Production of deuterated switchgrass by hydroponic cultivation.

MAIN CONCLUSION: The bioenergy crop switchgrass was grown hydroponically from tiller cuttings in 50 % D 2 O to obtain biomass with 34 % deuterium substitution and physicochemical properties similar to those of H 2 O-grown switchgrass controls. Deuterium enrichment of biological materials can potentially enable expanded experimental use of small angle neutron scattering (SANS) to investigate molecular structural transitions of complex systems such as plant cell walls. Two key advances have been made that facilitate cultivation of switchgrass, an important forage and biofuel crop, for controlled isotopic enrichment: (1) perfusion system with individual chambers and (2) hydroponic growth from tiller cuttings. Plants were grown and maintained for several months with periodic harvest. Photosynthetic activity was monitored by measurement of CO2 in outflow from the growth chambers. Plant morphology and composition appeared normal compared to matched controls grown with H2O. Using this improved method, gram quantities of switchgrass leaves and stems were produced by continuous hydroponic cultivation using growth medium consisting of basal mineral salts in 50 % D2O. Deuterium incorporation was confirmed by detection of the O-D and C-D stretching peaks with FTIR and quantified by (1)H- and (2)H-NMR. This capability to produce deuterated lignocellulosic biomass under controlled conditions will enhance investigation of cell wall structure and its deconstruction by neutron scattering and NMR techniques.

Deuterium incorporation in biomass cell wall components by NMR analysis.

A commercially available deuterated kale sample was analyzed for deuterium incorporation by ionic liquid solution (2)H and (1)H nuclear magnetic resonance (NMR). This protocol was found to effectively measure the percent deuterium incorporation at 33%, comparable to the 31% value determined by combustion. The solution NMR technique also suggested by a qualitative analysis that deuterium is preferentially incorporated into the carbohydrate components of the kale sample.

Small angle neutron scattering reveals pH-dependent conformational changes in Trichoderma reesei cellobiohydrolase I: implications for enzymatic activity.

Cellobiohydrolase I (Cel7A) of the fungus Trichoderma reesei (now classified as an anamorph of Hypocrea jecorina) hydrolyzes crystalline cellulose to soluble sugars, making it of key interest for producing fermentable sugars from biomass for biofuel production. The activity of the enzyme is pH-dependent, with its highest activity occurring at pH 4-5. To probe the response of the solution structure of Cel7A to changes in pH, we measured small angle neutron scattering of it in a series of solutions having pH values of 7.0, 6.0, 5.3, and 4.2. As the pH decreases from 7.0 to 5.3, the enzyme structure remains well defined, possessing a spatial differentiation between the cellulose binding domain and the catalytic core that only changes subtly. At pH 4.2, the solution conformation of the enzyme changes to a structure that is intermediate between a properly folded enzyme and a denatured, unfolded state, yet the secondary structure of the enzyme is essentially unaltered. The results indicate that at the pH of optimal activity, the catalytic core of the enzyme adopts a structure in which the compact packing typical of a fully folded polypeptide chain is disrupted and suggest that the increased range of structures afforded by this disordered state plays an important role in the increased activity of Cel7A through conformational selection.

SANS study of cellulose extracted from switchgrass.

Lignocellulosic biomass, which is an abundant renewable natural resource, has the potential to play a major role in the generation of renewable biofuels through its conversion to bioethanol. Unfortunately, it is a complex biological composite material that shows significant recalcitrance, making it a cost-ineffective feedstock for bioethanol production. Small-angle neutron scattering (SANS) was employed to probe the multi-scale structure of cellulosic materials. Cellulose was extracted from milled native switchgrass and from switchgrass that had undergone a dilute acid pretreatment method in order to disrupt the lignocellulose structure. The high-Q structural feature (Q > 0.07 Å(-1)) can be assigned to cellulose fibrils based on a comparison of cellulose purified by solvent extraction of native and dilute acid pretreated switchgrass and a commercial preparation of microcrystalline cellulose. Dilute acid pretreatment results in an increase in the smallest structural size, a decrease in the interconnectivity of the fibrils and no change in the smooth domain boundaries at length scales larger than 1000 Å.

Breakdown of cell wall nanostructure in dilute acid pretreated biomass.

The generation of bioethanol from lignocellulosic biomass holds great promise for renewable and clean energy production. A better understanding of the complex mechanisms of lignocellulose breakdown during various pretreatment methods is needed to realize this potential in a cost and energy efficient way. Here we use small-angle neutron scattering (SANS) to characterize morphological changes in switchgrass lignocellulose across molecular to submicrometer length scales resulting from the industrially relevant dilute acid pretreatment method. Our results demonstrate that dilute acid pretreatment increases the cross-sectional radius of the crystalline cellulose fibril. This change is accompanied by removal of hemicellulose and the formation of R(g) ∼ 135 A lignin aggregates. The structural signature of smooth cell wall surfaces is observed at length scales larger than 1000 A, and it remains remarkably invariable during pretreatment. This study elucidates the interplay of the different biomolecular components in the breakdown process of switchgrass by dilute acid pretreatment. The results are important for the development of efficient strategies of biomass to biofuel conversion.

Temporal and spatial fate of GFP-expressing motile and nonmotile Aeromonas hydrophila in the house fly digestive tract.

To gain insight into the transmissibility of bacteria by house flies, the temporal and spatial fate of green fluorescent protein (GFP)-expressing motile and nonmotile strains of Aeromonas hydrophila was examined within the alimentary canal. Liquid food consumed by house flies is first stored in the crop and then is regurgitated and/or passed into the midgut. Once within the midgut, food is contained inside a double-layered peritrophic matrix (PM), with the inner layer enveloping digested material and forming fecal pellets for excretion. Between 1 and 12 h after ingestion, and irrespective of motility, live GFP+ A. hydrophila adhered to the luminal surfaces of the crop and inner PM of bacteria-fed flies. However, some nonadherent, motile bacteria moved freely within the PM lumen in the anteromedial midgut, whereas fecal pellets (lysed bacteria) continued passing posteriorly. At 12-24 h, adhered bacteria of both strains were lysed in the distal midgut, compressed into fecal pellets, and excreted. Viable bacteria in the crop visually exceeded numbers within these fecal pellets. Culture recovery at the same time points showed a 1,000-fold increase of viable bacteria at 2 h, presumably in the crop, with a temporal decline thereafter. Further, viable bacteria were recovered from vomit specks and orally contaminated substrates up to 2 h after feeding but never from feces. These results suggest that A. hydrophila is a transient resident of the house fly alimentary canal and is only orally transmissible for a short time after ingestion. Thus, regurgitation may be more significant than fecal transmission in the spread of some house fly transmitted bacterial diseases.