a cellulosic bioenergy feedstock. This trial looked at dry matter (DM) and N yield dynamics of switchgrass overseeded with cool-season legumes and rye (Secale cereale L.), compared to switchgrass fertilized with 0, 56 and 112 kg N ha^-1 yr^-1 at an infertile and a fertile location. Optimal N fertilizer rate on switchgrass was 56 kg N ha^-1 at the infertile location. Legume yield was greater in the first season after planting, compared to subsequent years where annual legumes were allowed to reseed and alfalfa (Medicago sativa L.) was allowed to grow. This suggests that the reseeding model for annual legumes will not work in switchgrass swards grown for biomass unless soil seed banks are built up for more than one year, and that overseeding with alfalfa may have to be repeated in subsequent years to build up plant populations. Overseeding rye and legumes generally did not suppress or enhance switchgrass biomass production compared to unfertilized switchgrass. However, cumulative spring and fall biomass yields were generally greater due to winter and spring legume production, which could be beneficial for grazing or soil conservation systems, but not necessarily for once-yearly late autumn harvest biofuel production systems.     

Hydrological responses of land use change from cotton (Gossypium hirsutum L.) to cellulosic bioenergy crops in the Southern High Plains of Texas,USA

The Southern High Plains (SHP) region of Texas in the United States, where cotton is grown in a vast acreage, has the potential to grow cellulosic bioenergy crops such as perennial grasses and biomass sorghum (Sorghum bicolor). Evaluation of hydrological responses and biofuel production potential of hypothetical land use change from cotton (Gossypium hirsutum L.) to cellulosic bioenergy crops enables better understanding of the associated key agroecosystem processes and provides for the feasibility assessment of the targeted land use change in the SHP. The Soil and Water Assessment Tool (SWAT) was used to assess the impacts of replacing cotton with perennial Alamo switchgrass (Panicum virgatum L.), Miscanthus × giganteus (Miscanthus sinensis Anderss. [Poaceae]), big bluestem (Andropogon gerardii) and annual biomass sorghum on water balances, water use efficiency and biofuel production potential in the Double Mountain Fork Brazos watershed. Under perennial grass scenarios, the average (1994–2009) annual surface runoff from the entire watershed decreased by 6–8% relative to the baseline cotton scenario. In contrast, surface runoff increased by about 5% under the biomass sorghum scenario. Perennial grass land use change scenarios suggested an increase in average annual percolation within a range of 3–22% and maintenance of a higher soil water content during August to April compared to the baseline cotton scenario. About 19.1, 11.1, 3.2 and 8.8 Mg ha^?1 of biomass could potentially be produced if cotton area in the watershed would hypothetically be replaced by Miscanthus, switchgrass, big bluestem and biomass sorghum, respectively. Finally, Miscanthus and switchgrass were found to be ideal bioenergy crops for the dryland and irrigated systems, respectively, in the study watershed due to their higher water use efficiency, better water conservation effects, greater biomass and biofuel production potential, and minimum crop management requirements.     

Allelopathic potential of sweet potato (Ipomoea batatas) germplasm resources of Yunnan Province in southwest China

A laboratory bioassay was conducted to determine the allelopathic potentials of aqueous extracts from either roots or leaves of seventeen sweet potato [Ipomoea batatas L. (Lam)] cultivars (SP0, SP1, SP2, SP3, SP4, SP5, SP6, SP7, SP9, SP10, SP11, SP13, SP14, SP15, SP16, SP18, and SP19). Most inhibitory rates on Lactuca sativa calculated for leaf or root extracts from the seventeen sweet potato cultivars exhibited positive values and significantly increased with increasing concentration. Germination was totally inhibited at a concentration of 0.05 g·mL^?1 for leaf water extracts of SP13, SP15, SP18 and at a concentration of 0.05 g·mL^?1 for both leaf and root water extracts of SP19. Inhibition of root length was clearly greater than inhibition of shoot length for both leaf and root water extracts. Biomass inhibition increased with increasing concentration, but some cultivars showed stimulatory effects at low concentrations, and inhibition was generally more pronounced for root water extracts than for leaf water extracts. Moreover, most synthetical inhibitory rates for both leaf and root water extracts from the seventeen cultivars exhibited positive values and significantly increased with increasing concentration. Comparing the synthetical inhibitory rates for both leaf and root water extracts among the seventeen cultivars, SP19, SP6, SP11, and SP7 had the highest allelopathic inhibition. The inhibitory activity on germination index was the greatest, followed by germination rate, root length, biomass, and shoot length in all bioassays. Inhibition by leaf water extracts was generally greater than inhibition by root water extracts, except in the case of shoot length or biomass. Overall, we conclude that all seventeen sweet potato cultivars have strong inhibitory effects on L. sativa, but that these effects vary with cultivar and plant part, with SP19, SP6, SP11, and SP7 exhibiting the highest rates of allelopathic inhibition.     

