Sridhar Viamajala

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 °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 °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.

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.

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.

Toxic Effects of Chromium(VI) on Anaerobic and Aerobic Growth of Shewanella oneidensis MR‐1

Cr(VI) was added to early‐ and mid‐log‐phase Shewanella oneidensis ( S. oneidensis) MR‐1 cultures to study the physiological state‐dependent toxicity of Cr(VI). Cr(VI) reduction and culture growth were measured during and after Cr(VI) reduction. Inhibition of growth was observed when Cr(VI) was added to cultures of MR‐1 growing aerobically or anaerobically with fumarate as the terminal electron acceptor. Under anaerobic conditions, there was immediate cessation of growth upon addition of Cr(VI) in early‐ and mid‐log‐phase cultures. However, once Cr(VI) was reduced below detection limits (0.002 mM), the cultures resumed growth with normal cell yield values observed. In contrast to anaerobic MR‐1 cultures, addition of Cr(VI) to aerobically growing cultures resulted in a gradual decrease of the growth rate. In addition, under aerobic conditions, lower cell yields were also observed with Cr(VI)‐treated cultures when compared to cultures that were not exposed to Cr(VI). Differences in response to Cr(VI) between aerobically and anaerobically growing cultures indicate that Cr(VI) toxicity in MR‐1 is dependent on the physiological growth condition of the culture. Cr(VI) reduction has been previously studied in Shewanella spp., and it has been proposed that Shewanella spp.may be used in Cr(VI) bioremediation systems. Studies of Shewanella spp. provide valuable information on the microbial physiology of dissimilatory metal reducing bacteria; however, our study indicates that S. oneidensis MR‐1 is highly susceptible to growth inhibition by Cr(VI) toxicity, even at low concentrations [0.015 mM Cr(VI)].

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 PO  43− . 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. Biotechnol. Bioeng. 2011;108: 264–276.

Chromate reduction in Shewanella oneidensis MR-1 is an inducible process associated with anaerobic growth

Cr(VI) reduction was observed during tests with Shewanella oneidensis MR‐1 (previously named S. putrefaciens MR‐1) while being grown with nitrate or fumarate as electron acceptor and lactate as electron donor. From the onset of anoxic growth on fumarate, we measured a gradual and progressive increase in the specific Cr(VI) reduction rate with incubation time until a maximum was reached at late exponential/early stationary phase. Under denitrifying conditions, the specific Cr(VI) reduction rate was inhibited by nitrite, which is produced during nitrate reduction. However, once nitrite was consumed, the specific reduction rate increased until a maximum was reached, again during the late exponential/early stationary phase. Thus, under both fumarate‐ and nitrate‐reducing conditions, an increase in the specific Cr(VI) reduction rate was observed as the microorganisms transition from oxic to anoxic growth conditions, presumably as a result of induction of enzyme systems capable of reducing Cr(VI). Although Cr(VI) reduction has been studied in MR‐1 and in other facultative bacteria under both oxic and anoxic conditions, a transition in specific reduction rates based on physiological conditions during growth is a novel finding. Such physiological responses provide information required for optimizing the operation of in situ systems for remediating groundwater contaminated with heavy metals and radionuclides, especially those that are characterized by temporal variations in oxygen content. Moreover, such information may point the way to a better understanding of the cellular processes used by soil bacteria to accomplish Cr(VI) reduction.

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°C) enabled <1% of the thickness of secondary cell walls to be penetrated by enzyme, moderate severity pretreatment at (20 min at 120°C) allowed the enzymes to penetrate ∼20% of the cell wall, and the high severity (20 min pretreatment at 150°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. Biotechnol. Bioeng. 2009;103: 480–489.

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.

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.

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.