Plasticity, elasticity, and adhesion energy of plant cell walls: nanometrology of lignin loss using atomic force microscopy.

The complex organic polymer, lignin, abundant in plants, prevents the efficient extraction of sugars from the cell walls that is required for large scale biofuel production. Because lignin removal is crucial in overcoming this challenge, the question of how the nanoscale properties of the plant cell ultrastructure correlate with delignification processes is important. Here, we report how distinct molecular domains can be identified and how physical quantities of adhesion energy, elasticity, and plasticity undergo changes, and whether such quantitative observations can be used to characterize delignification. By chemically processing biomass, and employing nanometrology, the various stages of lignin removal are shown to be distinguished through the observed morphochemical and nanomechanical variations. Such spatially resolved correlations between chemistry and nanomechanics during deconstruction not only provide a better understanding of the cell wall architecture but also is vital for devising optimum chemical treatments.

Opto-nanomechanical spectroscopic material characterization.

The non-destructive, simultaneous chemical and physical characterization of materials at the nanoscale is an essential and highly sought-after capability. However, a combination of limitations imposed by Abbe diffraction, diffuse scattering, unknown subsurface, electromagnetic fluctuations and Brownian noise, for example, have made achieving this goal challenging. Here, we report a hybrid approach for nanoscale material characterization based on generalized nanomechanical force microscopy in conjunction with infrared photoacoustic spectroscopy. As an application, we tackle the outstanding problem of spatially and spectrally resolving plant cell walls. Nanoscale characterization of plant cell walls and the effect of complex phenotype treatments on biomass are challenging but necessary in the search for sustainable and renewable bioenergy. We present results that reveal both the morphological and compositional substructures of the cell walls. The measured biomolecular traits are in agreement with the lower-resolution chemical maps obtained with infrared and confocal Raman micro-spectroscopies of the same samples. These results should prove relevant in other fields such as cancer research, nanotoxicity, and energy storage and production, where morphological, chemical and subsurface studies of nanocomposites, nanoparticle uptake by cells and nanoscale quality control are in demand.

Karhunen-Loève treatment to remove noise and facilitate data analysis in sensing, spectroscopy and other applications.

Resolving weak spectral variations in the dynamic response of materials that are either dominated or excited by stochastic processes remains a challenge. Responses that are thermal in origin are particularly relevant examples due to the delocalized nature of heat. Despite its inherent properties in dealing with stochastic processes, the Karhunen-Loève expansion has not been fully exploited in measurement of systems that are driven solely by random forces or can exhibit large thermally driven random fluctuations. Here, we present experimental results and analysis of the archetypes (a) the resonant excitation and transient response of an atomic force microscope probe by the ambient random fluctuations and nanoscale photothermal sample response, and (b) the photothermally scattered photons in pump-probe spectroscopy. In each case, the dynamic process is represented as an infinite series with random coefficients to obtain pertinent frequency shifts and spectral peaks and demonstrate spectral enhancement for a set of compounds including the spectrally complex biomass. The considered cases find important applications in nanoscale material characterization, biosensing, and spectral identification of biological and chemical agents.

Nanometrology of delignified Populus using mode synthesizing atomic force microscopy.

The study of the spatially resolved physical and compositional properties of materials at the nanoscale is increasingly challenging due to the level of complexity of biological specimens such as those of interest in bioenergy production. Mode synthesizing atomic force microscopy (MSAFM) has emerged as a promising metrology tool for such studies. It is shown that, by tuning the mechanical excitation of the probe-sample system, MSAFM can be used to dynamically investigate the multifaceted complexity of plant cells. The results are argued to be of importance both for the characteristics of the invoked synthesized modes and for accessing new features of the samples. As a specific system to investigate, we present images of Populus, before and after a holopulping treatment, a crucial step in the biomass delignification process.

Optomechanical spectroscopy with broadband interferometric and quantum cascade laser sources.

The spectral tunability of semiconductor-metal multilayer structures can provide a channel for the conversion of light into useful mechanical actuation. Responses of suspended silicon, silicon nitride, chromium, gold, and aluminum microstructures are shown to be utilized as a detector for visible and IR spectroscopy. Both dispersive and interferometric approaches are investigated to delineate the potential use of the structures in spatially resolved spectroscopy and spectrally resolved microscopy. The thermoplasmonic, spectral absorption, interference effects, and the associated energy deposition that contributes to the mechanical response are discussed to describe the potential of optomechanical detection in future integrated spectrometers.

Spectroscopy and atomic force microscopy of biomass.

