In situ and Ex situ Catalytic Pyrolysis of Microalgae and Integration With Pyrolytic Fractionation.

Microalgae are attractive feedstocks for biofuel production and are especially suitable for thermochemical conversion due to the presence of thermally labile constituents-lipids, starch and protein. However, the thermal degradation of starch and proteins produces water as well as other O- and N-compounds that are mixed-in with energy-dense lipid pyrolysis products. To produce hydrocarbon-rich products from microalgae biomass, we assessed in situ and ex situ catalytic pyrolysis of a lipid-rich Chlorella sp. in the presence of the HZSM-5 zeolite catalyst over a temperature range of 450-550°C. Results show that product yields and compositions were similar under both in situ and ex situ conditions with benzene, toluene and xylene produced as the primary aromatic products. Yields of aromatics increased with increasing temperature and the highest aromatic yield (36.4% g aromatics/g ash-free microalgae) and selectivity (87% g aromatics/g bio-oil) was obtained at 550°C. Also, at this temperature, oxygenates and nitrogenous compounds were not detected among the liquid products during ex situ catalytic pyrolysis. We also assessed the feasibility of a two-step fractional pyrolysis approach integrated with vapor phase catalytic upgrading. In these experiments, the biomass was first pyrolyzed at 320°C to degrade and volatilize starch, protein and free fatty acids. Then, the residual biomass was pyrolyzed again at 450°C to recover products from triglyceride decomposition. The volatiles from each fraction were passed through an ex situ catalyst bed. Results showed that net product yields from the 2-step process were similar to the single step ex situ catalytic pyrolysis at 450°C indicating that tailored vapor phase upgrading can be applied to allow separate recovery of products from the chemically distinct biomass components-(1) lower calorific value starch and proteins and (2) energy-dense lipids.

Quantification of Lipid Content in Oleaginous Biomass Using Thermogravimetry.

Laboratory analytical techniques employed for triglyceride quantification in oleaginous biomass (e.g., microalgae and oilseeds) involve multiple steps and typically require use of volatile organic solvents. Here we describe a single-step approach for measurement of triglycerides using thermogravimetry (TG). We have observed that triglycerides undergo complete volatilization over a narrow temperature interval of 370-450 °C, with negligible solid residue under inert atmosphere, whereas other constituents of oleaginous biomass (such as proteins and carbohydrates) primarily degrade below 350 °C. As a result, triglyceride content of biomass can be estimated using TG by determining the mass loss of the sample in the temperature interval of 370-450 °C.

High-Yield Production of Fatty Nitriles by One-Step Vapor-Phase Thermocatalysis of Triglycerides.

Fatty nitriles are widely used as intermediate molecules in the pharmaceutical and polymer industries. In addition, hydrogenation of fatty nitriles produces fatty amines that are common surfactants. In the conventional fatty nitrile process, triglycerides are first hydrolyzed and the resulting fatty acids are catalytically reacted with NH3 in a liquid-phase reaction. In this study, we report a simpler one-step fatty nitrile production method that involves a direct vapor-phase reaction of triglycerides with NH3 in the presence of heterogeneous solid acid catalysts. The reactions were performed in a tubular reactor maintained at 400 °C into which triglycerides were injected through an atomizer to allow rapid volatilization and reaction; NH3 was fed as a gas. Several metal oxide catalysts were tested, and reactions in the presence of V2O5 resulted in near-theoretical fatty nitrile yields (84 wt % relative to the feed mass). In general, catalysts with higher acidity such as V2O5, Fe2O3, and ZnO showed higher fatty nitrile yields compared to low acidity catalysts such as ZrO, Al2O3, and CuO. Energy balance calculations indicate that the one-step reaction described here would require significantly lower energy than the conventional process primarily because of the elimination of the energy-intense triglyceride hydrolysis.

Using life cycle assessment and techno-economic analysis in a real options framework to inform the design of algal biofuel production facilities.

This study investigates the use of "real options analysis" (ROA) to quantify the value of greater product flexibility at algal biofuel production facilities. A deterministic optimization framework is integrated with a combined life cycle assessment/techno-economic analysis model and subjected to an ensemble of 30-year commodity price trajectories. Profits are maximized for two competing plant configurations: 1) one that sells lipid-extracted algae as animal feed only; and 2) one that can sell lipid-extracted algae as feed or use it to recover nutrients and energy, due to an up-front investment in anaerobic digestion/combined heat and power. Results show that added investment in plant flexibility does not result in an improvement in net present value, because current feed meal prices discourage use of lipid-extracted algae for nutrient and energy recovery. However, this study demonstrates that ROA provides many useful insights regarding plant design that cannot be captured via traditional techno-economic modeling.

