Down-regulation of the caffeic acid <Emphasis Type="Italic">O</Emphasis>-methyltransferase gene in switchgrass reveals a novel monolignol analog

Background

Down-regulation of the caffeic acid 3-O-methyltransferase EC 2.1.1.68 (COMT) gene in the lignin biosynthetic pathway of switchgrass (Panicum virgatum) resulted in cell walls of transgenic plants releasing more constituent sugars after pretreatment by dilute acid and treatment with glycosyl hydrolases from an added enzyme preparation and from Clostridium thermocellum. Fermentation of both wild-type and transgenic switchgrass after milder hot water pretreatment with no water washing showed that only the transgenic switchgrass inhibited C. thermocellum. Gas chromatography–mass spectrometry (GCMS)-based metabolomics were undertaken on cell wall aqueous extracts to determine the nature of the microbial inhibitors.

Results

GCMS confirmed the increased concentration of a number of phenolic acids and aldehydes that are known inhibitors of microbial fermentation. Metabolomic analyses of the transgenic biomass additionally revealed the presence of a novel monolignol-like metabolite, identified as trans-3, 4-dimethoxy-5-hydroxycinnamyl alcohol (iso-sinapyl alcohol) in both non-pretreated, as well as hot water pretreated samples. iso-Sinapyl alcohol and its glucoside were subsequently generated by organic synthesis and the identity of natural and synthetic materials were confirmed by mass spectrometric and NMR analyses. The additional novel presence of iso-sinapic acid, iso-sinapyl aldehyde, and iso-syringin suggest the increased activity of a para-methyltransferase, concomitant with the reduced COMT activity, a strict meta-methyltransferase. Quantum chemical calculations were used to predict the most likely homodimeric lignans generated from dehydration reactions, but these products were not evident in plant samples.

Conclusions

Down-regulation of COMT activity in switchgrass resulted in the accumulation of previously undetected metabolites resembling sinapyl alcohol and its related metabolites, but that are derived from para-methylation of 5-hydroxyconiferyl alcohol, and related precursors and products; the accumulation of which suggests altered metabolism of 5-hydroxyconiferyl alcohol in switchgrass. Given that there was no indication that iso-sinapyl alcohol was integrated in cell walls, it is considered a monolignol analog. Diversion of substrates from sinapyl alcohol to free iso-sinapyl alcohol, its glucoside, and associated upstream lignin pathway changes, including increased phenolic aldehydes and acids, are together associated with more facile cell wall deconstruction, and to the observed inhibitory effect on microbial growth. However, iso-sinapyl alcohol and iso-sinapic acid, added separately to media, were not inhibitory to C. thermocellum cultures.

     

Recalcitrance Assessment of the Agro-industrial Residues from Five <Emphasis Type="Italic">Agave</Emphasis> Species: Ionic Liquid Pretreatment,Saccharification and Structural Characterization

Agave has recently shown its potential as a bioenergy feedstock with promising features such as higher biomass productivity than leading bioenergy feedstock while at the same time being drought-resistant with low water requirements and high sugar to ethanol conversion using ionic liquid (IL) pretreatment. IL pretreatment was studied to develop the first direct side-by-side comparative recalcitrance assessment of the agro-industrial residues from five Agave species [Agave americana (AME), A. angustifolia (ANG), A. fourcroydes (FOU), A. salmiana (SAL), and A. tequilana (TEQ)] using compositional analysis, X-ray diffraction, and the lignin syringyl/guaiacyl subunit ratio (S/G) by pyrolysis molecular beam mass spectrometry (PyMBMS). Prominent calcium oxalate peaks were found only in unpretreated AME, SAL, and TEQ. The S/G ratios of all five unpretreated Agave species were between 1.27 and 1.57 while the IL-pretreated samples were from 1.39 to 1.72. The highest overall sugar production was obtained with IL-pretreated FOU with 492 mg glucose/g biomass and 157 mg xylose/g biomass at 120 °C and 3 h using 1-ethyl-3-methylimidazolium acetate ([C₂C₁Im][OAc]). An estimated theoretical ethanol yield from the studied agro-industrial residues from the five Agave species was in the range of 1060 to 5800 L ethanol/ha/year. These comparison results demonstrate the potential of the Agave spp. as a suitable biofuel feedstock which can be employed within a biorefinery scheme.     