Interspecific biodiversity enhances biomass and lipid productivity of microalgae as biofuel feedstock

Large improvements in biomass and lipid production are required to make massive scale algal biodiesel production an economic reality. The application of the biodiversity strategy to enhance algal biomass as biofuel feedstock is little. The algal diversity was manipulated in this study to investigate the effects of a combination of biodiversity complementarity and a new medium consisting of seawater and agricultural fertilizer on lipid productivity. The algae diverse community includes two strains of Dunaliella salina (Dunaliella salina 19/30 and 19/18) and three species of Nannochloropsis (Nannochloropsis oculata, Nannochloropsis salina, and Nannochloropsis gaditana). The results showed that the most diverse community (5 species) produced an average of sixfold more biomass in the new medium than did the standard f/2 culture medium. The most diverse polyculture had the highest growth rate (1.01 day^?1), biomass (1.2 g L^?1), and lipid productivity (0.31 g L^?1 day^?1). The assessment of algal polycultures relative to monocultures is particularly interesting and novel for this biofuel field, and the observations that these polycultures resulted in significant lipid productivity improvements are very useful addition to the biofuel research. The possible mechanism (resource diversity) to explain the synergy in mixed cultures warrants further investigation.     

Carbon and nitrogen dynamics in bioenergy ecosystems: 1. Model development,validation and sensitivity analysis

Biofuel made from conventional (e.g., maize (Zea mays L.)) and cellulosic crops (e.g., switchgrass (Panicum virgatum L.) and Miscanthus (Miscanthus × giganteus)) provides alternative energy to fossil fuels and has been considered to mitigate greenhouse gas emissions. To estimate the large‐scale carbon and nitrogen dynamics of these biofuel ecosystems, process‐based models are needed. Here, we developed an agroecosystem model (AgTEM) based on the Terrestrial Ecosystem Model for these ecosystems. The model was incorporated with biogeochemical and ecophysiological processes including crop phenology, biomass allocation, nitrification, and denitrification, as well as agronomic management of irrigation and fertilization. It was used to estimate crop yield, biomass, net carbon exchange, and nitrous oxide emissions at an ecosystem level. The model was first parameterized for maize, switchgrass, and Miscanthus ecosystems and then validated with field observation data. We found that AgTEM well reproduces the annual net primary production and nitrous oxide fluxes of most sites, with over 85% of total variation explained by the model. Local sensitivity analysis indicated that the model sensitivity varies among different ecosystems. Net primary production of maize is sensitive to temperature, precipitation, cloudiness, fertilizer, and irrigation and less sensitive to atmospheric CO₂ concentrations. In contrast, the net primary production of switchgrass and Miscanthus is most sensitive to temperature among all factors. Nitrous oxide fluxes are sensitive to management in maize ecosystems, and sensitive to climate factors in cellulosic ecosystems. The developed model should help advance our understanding of carbon and nitrogen dynamics of these biofuel ecosystems at both site and regional levels.     

Grain zinc, iron and protein concentrations and zinc-efficiency in wild emmer wheat under contrasting irrigation regimes