Evaluating the relative impacts of operational and financial factors on the competitiveness of an algal biofuel production facility.

Algal biofuels are becoming more economically competitive due to technological advances and government subsidies offering tax benefits and lower cost financing. These factors are linked, however, as the value of technical advances is affected by modeling assumptions regarding the growth conditions, process design, and financing of the production facility into which novel techniques are incorporated. Two such techniques, related to algal growth and dewatering, are evaluated in representative operating and financing scenarios using an integrated techno-economic model. Results suggest that these techniques can be valuable under specified conditions, but also that investment subsidies influence cost competitive facility design by incentivizing development of more capital intensive facilities (e.g., favoring hydrothermal liquefaction over transesterification-based facilities). Evaluating novel techniques under a variety of operational and financial scenarios highlights the set of site-specific conditions in which technical advances are most valuable, while also demonstrating the influence of subsidies linked to capital intensity.

Uptake of inorganic and organic nutrient species during cultivation of a Chlorella isolate in anaerobically digested dairy waste.

A natural assemblage of microalgae from a facultative lagoon system treating municipal wastewater was enriched for growth in the effluents of an anaerobic digester processing dairy waste. A green microalga with close resemblance to Chlorella sp. was found to be dominant after multiple cycles of sub-culturing. Subsequently, the strain (designated as LLAI) was isolated and cultivated in 20× diluted digester effluents under various incident light intensities (255-1,100 µmoles m-2 s-1 ) to systematically assess growth and nutrient utilization. Our results showed that LLAI production increased with increasing incident light and a maximum productivity of 0.34 g L-1 d-1 was attained when the incident irradiance was 1,100 µmoles m-2 s-1 . Lack of growth in the absence of light indicated that the cultures did not grow heterotrophically on the organic compounds present in the medium. However, the cultures were able to uptake organic N and P under phototrophic conditions and our calculations suggest that the carbon associated with these organic nutrients contributed significantly to the production of biomass. Overall, under high light conditions, LLAI cultures utilized half of the soluble organic nitrogen and >90% of the ammonium, orthophosphate, and dissolved organic phosphorus present in the diluted waste. Strain LLAI was also found to accumulate triacylglycerides (TAG) even before the onset of nutrient limitation and a lipid productivity of 37 mg-TAG L-1 d-1 was measured in cultures incubated at an incident irradiance of 1,100 µmoles m-2 s-1 . The results of this study suggest that microalgae isolates from natural environments are well-suited for nutrient remediation and biomass production from wastewater containing diverse inorganic and organic nutrient species. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1336-1342, 2016.

Hexavalent chromium reduction by Cellulomonas sp. strain ES6: the influence of carbon source, iron minerals, and electron shuttling compounds.

The reduction of hexavalent chromium, Cr(VI), to trivalent chromium, Cr(III), can be an important aspect of remediation processes at contaminated sites. Cellulomonas species are found at several Cr(VI) contaminated and uncontaminated locations at the Department of Energy site in Hanford, Washington. Members of this genus have demonstrated the ability to effectively reduce Cr(VI) to Cr(III) fermentatively and therefore play a potential role in Cr(VI) remediation at this site. Batch studies were conducted with Cellulomonas sp. strain ES6 to assess the influence of various carbon sources, iron minerals, and electron shuttling compounds on Cr(VI) reduction rates as these chemical species are likely to be present in, or added to, the environment during in situ bioremediation. Results indicated that the type of carbon source as well as the type of electron shuttle present influenced Cr(VI) reduction rates. Molasses stimulated Cr(VI) reduction more effectively than pure sucrose, presumably due to presence of more easily utilizable sugars, electron shuttling compounds or compounds with direct Cr(VI) reduction capabilities. Cr(VI) reduction rates increased with increasing concentration of anthraquinone-2,6-disulfonate (AQDS) regardless of the carbon source. The presence of iron minerals and their concentrations did not significantly influence Cr(VI) reduction rates. However, strain ES6 or AQDS could directly reduce surface-associated Fe(III) to Fe(II), which was capable of reducing Cr(VI) at a near instantaneous rate. These results suggest the rate limiting step in these systems was the transfer of electrons from strain ES6 to the intermediate or terminal electron acceptor whether that was Cr(VI), Fe(III), or AQDS.