Monitoring and Analyzing Process Streams Towards Understanding Ionic Liquid Pretreatment of Switchgrass (Panicum virgatum L.)

Fundamental understanding of biomass pretreatment and its influence on saccharification kinetics, total sugar yield, and inhibitor formation is essential to develop efficient next-generation biofuel strategies, capable of displacing fossil fuels at a commercial level. In this study, we investigated the effect of residence time and temperature during ionic liquid (IL) pretreatment of switchgrass using 1-ethyl-3-methyl imidazolium acetate. The primary metrics of pretreatment performance are biomass delignification, xylan and glucan depolymerization, porosity, surface area, cellulase kinetics, and sugar yields. Compositional analysis and quantification of process streams of saccharides and lignin demonstrate that delignification increases as a function of pretreatment temperature and is hypothesized to be correlated with the apparent glass transition temperature of lignin. IL pretreatment did not generate monosaccharides from hemicellulose. Compared to untreated switchgrass, Brunauer–Emmett–Teller surface area of pretreated switchgrass increased by a factor of ～30, with a corresponding increase in saccharification kinetics of a factor of ～40. There is an observed dependence of cellulase kinetics with delignification efficiency. Although complete biomass dissolution is observed after 3 h of IL pretreatment, the pattern of sugar release, saccharification kinetics, and total sugar yields are strongly correlated with temperature.     

Competitive effects of cultivar and wild switchgrass on other native grasses

There is mounting concern that selection and breeding of native grasses for greater biomass production could promote weediness. Yet little is known about the invasion potential or ecological impacts of such selectively bred native grasses. Here we focus on cultivars of native switchgrass (Panicum virgatum L.) that have undergone selection, breeding, and intraspecific hybridization to improve agronomic traits for biomass production. We evaluated the competitive effects of switchgrass cultivars (EG-2101 and ‘Trailblazer’) and wild switchgrass populations on two native prairie grasses [sideoats grama, Bouteloua curtipendula (Michx.) Torr., and Canada wild rye, Elymus canadensis L.] across a gradient of switchgrass density in a greenhouse. Cultivars produced 48–128% more biomass and reduced sideoats grama biomass by 25–59% more than wild switchgrass. Effects of switchgrass cultivars on Canada wild rye were minimal compared to sideoats grama. Later flowering and larger seed size of cultivars may be contributing to their greater biomass and competitive effects on sideoats grama. These data suggest that breeding switchgrass for enhanced biomass yield may increase competitive effects on some native grasses. Further studies are merited to test the potential for switchgrass biomass cultivars to spread and impact species diversity of restored and remnant native plant communities.     

Comparative evaluation of <Emphasis Type="Italic">Populus</Emphasis> variants total sugar release and structural features following pretreatment and digestion by two distinct biological systems

Background

Populus natural variants have been shown to realize a broad range of sugar yields during saccharification, however, the structural features responsible for higher sugar release from natural variants are not clear. In addition, the sugar release patterns resulting from digestion with two distinct biological systems, fungal enzymes and Clostridium thermocellum, have yet to be evaluated and compared. This study evaluates the effect of structural features of three natural variant Populus lines, which includes the line BESC standard, with respect to the overall process of sugar release for two different biological systems.

Results

Populus natural variants, SKWE 24-2 and BESC 876, showed higher sugar release from hydrothermal pretreatment combined with either enzymatic hydrolysis or Clostridium thermocellum fermentation compared to the Populus natural variant, BESC standard. However, C. thermocellum outperformed the fungal cellulases yielding 96.0, 95.5, and 85.9% glucan plus xylan release from SKWE 24-2, BESC 876, and BESC standard, respectively. Among the feedstock properties evaluated, cellulose accessibility and glycome profiling provided insights into factors that govern differences in sugar release between the low recalcitrant lines and the BESC standard line. However, because this distinction was more apparent in the solids after pretreatment than in the untreated biomass, pretreatment was necessary to differentiate recalcitrance among Populus lines. Glycome profiling analysis showed that SKWE 24-2 contained the most loosely bound cell wall glycans, followed by BESC 876, and BESC standard. Additionally, lower molecular weight lignin may be favorable for effective hydrolysis, since C. thermocellum reduced lignin molecular weight more than fungal enzymes across all Populus lines.