Scanning probe microscopy has emerged as a powerful approach to a broader understanding of the molecular architecture of cell walls, which may shed light on the challenge of efficient cellulosic ethanol production. We have obtained preliminary images of both Populus and switchgrass samples using atomic force microscopy (AFM). The results show distinctive features that are shared by switchgrass and Populus. These features may be attributable to the lignocellulosic cell wall composition, as the collected images exhibit the characteristic macromolecular globule structures attributable to the lignocellulosic systems. Using both AFM and a single case of mode synthesizing atomic force microscopy (MSAFM) to characterize Populus, we obtained images that clearly show the cell wall structure. The results are of importance in providing a better understanding of the characteristic features of both mature cells as well as developing plant cells. In addition, we present spectroscopic investigation of the same samples.

Reductive transformation of methyl parathion by the cyanobacterium Anabaena sp. strain PCC7120.

Organophosphorus compounds are toxic chemicals that are applied worldwide as household pesticides and for crop protection, and they are stockpiled for chemical warfare. As a result, they are routinely detected in air and water. Methods and routes of biodegradation of these compounds are being sought. We report that under aerobic, photosynthetic conditions, the cyanobacterium Anabaena sp. transformed methyl parathion first to o,o-dimethyl o-p-nitrosophenyl thiophosphate and then to o,o-dimethyl o-p-aminophenyl thiophosphate by reducing the nitro group. The process of methyl parathion transformation occurred in the light, but not in the dark. Methyl parathion was toxic to cyanobacteria in the dark but did not affect their viability in the light. Methyl parathion transformation was not affected by mutations in the genes involved in nitrate reduction in cyanobacteria.

Enzyme stabilization by covalent binding in nanoporous sol-gel glass for nonaqueous biocatalysis.

A unique nanoporous sol-gel glass possessing a highly ordered porous structure (with a pore size of 153 A in diameter) was examined for use as a support material for enzyme immobilization. A model enzyme, alpha-chymotrypsin, was efficiently bound onto the glass via a bifunctional ligand, trimethoxysilylpropanal, with an active enzyme loading of 0.54 wt%. The glass-bound chymotrypsin exhibited greatly enhanced stability both in aqueous solution and organic solvents. The half-life of the glass-bound alpha-chymotrypsin was >1000-fold higher than that of the native enzyme, as measured either in aqueous buffer or anhydrous methanol. The enhanced stability in methanol, which excludes the possibility of enzyme autolysis, particularly reflected that the covalent binding provides effective protection against enzyme inactivation caused by structural denaturation. In addition, the activity of the immobilized alpha-chymotrypsin was also much higher than that of the native enzyme in various organic solvents. From these results, it appears that the glass-enzyme complex developed in the present work can be used as a high-performance biocatalyst for various chemical processing applications, particularly in organic media. Published by John Wiley & Sons

Enhancement of the conversion of toluene by Pseudomonas putida F-1 using organic cosolvents.

Pseudomonas putida F-1 (ATCC700007) was used as a model organism in stirred tank reactors to study conversion enhancement of poorly soluble substrates by organic cosolvents. After a literature study, silicone oil was used as a solvent system to enhance the mass transfer rate. To study the benefits of the organic solvent addition, batch experiments were conducted in two side-by-side fermentation vessels (experimental and control) at three different levels of silicone oil (10, 30, and 50%). Results showed that the presence of silicone oil resulted in a 100% increase in the toluene mass transfer compared to the control. Experiments in continuous stirred-tank reactors showed that improved conversion could be obtained at higher agitation rates.

Ethanol production from glucose and xylose by immobilized Zymomonas mobilis CP4(pZB5).

Fermentation of glucose-xylose mixtures to ethanol was investigated in batch and continuous experiments using immobilized recombinant Zymomonas mobilis CP4(pZB5). This microorganism was immobilized by entrapment in kappa-carrageenan beads having a diameter of 1.5-2.5 mm. Batch experiments showed that the immobilized cells cofermented glucose and xylose to ethanol and that the presence of glucose improved the xylose utilization rate. Batch fermentation of rice straw hydrolysate containing 76 g/L of glucose and 33.8 g/L of xylose gave an ethanol concentration of 44.3 g/L after 24 h, corresponding to a yield of 0.46 g of ethanol/g of sugars. Comparable results were achieved with a synthetic sugar control. Continuous fermentation experiments were performed in a laboratory-scale fluidized-bed bioreactor (FBR). Glucose-xylose feed mixtures were pumped through the FBR at residence times of 2-4 h. Glucose conversion to ethanol was maintained above 98% in all experiments. Xylose conversion to ethanol was highest at 91.5% for a feed containing 50 g/L of glucose and 13 g/L of xylose at a dilution rate of 0.24/h. The xylose conversion to ethanol decreased with increasing feed xylose concentration, dilution rate, and age of the immobilized cells. Volumetric ethanol productivities in the range of 6.5-15.3 g/L.h were obtained. The improved productivities achieved in the FBR compared to other bioreactor systems can help in reducing the production costs of fuel ethanol from lignocellulosic sugars.