Polyhydroxyalkanoate quantification in organic wastes and pure cultures using a single-step extraction and 1H NMR analysis.

In this study, a proton nuclear magnetic resonance (1H NMR) method was developed to quantitatively analyze polyhydroxyalkanoate (PHA) content in Cupriavidus necator H16, Azotobacter vinelandii AvOP, and mixed microbial cultures from the effluent of an agricultural waste treatment anaerobic digester. In contrast to previous methods, a single-step PHA extractive method using deuterated chloroform was established, thereby facilitating direct 1H NMR analysis. The accuracy of the method was verified through comparison with well-established gas chromatography (GC) methanolysis techniques. Nile blue fluorescence staining was also carried out to serve as an independent and qualitative indicator of intracellular PHA content. The results indicate that the 1H NMR method is appropriate for rapid and non-destructive quantification of overall PHA content and determination of PHA copolymer composition in a variety of cultures. Notably, this technique was effective in measuring PHA content in full-strength waste samples where high concentrations of background impurities and organic compounds are present. The straightforward procedures minimize error-introducing steps, require less time and materials, and result in an accurate method suitable for routine analyses.

Reduction of environmental and energy footprint of microalgal biodiesel production through material and energy integration.

The life cycle impacts were assessed for an integrated microalgal biodiesel production system that facilitates energy- and nutrient- recovery through anaerobic digestion, and utilizes glycerol generated within the facility for additional heterotrophic biodiesel production. Results show that when external fossil energy inputs are lowered through process integration, the energy demand, global warming potential (GWP), and process water demand decrease significantly and become less sensitive to algal lipid content. When substitution allocation is used to assign additional credit for avoidance of fossil energy use (through utilization of recycled nutrients and biogas), GWP and water demand can, in fact, increase with increase in lipid content. Relative to stand-alone algal biofuel facilities, energy demand can be lowered by 3-14 GJ per ton of biodiesel through process integration. GWP of biodiesel from the integrated system can be lowered by up to 71% compared to petroleum fuel. Evaporative water loss was the primary water demand driver.

Comparative study of pyrolysis of algal biomass from natural lake blooms with lignocellulosic biomass.

Pyrolysis experiments were performed with algal and lignocellulosic feedstocks under similar reactor conditions for comparison of product (bio-oil, gas and bio-char) yields and composition. In spite of major differences in component bio-polymers, feedstock properties relevant to thermo-chemical conversions, such as overall C, H and O-content, C/O and H/C molar ratio as well as calorific values, were found to be similar for algae and lignocellulosic material. Bio-oil yields from algae and some lignocellulosic materials were similar; however, algal bio-oils were compositionally different and contained several N-compounds (most likely from protein degradation). Algal bio-char also had a significantly higher N-content. Overall, our results suggest that it is feasible to convert algal cultures deficient in lipids, such as nuisance algae obtained from natural blooms, into liquid fuels by thermochemical methods. As such, pyrolysis technologies being developed for lignocellulosic biomass may be directly applicable to algal feedstocks as well.

Influence of carbon sources and electron shuttles on ferric iron reduction by Cellulomonas sp. strain ES6.

Microbially reduced iron minerals can reductively transform a variety of contaminants including heavy metals, radionuclides, chlorinated aliphatics, and nitroaromatics. A number of Cellulomonas spp. strains, including strain ES6, isolated from aquifer samples obtained at the U.S. Department of Energy's Hanford site in Washington, have been shown to be capable of reducing Cr(VI), TNT, natural organic matter, and soluble ferric iron [Fe(III)]. This research investigated the ability of Cellulomonas sp. strain ES6 to reduce solid phase and dissolved Fe(III) utilizing different carbon sources and various electron shuttling compounds. Results suggest that Fe(III) reduction by and growth of strain ES6 was dependent upon the type of electron donor, the form of iron present, and the presence of synthetic or natural organic matter, such as anthraquinone-2,6-disulfonate (AQDS) or humic substances. This research suggests that Cellulomonas sp. strain ES6 could play a significant role in metal reduction in the Hanford subsurface and that the choice of carbon source and organic matter addition can allow for independent control of growth and iron reduction activity.

Multiple mechanisms of uranium immobilization by Cellulomonas sp. strain ES6.