Micronutrient malnutrition, and particularly deficiency in zinc (Zn) and iron (Fe), afflicts over three billion people worldwide, and nearly half of the world’s cereal-growing area is affected by soil Zn deficiency. Wild emmer wheat [Triticum turgidum ssp. dicoccoides (Körn.) Thell.], the progenitor of domesticated durum wheat and bread wheat, offers a valuable source of economically important genetic diversity including grain mineral concentrations. Twenty two wild emmer wheat accessions, representing a wide range of drought resistance capacity, as well as two durum wheat cultivars were examined under two contrasting irrigation regimes (well-watered control and water-limited), for grain yield, total biomass production and grain Zn, Fe and protein concentrations. The wild emmer accessions exhibited high genetic diversity for yield and grain Zn, Fe and protein concentrations under both irrigation regimes, with a considerable potential for improvement of the cultivated wheat. Grain Zn, Fe and protein concentrations were positively correlated with one another. Although irrigation regime significantly affected ranking of genotypes, a few wild emmer accessions were identified for their advantage over durum wheat, having consistently higher grain Zn (e.g., 125 mg kg^?1), Fe (85 mg kg^?1) and protein (250 g kg^?1) concentrations and high yield capacity. Plants grown from seeds originated from both irrigation regimes were also examined for Zn efficiency (Zn deficiency tolerance) on a Zn-deficient calcareous soil. Zinc efficiency, expressed as the ratio of shoot dry matter production under Zn deficiency to Zn fertilization, showed large genetic variation among the genotypes tested. The source of seeds from maternal plants grown under both irrigation regimes had very little effect on Zn efficiency. Several wild emmer accessions revealed combination of high Zn efficiency and drought stress resistance. The results indicate high genetic potential of wild emmer wheat to improve grain Zn, Fe and protein concentrations, Zn deficiency tolerance and drought resistance in cultivated wheat.     

Offsetting high water demands with high productivity: Sorghum as a biofuel crop in a high irradiance arid ecosystem

High irradiance arid environments are promising, yet understudied, areas for biofuel production. We investigated the productivity and environmental trade‐offs of growing sorghum (Sorghum bicolor) as a biofuel feedstock in the low deserts of California (CA). Using a 5.3 ha experimental field in the Imperial Valley, CA, we measured aboveground biomass production and net ecosystem exchange of CO₂ and H₂O via eddy covariance over three growing periods between February and November 2012. Environmental conditions were extreme, with high irradiance, vapor pressure deficit (VPD), and air temperature throughout the growing season. Air temperature peaked in August with a maximum of 45.7 °C. Sorghum produced an annual aboveground biomass yield of 43.7 Mg per hectare. Net ecosystem exchange (NEE) was highest during the summer growth period and reached a maximum of ?68 μmol CO₂ m^?2 s^?1. Water use efficiency, or biomass water ratio (BWR), was high (4.0 g dry biomass kg^?1 H₂O) despite high seasonal evapotranspiration (1094 kg H₂O m^?2). The BWR of sorghum surpassed that of many C4 biofuel candidate crops in the United States, as well as that of alfalfa which is currently widely grown in the Imperial Valley. Sorghum also outperformed many US biofuel crops in terms of radiation use efficiency (RUE), achieving 1.5 g dry biomass MJ^?1. We found no evidence of saturation of NEE at high levels of photosynthetically active radiation (PAR) (up to 2250 μmol m^?2 s^?1). In addition, we found no evidence that NEE was inhibited by either high VPD or air temperature during peak photosynthetic phases. The combination of high productivity, high BWR, and high RUE suggests that sorghum is well adapted to this extreme environment. The biomass production rates and efficiency metrics spanning three growing periods provide fundamental data for future Life Cycle Assessments (LCA), which are needed to assess the sustainability of this sorghum biofuel feedstock system.     

Potential for Production of Perennial Biofuel Feedstocks in Conservation Buffers on the Coastal Plain of Georgia,USA

With global increases in the production of cellulosic biomass for fuel, or “biofuel,” concerns over potential negative effects of using land for biofuel production have promoted attention to concepts of agricultural landscape design that sustainably balance tradeoffs between food, fuel, fiber, and conservation. The Energy Independence Security Act (EISA) of 2007 mandates an increase in advanced biofuels to 21 billion gallons in 2022. The southeastern region of the USA has been identified as a contributor to meeting half of this goal. We used a GIS-based approach to estimate the production and N-removal potential of three perennial biofeedstocks planted as conservation buffers (field borders associated with riparian buffers, and grassed waterways) on the Coastal Plain of Georgia, USA. Land cover, hydrology, elevation, and soils data were used to identify locations within agricultural landscapes that are most susceptible to runoff, erosion, and nutrient loss. We estimated potential annual biomass production from these areas to be: 2.5–3.5 Tg for giant miscanthus (Miscanthus?×?giganteus), 2–8.6 Tg for “Merkeron” napier grass (Pennisetum purpureum), and 1.9–7.5 Tg for “Alamo” switchgrass (Panicum virgatum). When production strategies were taken into consideration, we estimated total biomass yield of perennial grasses for the Georgia Coastal Plain at 2.2–9.4 Tg year^?1. Using published rates of N removal and ethanol conversion, we calculated the amount of potential N removal by these systems as 8100–51,000 Mg year^?1 and ethanol fuel production as 778–3296 Ml year^?1 (206 to 871 million gal. US).     

