Bioconversion of Pelletized Big Bluestem, Switchgrass, and Low-Diversity Grass Mixtures Into Sugars and Bioethanol

Three crops of warm-season grasses are being developed for biomass production on northern rain-fed marginal farmland: big bluestem (BBS), switchgrass (SG), and a low diversity mixture of grasses (LDM). In this study, biomass harvested from established fields were compared for pelletization and subsequent conversion to sugars and ethanol. Each biomass was successfully pelletized to similar bulk densities without adding a binder at a commercial feed operation. Pelletizing increased the bulk density by 407% on average and was equally effective on all three biomass samples (528–554 kg/m3). Chemical analysis of the samples indicated that glucan and xylan contents were slightly reduced during pelletizing (by 23 and 16 g/kg, respectively), as well as theoretical ethanol yields, which are based upon total carbohydrate contents. Pellets and milled straws were pre-treated with either liquid hot-water or low-moisture ammonium hydroxide (LMA) and subsequently hydrolyzed with cellulases. Glucose and total sugar yields were similar for non-pellets and pellets using either pre-treatment; carbohydrates present in pellets were more efficiently recovered compared to non-pellets. LMA pretreated samples were separately hydrolyzed and fermented to ethanol using Scheffersomyces stipitis yeast. Hydrolysis recovered 69.7–76.8% of the glucose and 66.5–73.3% of the xylose across all samples. Glucose yields were 251–279 g/kg, db and were significantly lower for SG as compared to the other biomass samples. Recovered sugars were fermented to ethanol at 77.7–86.7% of theoretical yield. Final ethanol yields (245.9–275.5 L/Mg, db) were similar for all of the grasses and estimated to equate to production levels for BBS, LDM, and SG of 1,952, 2,586, and 2,636 l of ethanol per ha, respectively

Bioconversion of Pelletized Big Bluestem, Switchgrass, and Low-Diversity Grass Mixtures Into Sugars and Bioethanol

Three crops of warm-season grasses are being developed for biomass production on northern rain-fed marginal farmland: big bluestem (BBS), switchgrass (SG), and a low diversity mixture of grasses (LDM). In this study, biomass harvested from established fields were compared for pelletization and subsequent conversion to sugars and ethanol. Each biomass was successfully pelletized to similar bulk densities without adding a binder at a commercial feed operation. Pelletizing increased the bulk density by 407% on average and was equally effective on all three biomass samples (528–554 kg/m3). Chemical analysis of the samples indicated that glucan and xylan contents were slightly reduced during pelletizing (by 23 and 16 g/kg, respectively), as well as theoretical ethanol yields, which are based upon total carbohydrate contents. Pellets and milled straws were pre-treated with either liquid hot-water or low-moisture ammonium hydroxide (LMA) and subsequently hydrolyzed with cellulases. Glucose and total sugar yields were similar for non-pellets and pellets using either pre-treatment; carbohydrates present in pellets were more efficiently recovered compared to non-pellets. LMA pretreated samples were separately hydrolyzed and fermented to ethanol using Scheffersomyces stipitis yeast. Hydrolysis recovered 69.7–76.8% of the glucose and 66.5–73.3% of the xylose across all samples. Glucose yields were 251–279 g/kg, db and were significantly lower for SG as compared to the other biomass samples. Recovered sugars were fermented to ethanol at 77.7–86.7% of theoretical yield. Final ethanol yields (245.9–275.5 L/Mg, db) were similar for all of the grasses and estimated to equate to production levels for BBS, LDM, and SG of 1,952, 2,586, and 2,636 l of ethanol per ha, respectively

Bioconversion of Pelletized Big Bluestem, Switchgrass, and Low-Diversity Grass Mixtures Into Sugars and Bioethanol