Conclusions

Low recalcitrant Populus natural variants, SKWE 24-2 and BESC 876, showed higher sugar yields than BESC standard when hydrothermal pretreatment was combined with biological digestion. However, C. thermocellum was determined to be a more robust and effective biological catalyst than a commercial fungal cellulase cocktail. As anticipated, recalcitrance was not readily predicted through analytical methods that determined structural properties alone. However, combining structural analysis with pretreatment enabled the identification of attributes that govern recalcitrance, namely cellulose accessibility, xylan content in the pretreated solids, and non-cellulosic glycan extractability.

     

Impact of Harvest Time and Cultivar on Conversion of Switchgrass to Bio-oils Via Fast Pyrolysis

The study of the effects of harvest time on switchgrass (Panicum virgatum L.) biomass and bioenergy production reported herein encompasses a large study evaluating the harvest of six switchgrass cultivars grown at three northern US locations over 3 years, harvested at upland peak crop (anthesis), post-frost, and post-winter. Delaying harvest of switchgrass until after frost and until after winter has resulted in decreased yields of switchgrass and reduced amounts of minerals in the biomass. This report examines how changes in biomass composition as a result of varying harvest time and other factors affect the distribution of products formed via fast pyrolysis. A subset (50) of the population (n = 864) was analyzed for fast pyrolysis and catalytic pyrolysis (zeolite catalyst) product yields using a pyrolysis-GC/MS system. The subset was used to build calibrations that were successful in predicting the pyrolysis product yield using near-infrared reflectance spectroscopy (NIRS), and partial least squares predictive models were applied to the entire sample set. The pyrolysis product yield was significantly affected by the field trial location, year of harvest, cultivar, and harvest time. Delaying harvest time of the switchgrass crop led to greater production of deoxygenated aromatics improving the efficiency of the catalytic fast pyrolysis and bio-oil quality. The changes in the pyrolysis product yield were related to biomass compositional changes, and key relationships between cell wall polymers, potassium concentration in the biomass, and pyrolysis products were identified. The findings show that the loss of minerals in the biomass as harvest time is delayed combined with the greater proportion in cellulose and lignin in the biomass has significant positive influences on conversion through fast pyrolysis.     

Effect of impaired twitching motility and biofilm dispersion on performance of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>-powered microbial fuel cells

Pseudomonas aeruginosa is a metabolically voracious bacterium that is easily manipulated genetically. We have previously shown that the organism is also highly electrogenic in microbial fuel cells (MFCs). Polarization studies were performed in MFCs with wild-type strain PAO1 and three mutant strains (pilT, bdlA and pilT bdlA). The pilT mutant was hyperpiliated, while the bdlA mutant was suppressed in biofilm dispersion chemotaxis. The double pilT bdlA mutant was expected to have properties of both mutations. Polarization data indicate that the pilT mutant showed 5.0- and 3.2-fold increases in peak power compared to the wild type and the pilT bdlA mutant, respectively. The performance of the bdlA mutant was surprisingly the lowest, while the pilT bdlA electrogenic performance fell between the pilT mutant and wild-type bacteria. Measurements of biofilm thickness and bacterial viability showed equal viability among the different strains. The thickness of the bdlA mutant, however, was twice that of wild-type strain PAO1. This observation implicates the presence of dead or dormant bacteria in the bdlA mutant MFCs, which increases biofilm internal resistance as confirmed by electrochemical measurements.     

Mechanistic insights into the effect of imidazolium ionic liquid on lipid production by <Emphasis Type="Italic">Geotrichum fermentans</Emphasis>

Background

Ionic liquid (IL) pretreatment has emerged as a promising technique that enables complete utilization of lignocellulosic biomass for biofuel production. However, imidazolium IL has recently been shown to exhibit inhibitory effect on cell growth and product formation of industrial microbes, such as oleaginous microorganisms. To date, the mechanism of this inhibition remains largely unknown.