Removal of hexavalent uranium (U(VI)) from aqueous solution was studied using a Gram-positive facultative anaerobe, Cellulomonas sp. strain ES6, under anaerobic, non-growth conditions in bicarbonate and PIPES buffers. Inorganic phosphate was released by cells during the experiments providing ligands for formation of insoluble U(VI) phosphates. Phosphate release was most probably the result of anaerobic hydrolysis of intracellular polyphosphates accumulated by ES6 during aerobic growth. Microbial reduction of U(VI) to U(IV) was also observed. However, the relative magnitudes of U(VI) removal by abiotic (phosphate-based) precipitation and microbial reduction depended on the buffer chemistry. In bicarbonate buffer, X-ray absorption fine structure (XAFS) spectroscopy showed that U in the solid phase was present primarily as a non-uraninite U(IV) phase, whereas in PIPES buffer, U precipitates consisted primarily of U(VI)-phosphate. In both bicarbonate and PIPES buffer, net release of cellular phosphate was measured to be lower than that observed in U-free controls suggesting simultaneous precipitation of U and PO4³â». In PIPES, U(VI) phosphates formed a significant portion of U precipitates and mass balance estimates of U and P along with XAFS data corroborate this hypothesis. High-resolution transmission electron microscopy (HR-TEM) and energy dispersive X-ray spectroscopy (EDS) of samples from PIPES treatments indeed showed both extracellular and intracellular accumulation of U solids with nanometer sized lath structures that contained U and P. In bicarbonate, however, more phosphate was removed than required to stoichiometrically balance the U(VI)/U(IV) fraction determined by XAFS, suggesting that U(IV) precipitated together with phosphate in this system. When anthraquinone-2,6-disulfonate (AQDS), a known electron shuttle, was added to the experimental reactors, the dominant removal mechanism in both buffers was reduction to a non-uraninite U(IV) phase. Uranium immobilization by abiotic precipitation or microbial reduction has been extensively reported; however, the present work suggests that strain ES6 can remove U(VI) from solution simultaneously through precipitation with phosphate ligands and microbial reduction, depending on the environmental conditions. Cellulomonadaceae are environmentally relevant subsurface bacteria and here, for the first time, the presence of multiple U immobilization mechanisms within one organism is reported using Cellulomonas sp. strain ES6.

Maximizing algal growth in batch reactors using sequential change in light intensity.

Algal growth requires optimal irradiance. In photobioreactors, optimal light requirements change during the growth cycle. At low culture densities, a high incident light intensity can cause photoinhibition, and in dense algal cultures, light penetration may be limited. Insufficient light supply in concentrated algae suspensions can create zones of dissimilar photon flux density inside the reactor, which can cause suboptimal algal growth. However, growth of dense cultures can also be impaired due to photoinhibition if cells are exposed to excessively high light intensities. In order to simultaneously maintain optimal growth and photon use efficiency, strategies for light supply must be based on cell concentrations in the culture. In this study, a lipid-producing microalgal strain, Neochloris oleoabundans, was grown in batch photobioreactors. Growth rates and biomass concentrations of cultures exposed to constant light were measured and compared with the growth kinetic parameters of cultures grown using sequentially increasing light intensities based on increasing culture densities during batch growth. Our results show that reactors operated under conditions of sequential increase in irradiance levels yield up to a 2-fold higher biomass concentration when compared with reactors grown under constant light without negatively impacting growth rates. In addition, this tailored light supply results in less overall photon use per unit mass of generated cells.

Detecting cellulase penetration into corn stover cell walls by immuno-electron microscopy.

In general, pretreatments are designed to enhance the accessibility of cellulose to enzymes, allowing for more efficient conversion. In this study, we have detected the penetration of major cellulases present in a commercial enzyme preparation (Spezyme CP) into corn stem cell walls following mild-, moderate- and high-severity dilute sulfuric acid pretreatments. The Trichoderma reesei enzymes, Cel7A (CBH I) and Cel7B (EG I), as well as the cell wall matrix components xylan and lignin were visualized within digested corn stover cell walls by immuno transmission electron microscopy (TEM) using enzyme- and polymer-specific antibodies. Low severity dilute-acid pretreatment (20 min at 100 degrees C) enabled <1% of the thickness of secondary cell walls to be penetrated by enzyme, moderate severity pretreatment at (20 min at 120 degrees C) allowed the enzymes to penetrate approximately 20% of the cell wall, and the high severity (20 min pretreatment at 150 degrees C) allowed 100% penetration of even the thickest cell walls. These data allow direct visualization of the dramatic effect dilute-acid pretreatment has on altering the condensed ultrastructure of biomass cell walls. Loosening of plant cell wall structure due to pretreatment and the subsequently improved access by cellulases has been hypothesized by the biomass conversion community for over two decades, and for the first time, this study provides direct visual evidence to verify this hypothesis. Further, the high-resolution enzyme penetration studies presented here provide insight into the mechanisms of cell wall deconstruction by cellulolytic enzymes.

