Herbert J. Strobel

Proteomic profile changes in membranes of ethanol-tolerant Clostridium thermocellum

Clostridium thermocellum, a cellulolytic, thermophilic anaerobe, has potential for commercial exploitation in converting fibrous biomass to ethanol. However, ethanol concentrations above 1% (w/v) are inhibitory to growth and fermentation, and this limits industrial application of the organism. Recent work with ethanol-adapted strains suggested that protein changes occurred during ethanol adaptation, particularly in the membrane proteome. A two-stage Bicine-doubled sodium dodecyl sulfate-polyacrylamide gel electrophoresis protocol was designed to separate membrane proteins and circumvent problems associated with membrane protein analysis using traditional gel-based proteomics approaches. Wild-type and ethanol-adapted C. thermocellum membranes displayed similar spot diversity and approximately 60% of proteins identified from purified membrane fractions were observed to be differentially expressed in the two strains. A majority (73%) of differentially expressed proteins were down-regulated in the ethanol-adapted strain. Based on putative identifications, a significant proportion of these down-regulated proteins were involved with carbohydrate transport and metabolism. Approximately one-third of the up-regulated proteins in the ethanol-adapted species were associated with chemotaxis and signal transduction. Overall, the results suggested that membrane-associated proteins in the ethanol-adapted strain are either being synthesized in lower quantities or not properly incorporated into the cell membrane.

Economic Analysis of Cellulase Production Methods for Bio-Ethanol

The cost of cellulase enzymes has limited the feasibility of producing ethanol from fibrous biomass. Traditional submerged fermentation (SmF) was compared to an alternative method of producing cellulase, solid state cultivation (SSC). Results from an economic analysis indicated that the unit costs for cellulase enzyme production were

Compressed solvents for the extraction of fermentation products within a hollow fiber membrane contactor

15.67 (The prices are all 2004 prices in this article, except otherwise stated. We deflated newer prices to 2004 prices using a deflation factor 0.9 per year and inflated older prices to 2004 prices using an inflation factor 1.1.) per kilogram (

Analysis of composition and structure of Clostridium thermocellum membranes from wild-type and ethanol-adapted strains

/kg) and

Growth of the thermophilic bacterium Clostridium thermocellum in continuous culture

40.36/kg, for the SSC and SmF methods, respectively, while the corresponding market price was over

Screening of thermophilic anaerobic bacteria for solid substrate cultivation on lignocellulosic substrates.

90.00/kg. A sensitivity analysis conducted using Monte Carlo simulation also suggests that the unit cost of production using the SSC method is lower than the unit cost of production using SmF with a certainty of 99.6% (9,959 out of 10,000 cases). These results indicate that the SSC method may be a more economical method of cellulase production, thereby reducing bio-ethanol production costs. SSC may increase the potential that bio-ethanol will become a viable supplemental fuel source in light of current economic, political, and environmental issues.

Cellobiose and cellodextrin metabolism by the ruminal bacterium Ruminococcus albus

Abstract The feasibility of extracting aqueous ethanol and acetone within a hollow fiber membrane contactor (HFC) has been examined using compressed CO 2 (69 bar), ethane (69 bar), and propane (34.5 bar) at ambient temperature. Ethanol and acetone were chosen as ‘model’ fermentation products to further examine the potential for extractive fermentation with compressed fluids. Aqueous and compressed solvent streams were contacted within a single hydrophobic isotactic polypropylene membrane fiber (0.6 mm ID; 106.7 cm in length; 75% porosity), providing a porous barrier between the two immiscible phases. The amount of solute extracted was determined as a function of the aqueous flowrate (tubeside) and molar solvent to feed ratio. The amount of aqueous ethanol (10 wt.%) and acetone (10 wt.%) extracted from binary feed solutions with compressed propane ranged from 6.4 to 14.3% and 21.8 to 90.6%, respectively, as a function of the aqueous flowrate (0.1 to 2 ml/min) and molar solvent to feed ratio ( S/F =1 to 10). Comparatively, ethanol extraction with compressed CO 2 ranged from 4.7 to 31.9% with similar variations in the aqueous flowrate (0.1 to 1 ml/min) and molar solvent to feed ratio (3 and 10). Acetone extracted with CO 2 ranged from 67.9 to 96.1% when varying the aqueous flowrate (0.1 to 1 ml/min) at a molar solvent to feed ratio of 3. Ternary ethanol/acetone/water mixtures were also examined to determine the effect of multi-solute aqueous solutions. The effect of aqueous and compressed fluid flows on extraction are interpreted based on the equilibrium distributions of the solutes between water and the compressed fluid (estimated using a group contribution association equation of state (GCA–EOS)) and the mass transfer characteristics of the compressed fluid.