Results

In this study, the feasibility of [Bmim][OAc]-pretreated rice straw hydrolysate as a substrate for microbial lipid production by Geotrichum fermentans, also known as Trichosporon fermentans, was evaluated. The residual [Bmim][OAc] present in the hydrolysate caused a reduction in biomass and lipid content (43.6 and 28.1%, respectively) of G. fermentans, compared with those of the control (7.8 g/L and 52.6%, respectively). Seven imidazolium ILs, [Emim][DEP], [Emim]Cl, [Amim]Cl, [Bmim]Cl, [Bzmim]Cl, [Emim][OAc], and [Bmim][OAc], capable of efficient pretreatment of lignocellulosic biomass were tested for their effects on the cell growth and lipid accumulation of G. fermentans to better understand the impact of imidazolium IL on the lipid production. All the ILs tested inhibited the cell growth and lipid accumulation. In addition, both the cation and the anion of IL contributed to IL toxicity. The side chain of IL cations showed a clear impact on toxicity. On examining IL anions, [OAc]^? was found to be more toxic than those of [DEP]^? and Cl^?. IL exhibited its toxicity by inhibiting sugar consumption and key enzyme (malic enzyme and ATP-citrate lyase) activities of G. fermentans. Cell membrane permeability was also altered to different extents in the presence of various ILs. Scanning electron microscopy revealed that IL induces fibrous structure on the surface of G. fermentans cell, which might represent an adaptive mechanism of the yeast to IL.

Conclusions

This work gives some mechanistic insights into the impact of imidazolium IL on the cell growth and lipid accumulation of oleaginous yeast, which is important for IL integration in lignocellulosic biofuel production, especially for microbial lipid production.

     

Laccase-Mediator Pretreatment of Wheat Straw Degrades Lignin and Improves Saccharification

Agricultural by-products such as wheat straw are attractive feedstocks for the production of second-generation bioethanol due to their high abundance. However, the presence of lignin in these lignocellulosic materials hinders the enzymatic hydrolysis of cellulose. The purposes of this work are to study the ability of a laccase-mediator system to remove lignin improving saccharification, as a pretreatment of wheat straw, and to analyze the chemical modifications produced in the remaining lignin moiety. Up to 48 % lignin removal from ground wheat straw was attained by pretreatment with Pycnoporus cinnabarinus laccase and 1-hydroxybenzotriazole (HBT) as mediator, followed by alkaline peroxide extraction. The lignin removal directly correlated with increases (～60 %) in glucose yields after enzymatic saccharification. The pretreatment using laccase alone (without mediator) removed up to 18 % of lignin from wheat straw. Substantial lignin removal (37 %) was also produced when the enzyme-mediator pretreatment was not combined with the alkaline peroxide extraction. Two-dimensional nuclear magnetic resonance (2D NMR) analysis of the whole pretreated wheat straw material swollen in dimethylsulfoxide-d ₆ revealed modifications of the lignin polymer, including the lower number of aliphatic side chains involved in main β-O-4′ and β-5′ inter-unit linkages per aromatic lignin unit. Simultaneously, the removal of p-hydroxyphenyl, guaiacyl, and syringyl lignin units and of p-coumaric and ferulic acids, as well as a moderate decrease of tricin units, was observed without a substantial change in the wood polysaccharide signals. Especially noteworthy was the formation of Cα-oxidized lignin units during the enzymatic treatment.     

Yield and Nutrient Removal by Bioenergy Grasses on Swine Effluent Spray Fields in the Coastal Plain Region of North Carolina

Bioenergy grasses such as giant miscanthus (Miscanthus × giganteus) and switchgrass (Panicum virgatum L.) are promising alternatives to the traditional coastal bermudagrass [Cynodon dactylon (L.) Pers.] at spray fields in Eastern North Carolina. The objective of this study was to determine the impact of different harvest practices on yield and nutrient removal of miscanthus and switchgrass in a swine (Sus scrofa domesticus) lagoon effluent spray field environment. Field trials of grasses under six single-cut and double-cut harvest practices (May/October, June/October, July/October, Aug/October, October only, and December only) were established at three commercial swine farms in Eastern North Carolina in either 2011 or 2012. Throughout the 4-year experimental period (2012–2015), both miscanthus and switchgrass produced significantly higher biomass yield than coastal bermudagrass. Two-cut harvest systems significantly improved the yields of miscanthus and switchgrass relative to a single harvest in December at spray fields. The maximum yields were 24 Mg ha^?1 year^?1 for miscanthus and 18 Mg ha^?1 year^?1 for switchgrass. Bioenergy grasses removed more nutrients under two-cut systems than under a single harvest. The significantly greater nutrient removals under two-cut harvest systems would result in lower requirements for receiver crop acreage and are more desirable from a spray field nutrient management perspective.     