Rheology of corn stover slurries at high solids concentrations--effects of saccharification and particle size.

The rheological characteristics of untreated and dilute acid pretreated corn stover (CS) slurries at high solids concentrations were studied under continuous shear using plate-plate type measurements. Slurry rheological behavior was examined as a function of insoluble solids concentration (10-40%), extent of pretreatment (0-75% removal of xylan) and particle size (-20 and -80 mesh). Results show that CS slurries exhibit shear-thinning behavior describable using a Casson model. Further, results demonstrate that the apparent viscosity and yield stress increase with increasing solids concentration (which corresponds to a decrease in free water). Dilute acid pretreatment leads to lower viscosity and yield stresses at equivalent solids concentrations, as does smaller particle size. Taken together, these findings are consistent with the hypothesis that the availability of free water in the slurry plays a significant role in determining its rheological behavior. In particular, as the free water content of the slurry decreases, e.g., with increasing solids concentration, the greater interaction among particles likely increases the apparent viscosity and yield stress properties of the slurry. The results also suggest that the availability of free water, and thereby slurry rheological properties, depend on the chemical composition of the corn stover as well as its physical characteristics such as particle size and porosity. Hydrophilic polymers within the cell wall, such as xylan or pectin, or larger pores within bigger particles, facilitate sequestration of water in the solid phase resulting in decreased availability of free water. Thus, dilute acid pretreated slurries, which contain smaller size particles having significantly lower xylan content than slurries of untreated milled stover, exhibit much lower viscosities and yield stresses than untreated slurries containing large particles at similar solid concentrations.

Permeable reactive biobarriers for in situ Cr(VI) reduction: bench scale tests using Cellulomonas sp. strain ES6.

Chromate (Cr(VI)) reduction studies were performed in bench scale flow columns using the fermentative subsurface isolate Cellulomonas sp. strain ES6. In these tests, columns packed with either quartz sand or hydrous ferric oxide (HFO)-coated quartz sand, were inoculated with strain ES6 and fed nutrients to stimulate growth before nutrient-free Cr(VI) solutions were injected. Results show that in columns containing quartz sand, a continuous inflow of 2 mg/L Cr(VI) was reduced to below detection limits in the effluent for durations of up to 5.7 residence times after nutrient injection was discontinued proving the ability of strain ES6 to reduce chromate in the absence of an external electron donor. In the HFO-containing columns, Cr(VI) reduction was significantly prolonged and effluent Cr(VI) concentrations remained below detectable levels for periods of up to 66 residence times after nutrient injection was discontinued. Fe was detected in the effluent of the HFO-containing columns throughout the period of Cr(VI) removal indicating that the insoluble Fe(III) bearing solids were being continuously reduced to form soluble Fe(II) resulting in prolonged abiotic Cr(VI) reduction. Thus, growth of Cellulomonas within the soil columns resulted in formation of permeable reactive barriers that could reduce Cr(VI) and Fe(III) for extended periods even in the absence of external electron donors. Other bioremediation systems employing Fe(II)-mediated reactions require a continuous presence of external nutrients to regenerate Fe(II). After depletion of nutrients, contaminant removal within these systems occurs by reaction with surface-associated Fe(II) that can rapidly become inaccessible due to formation of crystalline Fe-minerals or other precipitates. The ability of fermentative organisms like Cellulomonas to reduce metals without continuous nutrient supply in the subsurface offers a viable and economical alternative technology for in situ remediation of Cr(VI)-contaminated groundwater through formation of permeable reactive biobarriers (PRBB).

Deposition of lignin droplets produced during dilute acid pretreatment of maize stems retards enzymatic hydrolysis of cellulose.

Electron microscopy of lignocellulosic biomass following high-temperature pretreatment revealed the presence of spherical formations on the surface of the residual biomass. The hypothesis that these droplet formations are composed of lignins and possible lignin carbohydrate complexes is being explored. Experiments were conducted to better understand the formation of these "lignin" droplets and the possible implications they might have on the enzymatic saccharification of pretreated biomass. It was demonstrated that these droplets are produced from corn stover during pretreatment under neutral and acidic pH at and above 130 degrees C, and that they can deposit back onto the surface of residual biomass. The deposition of droplets produced under certain pretreatment conditions (acidic pH; T > 150 degrees C) and captured onto pure cellulose was shown to have a negative effect (5-20%) on the enzymatic saccharification of this substrate. It was noted that droplet density (per unit area) was greater and droplet size more variable under conditions where the greatest impact on enzymatic cellulose conversion was observed. These results indicate that this phenomenon has the potential to adversely affect the efficiency of enzymatic conversion in a lignocellulosic biorefinery.