Influence of high biomass concentrations on alkane solubilities.

Alkane solubilities were measured experimentally for high-density biomass. The resulting Henry's law constants for propane were found to decrease significantly for both dense yeast suspensions and an actual propane-degrading biofilm consortium. At the biomass densities of a typical biofilm, propane solubility was about an order of magnitude greater than that in pure water. For example, a dense biofilm had a propane Henry's law constant of 0.09+/-0.04 atm m(3) mol(-1) compared to 0.6+/-0.1 atm m(3) mol(-1) measured in pure water. The results were modeled with mixing rules and compared with octanol-water mixtures. Hydrogels (agar) and salts decreased the alkane solubility. By considering a theoretical solubility of propane in dry biomass, estimates were made of intrinsic Henry's law constants for propane in pure yeast and biomass, which were 13+/-2 and 5+/-2 atm kg biomass mol(-1) for yeast and biofilm consortium, respectively.

Ethanol production from corn starch in a fluidized-bed bioreactor.

The production of ethanol from industrial dry-milled corn starch was studied in a laboratory-scale fluidized-bed bioreactor using immobilized biocatalysts. Saccharification and fermentation were carried out either simultaneously or separately. Simultaneous saccharification and fermentation (SSF) experiments were performed using small, uniform kappa-carrageenan beads (1.5-2.5 mm in diameter) of co-immobilized glucoamylase and Zymomonas mobilis. Dextrin feeds obtained by the hydrolysis of 15% drymilled corn starch were pumped through the bioreactor at residence times of 1.5-4 h. Single-pass conversion of dextrins ranged from 54-89%, and ethanol concentrations of 23-36 g/L were obtained at volumetric productivities of 9-15 g/L-h. Very low levels of glucose were observed in the reactor, indicating that saccharification was the rate-limiting step. In separate hydrolysis and fermentation (SHF) experiments, dextrin feed solutions of 150-160 g/L were first pumped through an immobilized-glucoamylase packed column. At 55 degrees C and a residence time of 1 h, greater than 95% conversion was obtained, giving product streams of 162-172 g glucose/L. These streams were then pumped through the fluidized-bed bioreactor containing immobilized Z. mobilis. At a residence time of 2 h, 94% conversion and ethanol concentration of 70 g/L were achieved, resulting in an overall process productivity of 23 g/L-h. At residence times of 1.5 and 1 h, conversions of 75 and 76%, ethanol concentrations of 49 and 47 g/L, and overall process productivities of 19 and 25 g/L-h, respectively, were achieved.

Production of ethanol from starch by co-immobilized Zymomonas mobilis-glucoamylase in a fluidized-bed reactor.

The production of ethanol from starch was studied in a fluidized-bed reactor (FBR) using co-immobilized Zymomonas mobilis and glucoamylase. The FBR was a glass column of 2.54 cm in diameter and 120 cm in length. The Z. mobilis and glucoamylase were co-immobilized within small uniform beads (1.2-2.5 mm diameter) of kappa-carrageenan. The substrate for ethanol production was a soluble starch. Light steep water was used as the complex nutrient source. The experiments were performed at 35 degrees C and pH range of 4.0-5.5. The substrate concentrations ranged from 40 to 185 g/L, and the feed rates from 10 to 37 mL/min. Under relaxed sterility conditions, the FBR was successfully operated for a period of 22 d, during which no contamination or structural failure of the biocatalyst beads was observed. Volumetric productivity as high as 38 g ethanol/(Lh), which was 74% of the maximum expected value, was obtained. Typical ethanol volumetric productivity was in the range of 15-20 g/(Lh). The average yield was 0.49 g ethanol/g substrate consumed, which was 90% of the theoretical yield. Very low levels of glucose were observed in the reactor, indicating that starch hydrolysis was the rate-limiting step.

Microbial removal of alkanes from dilute gaseous waste streams: kinetics and mass transfer considerations.

Treatment of dilute gaseous hydrocarbon waste streams remains a current need for many industries, particularly as increasingly stringent environmental regulations and oversight force emission reduction. Biofiltration systems hold promise for providing low-cost alternatives to more traditional, energy-intensive treatment methods such as incineration and adsorption. Elucidation of engineering principles governing the behavior of such systems, including mass transfer limitations, will broaden their applicability. Our processes exploit a microbial consortium to treat a mixture of 0.5% n-pentane and 0.5% isobutane in air. Since hydrocarbon gases are sparingly soluble in water, good mixing and high surface area between the gas and liquid phases are essential for biodegradation to be effective. One liquid-continuous columnar bioreactor was operated for more than 30 months with continued degradation of n-pentane and isobutane as sole carbon and energy sources. The maximum degradation rate observed in this gas-recycle system was 2 g of volatile organic compounds (VOC)/(m3.h). A trickle-bed bioreactor was operated continuously for over 24 months to provide a higher surface area (using a structured packing) with increased rates. Degradation rates consistently achieved were approximately 50 g of VOC/(m3.h) via single pass in this gas-continuous columnar system. Effective mass transfer coefficients comparable to literature values were also measured for this reactor; these values were substantially higher than those found in the gas-recycle reactor. Control of biomass levels was implemented by limiting the level of available nitrogen in the recirculating aqueous media, enabling long-term stability of reactor performance.