Co-consumption of glucose and xylose for organic acid production by <Emphasis Type="Italic">Aspergillus carbonarius</Emphasis> cultivated in wheat straw hydrolysate

Aspergillus carbonarius exhibits excellent abilities to utilize a wide range of carbon sources and to produce various organic acids. In this study, wheat straw hydrolysate containing high concentrations of glucose and xylose was used for organic acid production by A. carbonarius. The results indicated that A. carbonarius efficiently co-consumed glucose and xylose and produced various types of organic acids in hydrolysate adjusted to pH 7. The inhibitor tolerance of A. carbonarius to the hydrolysate at different pH values was investigated and compared using spores and recycled mycelia. This comparison showed a slight difference in the inhibitor tolerance of the spores and the recycled mycelia based on their growth patterns. Moreover, the wild-type and a glucose oxidase deficient (Δgox) mutant were compared for their abilities to produce organic acids using the hydrolysate and a defined medium. The two strains showed a different pattern of organic acid production in the hydrolysate where the Δgox mutant produced more oxalic acid but less citric acid than the wild-type, which was different from the results obtained in the defined medium This study demonstrates the feasibility of using lignocellulosic biomass for the organic acid production by A. carbonarius.     

Front-end recovery of protein from lignocellulosic biomass and its effects on chemical pretreatment and enzymatic saccharification

Front-end protein recovery from biomass at different maturities, and its effects on chemical pretreatment and enzyme hydrolysis of partially deproteinized fiber were investigated. The protein recovery from alfalfa and switchgrass biomass using sodium dodecyl sulfate and potassium hydroxide treatments was ~50–65 % of initial biomass protein. When hot water was used as extraction media, the protein recovery was 52.9 and 43.7 % of total protein in switchgrass and alfalfa, respectively. For any treatment, relative protein recovery was higher from switchgrass than from alfalfa. Only approximately half the total protein was recovered from relatively mature (early fall) biomass compared with midsummer harvested biomass. When protein was recovered partially using sodium dodecyl sulfate or potassium hydroxide, and leftover fiber pretreated, aqueous ammonia pretreatment removed 58.5–60.1 % of lignin and retained more cellulose in the fiber compared with acid pretreatment (nearly no lignin removal). Protein removal was helpful in the enzyme digestibility of fibers. Delignification of ammonia pretreated partially deproteinized alfalfa fiber was in the range of 34.4–45 %, while dilute sulfuric acid did not remove lignin effectively. Overall, the higher delignification and enzyme digestibilities were observed in aqueous ammonia pretreated partially deproteinized alfalfa fibers regardless of biomass type.     

The <Emphasis Type="Italic">ecdysoneless</Emphasis><Superscript>1</Superscript> Gene Regulates Metabolism of the Juvenile Hormone and Dopamine in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

The dopamine (DA) content and the level of juvenile hormone (JH) degradation were studied in females of the wild-type Canton S strain and the ecdysoneless ¹ (ecd ¹) mutant, which does not produce ecdysone at a restrictive temperature (29°C). Exposure at the restrictive temperature considerably increased the JH-hydrolyzing activity and the DA content in five-day ecd ¹ females compared with flies of both strains growing at 19°C and Canton S females exposed at 29°C. In one-day ecd ¹ females, the level of JH degradation also increased at the restrictive temperature, but the DA content was low. The effect of ecdysone deficiency on the stress reaction in Drosophila melanogaster females was studied using changes in DA content and JH degradation as the reaction indicators. The ecd ¹ mutation did not prevent the initiation of the stress reaction in females exposed at the restrictive temperature, but changed its intensity (stress reactivity). The interaction of 20-hydroxyecdysone with JH and DA in regulating Drosophila reproduction under normal conditions and in stress is discussed.     