Investigation of the metabolic inhibition observed in solid-substrate cultivation of Clostridium thermocellum on cellulose

Clostridium thermocellum is a candidate organism for consolidated bioprocessing of lignocellulosic biomass into ethanol. However, commercial use is limited due to growth inhibition at modest ethanol concentrations. Recently, an ethanol-adapted strain of C. thermocellum was produced. Since ethanol adaptation in microorganisms has been linked to modification of membrane lipids, we tested the hypothesis that ethanol adaptation in C. thermocellum involves lipid modification by comparing the fatty acid composition and membrane anisotropy of wild-type and ethanol-adapted strains. Derivatization to fatty acid methyl esters provided quantitative lipid analysis. Compared to wild-type, the ethanol-adapted strain had a larger percentage of fatty acids with chain lengths >16:0 and showed a significant increase in the percentage of 16:0 plasmalogens. Structural identification of fatty acids was confirmed through mass spectral fragmentation patterns of picolinyl esters. Ethanol adaptation did not involve modification at sites of methyl branching or the unsaturation index. Comparison of steady-state fluorescence anisotropy experiments, in the absence and presence of ethanol, provided evidence for the effects of ethanol on membrane fluidity. In the presence of ethanol, both strains displayed increased fluidity by approximately 12%. These data support the model that ethanol adaptation was the result of fatty acid changes that increased membrane rigidity that counter-acted the fluidizing effect of ethanol.

High-Affinity Maltose Binding and Transport by the Thermophilic Anaerobe Thermoanaerobacter ethanolicus 39E

Clostridium thermocellum is an anaerobic thermophilic bacterium that produces enthanol from cellulosic substrates. When the organism was grown in continuous culture at dilution rates ranging from 0.04 to 0.25 h-1, growth yields on cellobiose were higher than on glucose, and even higher yields were observed on cellotetraose. However, differences in bacterial yield were much greater at slow growth rates, and it appeared that glucose-grown cells had a fourfold higher (0.41 g substrate/g protein/h) maintenance energy requirement than cellobiose-grown cultures. Cellobiose and glucose were co-utilized in dual substrate continuous culture, and this was in contrast to batch culture experiments which indicated that the organism preferred the disaccharide. These experiments demonstrate that carbohydrate utilization patterns in continuous culture are different from those in batch culture and that submaximal growth rates affect substrate preference and bioenergetic parameters. The mechanisms regulating carbohydrate use may be different in batch versus continuous culture.

Molecular analysis of the xylFGH operon, coding for xylose ABC transport, in Thermoanaerobacter ethanolicus

Interest in solid substrate cultivation (SSC) techniques is gaining for biochemical production from renewable resources; however, heat and mass transfer problems may limit application of this technique. The use of anaerboic thermophiles in SSC offers a unique solution to overcoming these challenges. The production potential of nine thermophilic anaerobic bacteria was examined on corn stover, sugar cane bagasse, paper pulp sludge, and wheat bran in submerged liquid cultivation (SmC) and SSC. Production of acetate, ethanol, and lactate was measured over a 10 day period, and total product concentrations were used to compare the performance of different organism‐substrate combinations using the two cultivation methods. Overall microbial activity in SmC and SSC was dependent on the organism and growth substrate. Clostridium thermocellum strains JW20, LQRI, and 27405 performed significantly better in SSC when grown on sugar cane bagasse and paper pulp sludge, producing at least 70 and 170 mM of total products, respectively. Growth of C. thermocellum strains in SSC on paper pulp sludge proved to be most favorable, generating at least twice the concentration of total products produced in SmC (p‐value < 0.05). Clostridium thermolacticum TC21 demonstrated growth on all substrates producing 30–80 and 60–116 mM of total product in SmC and SSC, respectively. Bacterial species with optimal growth temperatures of 70 °C grew best on wheat bran in SmC, producing total product concentrations of 45–75 mM. For some of the organism‐substrate combinations total end product concentrations in SSC exceeded those in SmC, indicating that SSC may be a promising alternative for microbial activity and value‐added biochemical production.