Three crops of warm-season grasses are being developed for biomass production on northern rain-fed marginal farmland: big bluestem (BBS), switchgrass (SG), and a low diversity mixture of grasses (LDM). In this study, biomass harvested from established fields were compared for pelletization and subsequent conversion to sugars and ethanol. Each biomass was successfully pelletized to similar bulk densities without adding a binder at a commercial feed operation. Pelletizing increased the bulk density by 407% on average and was equally effective on all three biomass samples (528–554 kg/m3). Chemical analysis of the samples indicated that glucan and xylan contents were slightly reduced during pelletizing (by 23 and 16 g/kg, respectively), as well as theoretical ethanol yields, which are based upon total carbohydrate contents. Pellets and milled straws were pre-treated with either liquid hot-water or low-moisture ammonium hydroxide (LMA) and subsequently hydrolyzed with cellulases. Glucose and total sugar yields were similar for non-pellets and pellets using either pre-treatment; carbohydrates present in pellets were more efficiently recovered compared to non-pellets. LMA pretreated samples were separately hydrolyzed and fermented to ethanol using Scheffersomyces stipitis yeast. Hydrolysis recovered 69.7–76.8% of the glucose and 66.5–73.3% of the xylose across all samples. Glucose yields were 251–279 g/kg, db and were significantly lower for SG as compared to the other biomass samples. Recovered sugars were fermented to ethanol at 77.7–86.7% of theoretical yield. Final ethanol yields (245.9–275.5 L/Mg, db) were similar for all of the grasses and estimated to equate to production levels for BBS, LDM, and SG of 1,952, 2,586, and 2,636 l of ethanol per ha, respectively

Random UV-C mutagenesis of Scheffersomyces (formerly Pichia) stipitis NRRL Y-7124 to improve anaerobic growth on lignocellulosic sugars

Scheffersomyces (formerly Pichia) stipitis NRRL Y-7124 was mutagenized using UV-C irradiation to produce yeast strains for anaerobic conversion of lignocellulosic sugars to ethanol. UV-C irradiation potentially produces large numbers of random mutations broadly and uniformly over the whole genome to generate unique strains. Wild-type cultures of S. stipitis NRRL Y-7124 were subjected to UV-C (234 nm) irradiation targeted at approximately 40% cell survival. When surviving cells were selected in sufficient numbers via automated plating strategies and cultured anaerobically on xylose medium for 5 months at 28°C, five novel mutagenized S. stipitis strains were obtained. Variable number tandem repeat analysis revealed that mutations had occurred in the genome, which may have produced genes that allowed the anaerobic utilization of xylose. The mutagenized strains were capable of growing anaerobically on xylose/glucose substrate with higher ethanol production during 250- to 500-h growth than a Saccharomyces cerevisiae yeast strain that is the standard for industrial fuel ethanol production. The S. stipitis strains resulting from this intense multigene mutagenesis strategy have potential application in industrial fuel ethanol production from lignocellulosic hydrolysates

Simple, Rapid and Cost-Effective Method for High Quality Nucleic Acids Extraction from Different Strains of Botryococcus braunii

This study deals with an effective nucleic acids extraction method from various strains of Botryococcus braunii which possesses an extensive extracellular matrix. A method combining freeze/thaw and bead-beating with heterogeneous diameter of silica/zirconia beads was optimized to isolate DNA and RNA from microalgae, especially from B. braunii. Eukaryotic Microalgal Nucleic Acids Extraction (EMNE) method developed in this study showed at least 300 times higher DNA yield in all strains of B. braunii with high integrity and 50 times reduced working volume compared to commercially available DNA extraction kits. High quality RNA was also extracted using this method and more than two times the yield compared to existing methods. Real-time experiments confirmed the quality and quantity of the input DNA and RNA extracted using EMNE method. The method was also applied to other eukaryotic microalgae, such as diatoms, Chlamydomonas sp., Chlorella sp., and Scenedesmus sp. resulting in higher efficiencies. Cost-effectiveness analysis of DNA extraction by various methods revealed that EMNE method was superior to commercial kits and other reported methods by >15%. This method would immensely contribute to area of microalgal genomics

The influence of initial xylose concentration, agitation, and aeration on ethanol production by Pichia stipitis from rice straw hemicellulosic hydrolysate