Solubilization, solution equilibria, and biodegradation of PAH's under thermophilic conditions.

Biodegradation rates of PAHs are typically low at mesophilic conditions and it is believed that the kinetics of degradation is controlled by PAH solubility and mass transfer rates. Solubility tests were performed on phenanthrene, fluorene and fluoranthene at 20 degrees C, 40 degrees C and 60 degrees C and, as expected, a significant increase in the equilibrium solubility concentration and of the rate of dissolution of these polycyclic aromatic hydrocarbons (PAHs) was observed with increasing temperature. A first-order model was used to describe the PAH dissolution kinetics and the thermodynamic property changes associated with the dissolution process (enthalpy, entropy and Gibb's free energy of solution) were evaluated. Further, other relevant thermodynamic properties for these PAHs, including the activity coefficients at infinite dilution, Henry's law constants and octanol-water partition coefficients, were calculated in the temperature range 20-60 degrees C. In parallel with the dissolution studies, three thermophilic Geobacilli were isolated from compost that grew on phenanthrene at 60 degrees C and degraded the PAH more rapidly than other reported mesophiles. Our results show that while solubilization rates of PAHs are significantly enhanced at elevated temperatures, the biodegradation of PAHs under thermophilic conditions is likely mass transfer limited due to enhanced degradation rates.

Isolation and characterization of Cr(VI) reducing Cellulomonas spp. from subsurface soils: implications for long-term chromate reduction.

Microbial enrichments from Cr(VI) contaminated and uncontaminated US Department of Energy Hanford Site sediments produced Cr(VI) reducing consortia when grown in the presence of Cr(VI) with acetate, D-xylose or glycerol as a carbon and energy source. Eight of the nine isolates from the consortia were Gram positive and four of these were identified by 16S rRNA sequence homology and membrane fatty acid composition as belonging to the genus Cellulomonas. Two strains, ES6 and WS01, were further examined for their ability to reduce Cr(VI) under growth and non-growth conditions. During fermentative growth on D-xylose, ES6 and WS01 decreased aqueous Cr(VI) concentrations from 0.04 mM Cr(VI) to below the detection limit (0.002 mM Cr(VI)) in less than three days and retained their ability to reduce Cr(VI) even after four months of incubation. Washed ES6 and WS01 cells also reduced Cr(VI) under non-growth conditions for over four months, both with and without the presence of an exogenous electron donor. K-edge XANES spectroscopy confirmed the reduction of Cr(VI) to Cr(III). The ability to reduce Cr(VI) after growth had stopped and in the absence of an external electron donor, suggests that stimulation of these types of organisms may lead to effective long-term, in situ passive reactive barriers for Cr(VI) removal. Our results indicate that Cr(VI) reduction by indigenous Cellulomonas spp. may be a potential method of in situ bioremediation of Cr(VI) contaminated sediment and groundwater.

Catalyst transport in corn stover internodes: elucidating transport mechanisms using Direct Blue-I.

The transport of catalysts (chemicals and enzymes) within plant biomass is believed to be a major bottleneck during thermochemical pretreatment and enzymatic conversion of lignocellulose. Subjecting biomass to size reduction and mechanical homogenization can reduce catalyst transport limitations; however, such processing adds complexity and cost to the overall process. Using high-resolution light microscopy, we have monitored the transport of an aqueous solution of Direct Blue-I (DB-I) dye through intact corn internodes under a variety of impregnation conditions. DB-I is a hydrophilic anionic dye with affinity for cellulose. This model system has enabled us to visualize likely barriers and mechanisms of catalyst transport in corn stems. Microscopic images were compared with calculated degrees of saturation (i.e., volume fraction of internode void space occupied by dye solution) to correlate impregnation strategies with dye distribution and transport mechanisms. Results show the waxy rind exterior and air trapped within individual cells to be the major barriers to dye transport, whereas the vascular bundles, apoplastic continuum (i.e., the intercellular void space at cell junctions), and fissures formed during the drying process provided the most utilized pathways for transport. Although representing only 20-30% of the internode volume, complete saturation of the apoplast and vascular bundles by fluid allowed dye contact with a majority of the cells in the internode interior.