A precise and consistent assay for major wall polymer features that distinctively determine biomass saccharification in transgenic rice by near-infrared spectroscopy

Background

The genetic modification of plant cell walls has been considered to reduce lignocellulose recalcitrance in bioenergy crops. As a result, it is important to develop a precise and rapid assay for the major wall polymer features that affect biomass saccharification in a large population of transgenic plants. In this study, we collected a total of 246 transgenic rice plants that, respectively, over-expressed and RNAi silenced 12 genes of the OsGH9 and OsGH10 family that are closely associated with cellulose and hemicellulose modification. We examined the wall polymer features and biomass saccharification among 246 transgenic plants and one wild-type plant. The samples presented a normal distribution applicable for statistical analysis and NIRS modeling.

Results

Among the 246 transgenic rice plants, we determined largely varied wall polymer features and the biomass enzymatic saccharification after alkali pretreatment in rice straws, particularly for the fermentable hexoses, ranging from 52.8 to 95.9%. Correlation analysis indicated that crystalline cellulose and lignin levels negatively affected the hexose and total sugar yields released from pretreatment and enzymatic hydrolysis in the transgenic rice plants, whereas the arabinose levels and arabinose substitution degree (reverse xylose/arabinose ratio) exhibited positive impacts on the hexose and total sugars yields. Notably, near-infrared spectroscopy (NIRS) was applied to obtain ten equations for predicting biomass enzymatic saccharification and seven equations for distinguishing major wall polymer features. Most of the equations exhibited high R ²/R ² _cv/R ² _ev and RPD values for a perfect prediction capacity.

Conclusions

Due to large generated populations of transgenic rice lines, this study has not only examined the key wall polymer features that distinctively affect biomass enzymatic saccharification in rice but has also established optimal NIRS models for a rapid and precise screening of major wall polymer features and lignocellulose saccharification in biomass samples. Importantly, this study has briefly explored the potential roles of a total of 12 OsGH9 and OsGH10 genes in cellulose and hemicellulose modification and cell wall remodeling in transgenic rice lines. Hence, it provides a strategy for genetic modification of plant cell walls by expressing the desired OsGH9 and OsGH10 genes that could greatly improve biomass enzymatic digestibility in rice.

     

New insight into the species diversity and life cycles of rust fungi (Pucciniales) affecting bioenergy switchgrass (<Emphasis Type="Italic">Panicum virgatum</Emphasis>) in the Eastern and Central United States

Research was undertaken to clarify the taxonomic identity of leaf rust (Pucciniales) fungi on bioenergy switchgrass in the Eastern and Central U.S. We integrated internal transcribed spacer 2 (ITS2) and partial 28S ribosomal RNA gene sequence data from collections taken from cultivated switchgrass and herbarium specimens, including purported aecial and telial states of Puccinia graminicola and Puccinia pammelii. Maximum likelihood and Bayesian analyses revealed four monophyletic clades: Puccinia emaculata sensu stricto (s.s.), P. pammelii, P. graminicola, and Puccinia novopanici. Results also indicated that P. emaculata s.s. was not affecting cultivated, bioenergy switchgrass. Aecidium pammelii and P. pammelii were distinct phylogenetically from P. emaculata s.s. and grouped within a well-supported clade, demonstrating aecial-telial host alternation for P. pammelii between Euphorbia corollata and switchgrass. Aecidium stillingiae on queen’s delight (Stillingia sylvatica)—a purported aecial state host for P. graminicola—shared identical sequences with the recently described species Puccinia pascua. The latter fungus, however, was recovered within a subclade of P. graminicola. Hence, queen’s delight likely is not an aecial host to P. graminicola s.s. Additional molecular studies are warranted to determine species boundaries within the P. graminicola complex. The majority of contemporary collections from cultivated switchgrass were recognized as P. novopanici. Collectively, bioenergy switchgrass is host to at least three phylogenetically distinct species, presenting a significant challenge to the future selection and breeding of switchgrass with improved rust resistance.