Degradation of phenolic compounds by the lignocellulose deconstructing thermoacidophilic bacterium Alicyclobacillus Acidocaldarius.

Alicyclobacillus acidocaldarius, a thermoacidophilic bacterium, has a repertoire of thermo- and acid-stable enzymes that deconstruct lignocellulosic compounds. The work presented here describes the ability of A. acidocaldarius to reduce the concentration of the phenolic compounds: phenol, ferulic acid, ρ-coumaric acid and sinapinic acid during growth conditions. The extent and rate of the removal of these compounds were significantly increased by the presence of micro-molar copper concentrations, suggesting activity by copper oxidases that have been identified in the genome of A. acidocaldarius. Substrate removal kinetics was first order for phenol, ferulic acid, ρ-coumaric acid and sinapinic acid in the presence of 50 µM copper sulfate. In addition, laccase enzyme assays of cellular protein fractions suggested significant activity on a lignin analog between the temperatures of 45 and 90 °C. This work shows the potential for A. acidocaldarius to degrade phenolic compounds, demonstrating potential relevance to biofuel production and other industrial processes.

Effect of pelleting on the enzymatic digestibility of corn stover.

Pelleting of lignocellulosic biomass to improve its transportation, storage and handling impacts subsequent processing and conversion. This work reports the role of high moisture pelleting in the enzymatic digestibility of corn stover prior to pretreatment, together with associated substrate characteristics. Pelleting increases the digestibility of unpretreated corn stover, from 8.2 to 15.5% glucan conversion, at 5% solid loading using 1 FPU Cellic® CTec2 per g solids. Compositional analysis indicates that loose and pelleted corn stover have similar non-dissolvable compositions, although their extractives are different. Enzymatic hydrolysis of corn stover after size reduction to normalize particle sizes and removal of extractives confirms that pelleting improves corn stover digestibility. Such differences may be explained by the decreased particle size, improved substrate accessibility, and hydrolysis of cross-linking structures induced by pelleting. These findings are useful for the development of processing schemes for sustainable and efficient use of lignocellulose.

New strategy for liquefying corn stover pellets.

The movement of solid material into and between unit operations within a biorefinery is a bottleneck in reaching design capacity, with formation of biomass slurries needed to introduce feedstock. Corn stover slurries have been achieved from dilute acid, pretreated materials resulting in slurry concentrations of up to about 150 g/L, above which flowability is compromised. We report a new strategy to liquefy corn stover at higher solids concentration (300 g/L) by initially cooking it with the enzyme mimetic maleic acid at 40 mM and 150 °C. This is followed by 6 h of enzymatic modification at 1 FPU (2.2 mg protein)/g solids, resulting in a yield stress of 171 Pa after 6 h and 58 Pa in 48 h compared to 6806 Pa for untreated stover. Mimetic treatment of corn stover pellets minimizes the inhibitory effect of xylo-oligomers on hydrolytic enzymes. This strategy allows for the delivery of solid lignocellulosic slurry into a pretreatment reactor by pumping, improving operability of a biorefinery.

Toxicity of select organic acids to the slightly thermophilic acidophile Acidithiobacillus caldus.

Acidithiobacillus caldus is a thermophilic acidophile found in commercial biomining, acid mine drainage systems, and natural environments. Previous work has characterized A. caldus as a chemolithotrophic autotroph capable of utilizing reduced sulfur compounds under aerobic conditions. Organic acids are especially toxic to chemolithotrophs in low-pH environments, where they diffuse more readily into the cell and deprotonate within the cytoplasm. In the present study, the toxic effects of oxaloacetate, pyruvate, 2-ketoglutarate, acetate, malate, succinate, and fumarate on A. caldus strain BC13 were examined under batch conditions. All tested organic acids exhibited some inhibitory effect. Oxaloacetate was observed to inhibit growth completely at a concentration of 250 microM, whereas other organic acids were completely inhibitory at concentrations of between 1,000 and 5,000 microM. In these experiments, the measured concentrations of organic acids decreased with time, indicating uptake or assimilation by the cells. Phospholipid fatty acid analyses indicated an effect of organic acids on the cellular envelope. Notable differences included an increase in cyclic fatty acids in the presence of organic acids, indicating possible instability of the cellular envelope. This was supported by field emission scanning-electron micrographs showing blebbing and sluffing in cells grown in the presence of organic acids.

Response of Halomonas campisalis to saline stress: changes in growth kinetics, compatible solute production and membrane phospholipid fatty acid composition.

The haloalkaliphile Halomonas campisalis, isolated near Soap Lake, Washington, was grown under both aerobic and denitrifying conditions from 0 to 260 g L(-1) NaCl, with optimal growth occurring at 20 and 30 g L(-1) NaCl, respectively. Halomonas campisalis was observed to produce high concentrations of compatible solutes, most notably ectoine (up to 500 mM within the cytoplasm), but hydroxyectoine and glycine betaine were also detected. The types and amounts of compatible solutes produced depended on salinity and specific growth rate, as well as on the terminal electron acceptor available (O(2) or NO(3) (-)). A decrease in ectoine production was observed with NO(3) (-) as compared with O(2) as the terminal electron acceptor. In addition, changes in the phospholipid fatty acid composition were measured with changing salinity. An increase in trans fatty acids was observed in the absence of salinity, and may be a response to membrane instability. Cyclic fatty acids were also observed to increase, both in the absence of salinity, and at very high salinities, indicating cell stress at these conditions.