Grass Invasion into Switchgrass Managed for Biomass Energy

Switchgrass (Panicum virgatum) is a C₄ perennial grass and is the model herbaceous perennial bioenergy feedstock. Although it is indigenous to North American grasslands east of the Rocky Mountains and has been planted for forage and conservation purposes for more than 75 years, there is concern that switchgrass grown as a biofuel crop could become invasive. Our objective is to report on the invasion of C₄ and C₃ grasses into the stands of two switchgrass cultivars following 10 years of management for biomass energy under different N and harvest management regimes in eastern Nebraska. Switchgrass stands were invaded by big bluestem (Andropogon gerardii), smooth bromegrass (Bromus inermis), and other grasses during the 10 years. The greatest invasion by grasses occurred in plots to which 0 N had been applied and with harvests at anthesis. In general, less grass encroachment occurred in plots receiving at least 60 kg of N ha^?1 or in plots harvested after frost. There were differences among cultivars with Cave-in-Rock being more resistant to invasion than Trailblazer. There was no observable evidence of switchgrass from this study invading into border areas or adjacent fields after 10 years of management for biomass energy. Results indicate that switchgrass is more likely to be invaded by other grasses than to encroach into native prairies or perennial grasslands seeded on marginally productive cropland in the western Corn Belt of the USA.     

Variation due to Growth Environment in Alfalfa Yield, Cellulosic Ethanol Traits, and Paper Pulp Characteristics

Alfalfa (Medicago sativa L.) is a promising bioenergy and bioproduct feedstock because of its high yield, N-fixation capacity, potential for planting in rotation with corn (Zea mays L.), and valuable protein co-product (leaf meal). Our objective was to examine the effect of growth environment on biomass yield, cellulosic ethanol traits, and paper pulp fiber characteristics of alfalfa stems. Landscape position (summit and mild slope), season of harvest (four harvests per season), and multiple years (2005 and 2006) provided environmental variation. Alfalfa stem samples were analyzed for cell wall carbohydrate and lignin concentration. Stems were subjected to dilute acid pre-treatment, enzymatic saccharification, and pulping processes to measure relevant cellulosic ethanol and paper production traits. Landscape position was not a significant source of variation for yield or any biomass quality trait. Yields varied among harvests in 2005 (1,410–3,265 kg ha^?1) and 2006 (1,610–3,795 kg ha^?1). All cell wall, conversion test, and paper production traits exhibited year by harvest interactions with no clear pattern. Total carbohydrates and lignin ranged from 440 to 531 g?kg^?1 DM and from 113 to 161 g?kg^-1 DM, respectively. Release of cell wall sugars by the conversion test ranged widely (419 to 962 g?kg^?1 DM). Fiber traits were similarly variable with length and fine content ranging from 1.24 to 1.59 mm and from 15.2% to 21.9%, respectively. Utilizing alfalfa biomass for cellulosic ethanol and paper pulp production will involve dealing with significant feedstock quality variation due to growth environment.     

Seasonal variation in light utilisation,biomass production and nutrient removal by wastewater microalgae in a full-scale high-rate algal pond

There has been renewed interest in the combined use of high-rate algal ponds (HRAP) for wastewater treatment and biofuel production. Successful wastewater treatment requires year-round efficient nutrient removal while high microalgal biomass yields are required to make biofuel production cost-effective. This paper investigates the year-round performance of microalgae in a 5-ha demonstration HRAP system treating primary settled wastewater in Christchurch, New Zealand. Microalgal performance was measured in terms of biomass production, nutrient removal efficiency, light absorption and photosynthetic potential on seasonal timescales. Retention time-corrected microalgal biomass (chlorophyll a) varied seasonally, being lowest in autumn and winter (287 and 364 mg m^?3day^?1, respectively) and highest in summer (703 mg m^?3day^?1), while the conversion efficiency of light to biomass was greatest in winter (0.39 mg Chl- a per μmol) and lowest in early summer (0.08 mg Chl- a per μmol). The percentage of ammonium (NH₄–N) removed was highest in spring (79 %) and summer (77 %) and lowest in autumn (47 %) and winter (53 %), while the efficiency of NH₄–N removal per unit biomass was highest in autumn and summer and lowest in winter and spring. Chlorophyll-specific light absorption per unit biomass decreased as total chlorophyll increased, partially due to the package effect, particularly in summer. The proportional increase in the maximum electron transport rate from winter to summer was significantly lower than the proportional increase in the mean light intensity of the water column. We concluded that microalgal growth and nutrient assimilation was constrained in spring and summer and carbon limitation may be the likely cause.     