Rice straw hemicellulosic hydrolysate was used as fermentation medium for ethanol production by Pichia stipitis NRRL Y-7124. Shaking bath experiments were initially performed aiming to establish the best initial xylose concentration to be used in this bioconversion process. In the sequence, assays were carried out under different agitation (100 to 200 rpm) and aeration (V flask/V medium ratio varying from 2.5 to 5.0) conditions, and the influence of these variables on the fermentative parameters values (ethanol yield factor, Y P/S; cell yield factor, Y X/S; and ethanol volumetric productivity, Q P) was investigated through a 22 full-factorial design. Initial xylose concentration of about 50 g/l was the most suitable for the development of this process, since the yeast was able to convert substrate in product with high efficiency. The factorial design assays showed a strong influence of both process variables in all the evaluated responses. The agitation and aeration increase caused a deviation in the yeast metabolism from ethanol to biomass production. The best results (Y P/S = 0.37 g/g and Q P = 0.39 g/l.h) were found when the lowest aeration (2.5 V flask/V medium ratio) and highest agitation (200 rpm) levels were employed. Under this condition, a process efficiency of 72.5% was achieved. These results demonstrated that the establishment of adequate conditions of aeration is of great relevance to improve the ethanol production from xylose by Pichia stipitis, using rice straw hemicellulosic hydrolysate as fermentation medium.The financial support from Fapesp (Brazil) is gratefully acknowledged

Metabolism-dependent bioaccumulation of uranium by Rhodosporidium toruloides isolated from the flooding water of a former uranium mine

Remediation of former uranium mining sites represents one of the biggest challenges worldwide that have to be solved in this century. During the last years, the search of alternative strategies involving environmentally sustainable treatments has started. Bioremediation, the use of microorganisms to clean up polluted sites in the environment, is considered one the best alternative. By means of culture-dependent methods, we isolated an indigenous yeast strain, KS5 (Rhodosporidium toruloides), directly from the flooding water of a former uranium mining site and investigated its interactions with uranium. Our results highlight distinct adaptive mechanisms towards high uranium concentrations on the one hand, and complex interaction mechanisms on the other. The cells of the strain KS5 exhibit high a uranium tolerance, being able to grow at 6 mM, and also a high ability to accumulate this radionuclide (350 mg uranium/g dry biomass, 48 h). The removal of uranium by KS5 displays a temperature- and cell viability-dependent process, indicating that metabolic activity could be involved. By STEM (scanning transmission electron microscopy) investigations, we observed that uranium was removed by two mechanisms, active bioaccumulation and inactive biosorption. This study highlights the potential of KS5 as a representative of indigenous species within the flooding water of a former uranium mine, which may play a key role in bioremediation of uranium contaminated sites.This work was supported by the Bundesministerium für Bildung und Forschung grand nº 02NUK030F (TransAqua). Further support took place by the ERDF-co-financed Grants CGL2012-36505 and 315 CGL2014-59616R, Ministerio de Ciencia e Innovación, Spain

Endophytes vs tree pathogens and pests: can they be used as biological control agents to improve tree health?

Like all other plants, trees are vulnerable to attack by a multitude of pests and pathogens. Current control measures for many of these diseases are limited and relatively ineffective. Several methods, including the use of conventional synthetic agro-chemicals, are employed to reduce the impact of pests and diseases. However, because of mounting concerns about adverse effects on the environment and a variety of economic reasons, this limited management of tree diseases by chemical methods is losing ground. The use of biological control, as a more environmentally friendly alternative, is becoming increasingly popular in plant protection. This can include the deployment of soil inoculants and foliar sprays, but the increased knowledge of microbial ecology in the phytosphere, in particular phylloplane microbes and endophytes, has stimulated new thinking for biocontrol approaches. Endophytes are microbes that live within plant tissues. As such, they hold potential as biocontrol agents against plant diseases because they are able to colonize the same ecological niche favoured by many invading pathogens. However, the development and exploitation of endophytes as biocontrol agents will have to overcome numerous challenges. The optimization and improvement of strategies employed in endophyte research can contribute towards discovering effective and competent biocontrol agents. The impact of environment and plant genotype on selecting potentially beneficial and exploitable endophytes for biocontrol is poorly understood. How endophytes synergise or antagonise one another is also an important factor. This review focusses on recent research addressing the biocontrol of plant diseases and pests using endophytic fungi and bacteria, alongside the challenges and limitations encountered and how these can be overcome. We frame this review in the context of tree pests and diseases, since trees are arguably the most difficult plant species to study, work on and manage, yet they represent one of the most important organisms on Earth

Endophytes vs tree pathogens and pests: can they be used as biological control agents to improve tree health?