Performance of coimmobilized yeast and amyloglucosidase in a fluidized bed reactor for fuel ethanol production.

The performance of coimmobilized Saccharomyces cerevisiae and amyloglucosidase (AG) was evaluated in a fluidized-bed reactor. Soluble starch and yeast extracts were used as feed stocks. Conversion of soluble starch streams to ethanol has potential practical applications in corn dry and wet milling and in developmental lignocellulosic processes. The biocatalyst performed well, and demonstrated no significant loss of activity or physical integrity during 10 wk of continuous operation. The reactor was easily operated and required no pH control. No operational problems were encountered from bacterial contaminants even though the reactor was operated under nonsterile conditions over the entire course of experiments. Productivities ranged between 25 and 44 g ethanol/L/h/. The experiments demonstrated that ethanol inhibition and bed loading had significant effects on reactor performance.

Production of succinic acid by Anaerobiospirillum succiniciproducens.

The effect of an external supply of carbon dioxide and pH on the production of succinic acid by Anaerobiospirillum succiniciproducens was studied. In a rich medium containing yeast extract and peptone, when the external carbon dioxide supply was provided by a 1.5M Na2CO3 solution that also was used to maintain the pH at 6.0, no additional carbon dioxide supply was needed. In fact, sparging CO2 gas into the fermenter at 0.025 L/L-min or higher rates resulted in significant decreases in both production rate and yield of succinate. Under the same conditions, the production of the main by-product acetate was not affected by sparging CO2 gas into the fermenter. The optimum pH (pH 6.0) for the production of succinic acid was found to be in agreement with results previously reported in the literature. Succinic acid production also was studied in an industrial-type inexpensive medium in which light steep water was the only source of organic nutrients. At pH 6.0 and with a CO2 gas sparge rate of 0.08 L/L-min, succinate concentration reached a maximum of 32 g/L in 27 h with a yield of 0.99 g succinate/g glucose consumed.

Development of a predictive description of an immobilized-cell, three-phase, fluidized-bed bioreactor.

A fully predictive mathematical description of a three-phase, tapered, fluidized-bed bioreactor is developed. This mathematical model includes the effects of the tapered bed, variable dispersion coefficient, and variable solid holdup upon the concentration profiles developed in the bed. In addition, the effect of the concentration profile which is developed inside the biocatalyst bead is included by means of an effectiveness factor calculation. Using accepted correlations for the dispersion coefficient and for the liquid, gas, and solid holdup in the bed, the model is fully predictive. The model was found to adequately predict experimental obtained concentration profiles. Then, the model was used to examine the various phase holdups through the bed and the degree to which the dispersion coefficient varied through the bed. The effect of changes in these calculated variables upon the reaction rate is discussed. (c) 1995 John Wiley & Sons, Inc.

A proposed biparticle fluidized-bed for lactic acid fermentation and simultaneous adsorption.

A bioreactor configuration is proposed for simultaneous fermentation and separation of the desired product. The bioreactor consists of a columnar fluidized bed of immobilized microorganisms. Denser adsorbent particles are added to this column. These adsorbent particles fall through the bed, absorb the product, and are removed from the base of the columnar reactor. The system hydrodynamics and the separability of the two types of particles were confirmed for low-density gel beads. The addition of the adsorbent, activated carbon, to a fermentation of Lactobacillus delbreuckii absorbed lactic acid. The addition of adsorbent enhanced the fermentation and controlled the pH.

Size changes associated with metal adsorption onto modified bone gelatin beads.

Significant quantities of heavy metals will adsorb onto modified bone gelatin beads. As this adsorption occurs, the bead can undergo a substantial volume change. Research has shown that the equilibrium bead diameter was a function of the solution pH and the ion concentration in the solution. Here, we demonstrate that under certain conditions, the volume of the beads that absorbed the metal was only 35% of the bead volume when no metal was adsorbed. By taking advantages of these size changes, a fluidized-bed separator can be operated such that natural segregation of loaded beads occurs. This phenomenon may facilitate the design of continous separators for the recovery and concentration of heavy-metal-contaminated waters. These concepts are demonstrated using Cu(2+) adsorption onto such beads.