Non-destructive hydrocarbon extraction from Botryococcus braunii BOT-22 (race B)

There is worldwide interest in developing algal biofuel. One main reason for the lack of success so far in producing a sustainable transport fuel from microalgae is the high cost of biomass processing, especially dewatering and oil extraction. There is also a significant cost involved in the energy content of the nutrient fertilisers required for biomass production. Non-destructive oil extraction or “milking” from algae biomass has the potential to bypass all of these hurdles. Using a “milking” strategy means that there would be no need for (a) biomass dewatering, (b) breaking cells for oil extraction and (c) addition of nutrients to the culture, resulting in a significant reduction in energy and fertiliser cost involved in production of biofuel from algae. We make use of the natural tendency of Botryococcus to produce external hydrocarbon in the extracellular matrix. In current study, we showed that external hydrocarbon from Botryococcus braunii BOT-22 can be non-destructively extracted using n-heptane (optimum contact time with n-heptane?=?20 min). We were able to recover almost the entire de novo-produced external hydrocarbons at 5- and 11-day intervals when the culture was maintained with or without 1 % CO₂ addition, respectively. This repeated non-destructive extraction of external hydrocarbon of B. braunii was possible for up to 70 days when 1 % CO₂ was supplied to the culture. When CO₂ was limited, a 70 % lower external hydrocarbon productivity was achieved using the same process. Although the productivity of external hydrocarbon of 9.33 mg L^?1 day^?1 of the “milked” culture is low in these un-optimised cultures, it was 1.3?±?0.2-fold higher compared with that of a conventional semicontinuous culture, showing the potential of this method.     

Biomass and Lipid Production of Dinoflagellates and Raphidophytes in Indoor and Outdoor Photobioreactors

The principal fatty acids from the lipid profiles of two autochthonous dinoflagellates (Alexandrium minutum and Karlodinium veneficum) and one raphidophyte (Heterosigma akashiwo) maintained in bubble column photobioreactors under outdoor culture conditions are described for the first time. The biomass production, lipid content and lipid productivity of these three species were determined and the results compared to those obtained when the strains were cultured indoors. Under the latter condition, the biotic values did not significantly differ among species, whereas under outdoor conditions, differences in both duplication time and fatty acids content were observed. Specifically, A. minutum had higher biomass productivity (0.35 g·L^?1 day^?1), lipid productivity (80.7 mg lipid·L^?1 day^?1) and lipid concentration (252 mg lipid·L^?1) at harvest time (stationary phase) in outdoor conditions. In all three strains, the growth rate and physiological response to the light and temperature fluctuations of outdoor conditions greatly impacted the production parameters. Nonetheless, the species could be successfully grown in an outdoor photobioreactor and were of sufficient robustness to enable the establishment of long-term cultures yielding consistent biomass and lipid production.     

Infestation Rates and Tiller Morphology Effects by the Switchgrass Moth on Six Cultivars of Switchgrass

Switchgrass (Panicum virgatum L.) is a potential biomass crop for native species-based biofuel systems in North America. A recently identified pest of switchgrass, the switchgrass moth, Blastobasis repartella (Dietz) (Lepidoptera: Coleophoridae), feeds in the basal above-ground internodes and below-ground in the proaxis and rhizomes, causing premature tiller and rhizome loss. Our goal was to determine genetic and temporal variation among six upland cultivars for frequency of tiller infestation by larvae of the switchgrass moth in mature stands in the northern Great Plains and if variation in biomass production was associated with variation in frequency of infestation. Data were collected in 2011 and 2012 for tiller density, biomass, frequency of infestation, number of leaves per healthy and infested tiller, and weights of healthy and infested tillers. Differences were found among cultivars for tiller density, biomass yield, and numbers of leaves per healthy and infested tillers. ‘Summer’, ‘Sunburst’, ‘Pathfinder’, and ‘Cave-In-Rock’ were the highest yielding cultivars. Mean frequency of infestation was different between 2011 (6.7 %) and 2012 (9.6 %). Infested tillers had one less collared leaf than healthy tillers. The weights of healthy tillers were ca. 3× those of infested tillers in both years, suggesting an impact on biomass accumulation and economic value. Levels of infestation were similar for all six cultivars, indicating no feeding preference by the switchgrass moth larva among genetically diverse cultivars of switchgrass. Regression of biomass yield on frequency of infestation showed negative linear relationships for ‘Carthage’ and ‘Kentucky 1625’.     