Effects of ferrous sulfate, inoculum history, and anionic form on lead, zinc, and copper toxicity to Acidithiobacillus caldus strain BC13.

The current study reports the single and combined toxicities of Pb, Zn, and Cu to Acidithiobacillus caldus strain BC13. The observed half-maximal inhibitory concentrations (IC50), ± 95% confidence intervals, for Pb, Zn, and Cu were 0.9 ± 0.1 mM, 39 ± 0.5 mM, and 120 ± 8 mM, respectively. The observed minimum inhibitory concentrations (MIC) for Pb, Zn, and Cu were 7.5 mM, 75 mM, and 250 mM, respectively. When metals were presented in binary mixtures, the toxicities were less than additive. For example, when 50% of the Pb MIC and 50% of the Cu MIC were presented together, the specific growth rate was inhibited by only 59 ± 3%, rather than 100%. In addition, the presence of ferrous iron in the growth media decreased Pb and Zn toxicity to A. caldus strain BC13. The importance of inoculum history was evaluated by pre-adapting cultures through subsequent transfers in the presence of Pb, Zn, and Cu at their respective IC50s. After pre-adaptation, cultures had specific growth rates 39 ± 11, 32 ± 7, and 28 ± 12% higher in the presence of Pb, Zn, and Cu IC50s, respectively, compared with cultures that had not been pre-adapted. In addition, when cells exposed to the MICs of Pb, Zn, and Cu were harvested, washed, and re-inoculated into fresh, metal-free medium, they grew, showing that the cells remained viable with little residual toxicity. Finally, metal chlorides showed more toxicity than metal sulfates, and studies using sodium chloride or a mixture of metal sulfates and sodium chloride suggested that this was attributable to an additive combination of the metal and chloride toxicities.

Effects of cell condition, pH, and temperature on lead, zinc, and copper sorption to Acidithiobacillus caldus strain BC13.

This study describes the effects of cell condition, pH, and temperature on lead, zinc, and copper sorption to Acidithiobacillus caldus strain BC13 with a Langmuir model. Copper exhibited the highest loading capacity, 4.76 ± 0.28 mmol g(-1), to viable cells at pH 5.5. The highest k(L) (binding-site affinity) observed was 61.2 ± 3.0 L mmol(-1) to dehydrated cells at pH 4.0. The pHs that maximized loading capacities and binding-site affinities were generally between 4.0 and 5.5, where the sum of free-proton and complexed-metal concentrations was near a minimum. Of additional importance, lead, zinc, and copper sorbed to viable cells at pH values as low as 1.5. Previous studies with other acidithiobacilli did not measure viable-cell sorption below pH 4.0. In separate experiments, desorption studies showed that far less copper was recovered from viable cells than any other metal or cell condition, suggesting that uptake may play an important role in copper sorption by At. caldus strain BC13. To reflect an applied system, the sorption of metal mixtures was also studied. In these experiments, lead, zinc, and copper sorption from a tertiary mixture were 40.2 ± 4.3%, 28.7 ± 3.8%, and 91.3 ± 3.0%, respectively, of that sorbed in single-metal systems.

In silico approaches to study mass and energy flows in microbial consortia: a syntrophic case study.

BACKGROUND: Three methods were developed for the application of stoichiometry-based network analysis approaches including elementary mode analysis to the study of mass and energy flows in microbial communities. Each has distinct advantages and disadvantages suitable for analyzing systems with different degrees of complexity and a priori knowledge. These approaches were tested and compared using data from the thermophilic, phototrophic mat communities from Octopus and Mushroom Springs in Yellowstone National Park (USA). The models were based on three distinct microbial guilds: oxygenic phototrophs, filamentous anoxygenic phototrophs, and sulfate-reducing bacteria. Two phases, day and night, were modeled to account for differences in the sources of mass and energy and the routes available for their exchange. RESULTS: The in silico models were used to explore fundamental questions in ecology including the prediction of and explanation for measured relative abundances of primary producers in the mat, theoretical tradeoffs between overall productivity and the generation of toxic by-products, and the relative robustness of various guild interactions. CONCLUSION: The three modeling approaches represent a flexible toolbox for creating cellular metabolic networks to study microbial communities on scales ranging from cells to ecosystems. A comparison of the three methods highlights considerations for selecting the one most appropriate for a given microbial system. For instance, communities represented only by metagenomic data can be modeled using the pooled method which analyzes a community's total metabolic potential without attempting to partition enzymes to different organisms. Systems with extensive a priori information on microbial guilds can be represented using the compartmentalized technique, employing distinct control volumes to separate guild-appropriate enzymes and metabolites. If the complexity of a compartmentalized network creates an unacceptable computational burden, the nested analysis approach permits greater scalability at the cost of more user intervention through multiple rounds of pathway analysis.