Like all other plants, trees are vulnerable to attack by a multitude of pests and pathogens. Current control measures for many of these diseases are limited and relatively ineffective. Several methods, including the use of conventional synthetic agro-chemicals, are employed to reduce the impact of pests and diseases. However, because of mounting concerns about adverse effects on the environment and a variety of economic reasons, this limited management of tree diseases by chemical methods is losing ground. The use of biological control, as a more environmentally friendly alternative, is becoming increasingly popular in plant protection. This can include the deployment of soil inoculants and foliar sprays, but the increased knowledge of microbial ecology in the phytosphere, in particular phylloplane microbes and endophytes, has stimulated new thinking for biocontrol approaches. Endophytes are microbes that live within plant tissues. As such, they hold potential as biocontrol agents against plant diseases because they are able to colonize the same ecological niche favoured by many invading pathogens. However, the development and exploitation of endophytes as biocontrol agents will have to overcome numerous challenges. The optimization and improvement of strategies employed in endophyte research can contribute towards discovering effective and competent biocontrol agents. The impact of environment and plant genotype on selecting potentially beneficial and exploitable endophytes for biocontrol is poorly understood. How endophytes synergise or antagonise one another is also an important factor. This review focusses on recent research addressing the biocontrol of plant diseases and pests using endophytic fungi and bacteria, alongside the challenges and limitations encountered and how these can be overcome. We frame this review in the context of tree pests and diseases, since trees are arguably the most difficult plant species to study, work on and manage, yet they represent one of the most important organisms on Earth

Fumarate Reductase Activity Maintains an Energized Membrane in Anaerobic Mycobacterium tuberculosis

Oxygen depletion of Mycobacterium tuberculosis engages the DosR regulon that coordinates an overall down-regulation of metabolism while up-regulating specific genes involved in respiration and central metabolism. We have developed a chemostat model of M. tuberculosis where growth rate was a function of dissolved oxygen concentration to analyze metabolic adaptation to hypoxia. A drop in dissolved oxygen concentration from 50 mmHg to 0.42 mmHg led to a 2.3 fold decrease in intracellular ATP levels with an almost 70-fold increase in the ratio of NADH/NAD+. This suggests that re-oxidation of this co-factor becomes limiting in the absence of a terminal electron acceptor. Upon oxygen limitation genes involved in the reverse TCA cycle were upregulated and this upregulation was associated with a significant accumulation of succinate in the extracellular milieu. We confirmed that this succinate was produced by a reversal of the TCA cycle towards the non-oxidative direction with net CO2 incorporation by analysis of the isotopomers of secreted succinate after feeding stable isotope (13C) labeled precursors. This showed that the resulting succinate retained both carbons lost during oxidative operation of the TCA cycle. Metabolomic analyses of all glycolytic and TCA cycle intermediates from 13C-glucose fed cells under aerobic and anaerobic conditions showed a clear reversal of isotope labeling patterns accompanying the switch from normoxic to anoxic conditions. M. tuberculosis encodes three potential succinate-producing enzymes including a canonical fumarate reductase which was highly upregulated under hypoxia. Knockout of frd, however, failed to reduce succinate accumulation and gene expression studies revealed a compensatory upregulation of two homologous enzymes. These major realignments of central metabolism are consistent with a model of oxygen-induced stasis in which an energized membrane is maintained by coupling the reductive branch of the TCA cycle to succinate secretion. This fermentative process may offer unique targets for the treatment of latent tuberculosis