Switchgrass Biofuel Production on Reclaimed Surface Mines: II. Feedstock Quality and Theoretical Ethanol Production

Switchgrass (Panicum virgatum L.), a warm-season perennial grass, is an important bioenergy crop candidate because it produces high biomass yields on marginal lands and on reclaimed surface mined sites. In companion studies, dry matter (DM) yields for Cave-in-Rock, Shawnee, and Carthage cultivars varied from 4.2 to 13.0 Mg ha^?1averaged over 6 years at the reclaimed Hampshire site, and fertilization increased yields of Cave-in-Rock at Black Castle and Coal Mac sites from 0.3 to 2 Mg ha^?1 during the first 3 years. The objective of these experiments was to compare the impacts of cultivar and soil amendments on biomass quality and theoretical ethanol production of switchgrass grown on surface mines with differing soil characteristics. Biomass quality was determined for fiber, ash, lignin, digestibility, and carbohydrate contents via near-infrared reflectance spectroscopy, and carbohydrates were used to calculate theoretical ethanol yield (TEY; L Mg^?1) and multiplied by biomass yield to calculate theoretical ethanol production (TEP; L ha^?1). Cultivars at the Hampshire site did not differ in TEY and ranged from 426 to 457 L Mg^?1. Theoretical ethanol production from Cave-in-Rock at Hampshire was 7350 L ha^?1, which was higher than other cultivars because of its greater biomass production. This TEP was higher than in other studies which predicted 4000 to 5000 L ha^?1. At the Black Castle and Coal Mac sites, fertilizer applications slightly affected biomass quality of switchgrass and TEY, but provided greater TEP as a function of increased yield. Similar to other findings, total switchgrass biomass production has more impact than compositional differences on TEP, so maximizing biomass production is critical for maximizing potential biofuel production. With appropriate soil substrates, fertilization, planning, and management, large areas of reclaimed surface mines can be converted to switchgrass stands to produce high biomass quality and yields to support a bioethanol industry.     

A holistic high-throughput screening framework for biofuel feedstock assessment that characterises variations in soluble sugars and cell wall composition in <Emphasis Type="Italic">Sorghum bicolor</Emphasis>

Background

A major hindrance to the development of high yielding biofuel feedstocks is the ability to rapidly assess large populations for fermentable sugar yields. Whilst recent advances have outlined methods for the rapid assessment of biomass saccharification efficiency, none take into account the total biomass, or the soluble sugar fraction of the plant. Here we present a holistic high-throughput methodology for assessing sweet Sorghum bicolor feedstocks at 10 days post-anthesis for total fermentable sugar yields including stalk biomass, soluble sugar concentrations, and cell wall saccharification efficiency.

Results

A mathematical method for assessing whole S. bicolor stalks using the fourth internode from the base of the plant proved to be an effective high-throughput strategy for assessing stalk biomass, soluble sugar concentrations, and cell wall composition and allowed calculation of total stalk fermentable sugars. A high-throughput method for measuring soluble sucrose, glucose, and fructose using partial least squares (PLS) modelling of juice Fourier transform infrared (FTIR) spectra was developed. The PLS prediction was shown to be highly accurate with each sugar attaining a coefficient of determination (R ² ) of 0.99 with a root mean squared error of prediction (RMSEP) of 11.93, 5.52, and 3.23 mM for sucrose, glucose, and fructose, respectively, which constitutes an error of <4% in each case. The sugar PLS model correlated well with gas chromatography–mass spectrometry (GC-MS) and brix measures. Similarly, a high-throughput method for predicting enzymatic cell wall digestibility using PLS modelling of FTIR spectra obtained from S. bicolor bagasse was developed. The PLS prediction was shown to be accurate with an R ² of 0.94 and RMSEP of 0.64 μg.mgDW^-1.h^-1.

Conclusions

This methodology has been demonstrated as an efficient and effective way to screen large biofuel feedstock populations for biomass, soluble sugar concentrations, and cell wall digestibility simultaneously allowing a total fermentable yield calculation. It unifies and simplifies previous screening methodologies to produce a holistic assessment of biofuel feedstock potential.