Metabolic inhibition of Clostridium thermocellum, when grown in a high solids environment, was investigated by comparing submerged fermentation (SmF), solid-substrate cultivation (SSC) and solid-substrate cultivation with media replacement by periodic flushing (FSSC). Cellulose conversion extent and end-product concentrations were measured over time. SmF converted ∼65% of the cellulose in 240h (10days), whereas SSC converted <8% in the same period. FSSC converted approximately 25% and 47% of initial substrate after 240h; 45% and 71% of initial substrate after 25days, with media replacement every 24 and 12h, respectively. The SSC experienced higher initial production rates for all fermentation products, but could not sustain production rates. When acetate concentrations reached a critical point, the acetate decreased the intracellular volume of C. thermocellum cell suspensions at pH values similar to those observed in SSC. Acids produced by fermentation exacerbated the already unfavorable osmotic condition of SSC, resulting in metabolic inhibition. Consistent with this finding, approximately constant amounts of ethanol, acetate and lactate were produced during each flush of the FSSC. Flushed solid-substrate cultivation maintained favorable growth conditions for C. thermocellum even up to 25days, allowing more total product to be formed than in the other cultivation methods.

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.

Oxygen is either limiting or absent in many ecosystems. Anaerobic bacteria are often key players in such environments and these organisms have important roles in geo-elemental cycling, agriculture, and medicine. The metabolic versatility of anaerobes is exploited in a variety of industrial processes including fermented food production, biochemical synthesis, and bioremediation. There has been recent considerable interest in developing and enhancing technologies that employ anaerobes as biocatalysts. The study of anaerobic bacteria requires specialized techniques, and specific methods are described for the culture and manipulation of these microbes.

Avicel serves as a model microcrystalline cellulose substrate for investigations of cellulolytic microbial performance and cellulase enzyme systems in submerged liquid cultures. Clostridium thermocellum is a thermophilic, anaerobic bacterium capable of degrading lignocellulose and fermenting it to ethanol and other products, suggesting the native growth environment is similar to that supported by solid substrate cultivation. Few studies have examined the effects of process parameters on the metabolism of thermophilic anaerobes in solid substrate cultivation, however. The effects of solid substrate cultivation (SSC) substrate moisture content (30%, 50% and 70% wet-basis) and cultivation duration (2, 4 and 8 days) on the metabolic activity of C. thermocellum 27405 on Avicel was studied. The 70% substrate moisture content SSC culture yielded total end-product concentrations that were comparable to submerged liquid cultures. The SSC cultivation conditions with the highest end-product formation on Avicel were the combination of 70% substrate moisture content and cultivation duration period of 4 days, producing approximately 100 mM of total end-products. The ethanol and lactate concentrations were fairly constant and did not change significantly over time in SSC. Acetate production was more dependent on the cultivation conditions in SSC and was significant for both the 70% substrate moisture content SSC and liquid cultivation experiments, making up on average 56% and 86% of total end-products, respectively. Performance of C. thermocellum 27405 in SSC was more dependent on the kinetic properties rather than the thermodynamic properties of substrate moisture content. High substrate loadings in C. thermocellum cultivation affected product ratios, resulting in the higher observed acetate production. In addition, cessation of metabolism was observed prior to complete Avicel conversion; the mechanisms involved need further investigation.

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.

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 $15.67 (The prices are all 2004 prices in this article, except otherwise stated. We deflated newer prices to 2004 prices using a deflation factor 0.9 per year and inflated older prices to 2004 prices using an inflation factor 1.1.) per kilogram ($/kg) and $40.36/kg, for the SSC and SmF methods, respectively, while the corresponding market price was over $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.

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 anaerobic 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 degrees 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.

Solid substrate cultivation of thermophilic, anaerobic bacteria offers an alternative production method for many bio-based chemicals; however the process must be optimized for each substrate-organism fermentation. The effects of initial substrate moisture content (SMC, 30%, 50% and 70% wet-basis), supplemental nutrient concentration (SNC, 12%, 50% and 100%) and duration of cultivation time (6, 10 and 14 days), on product formation (lactate, ethanol and acetate) by Clostridium thermocellum 27405 were examined during growth on paper pulp sludge. Water activities at moisture contents above 30% wet-basis were essentially identical (∼0.99), yet the water contents differed significantly, and affected the metabolic activity of C. thermocellum. Increases in initial substrate moisture content from 50% to 70% for cultures supplemented with 50% or 100% nutrients resulted in a 75–145mM increase in total end products. At 70% SMC, the addition of 100% SNC generated a 56% increase in product formation above the addition of 50% nutrient supplementation. Increases in the quantity of free water present in the solid substrate cultivation system up to the water holding capacity of the paper pulp sludge led to improved performance of this anaerobic bacterium. While nutrient supplementation is common in the form of salts for many aerobic microorganisms, efficient metabolism for anaerobic C. thermocellum grown in SSC was highly dependent on added salts, vitamins and reducing agents. Further studies are needed to determine if this is a general effect for other anaerobes grown in solid substrate cultures.

Fluidization and melting point depression of aqueous dipalmitoylphosphatidylcholine (DPPC, Tm≈315K) liposomes by compressed/supercritical n-alkanes (C2 and C3; 0.8–20.7MPa) was quantified and compared to fluidization by liquid n-alkanes (C5–C10) using fluorescence anisotropy. The adsorption of ethane and propane into DPPC bilayers resulted in significant depression of the melting temperature (up to ΔTm=−16.2K at 7.0MPa ethane and propane). Pressure-dependent fluidization by gaseous n-alkanes (1.8–20.7MPa) and pressure-induced reversal of this fluidization was observed at temperatures corresponding to gel (295K) and fluid (333K) phases at atmospheric pressure. The ability to manipulate the degree of bilayer ordering, melting temperature, and width of the phase transition region is discussed in terms of the n-alkane concentration in the bilayer, effect of n-alkane molecular size, and van’t Hoff enthalpy of the gel–fluid phase transition. The permeability, phase, and solvent properties of the bilayer can be altered with pressurized ethane or propane, which may provide enhanced control for liposome formation and processing.

A novel, Bicine‐based SDS‐PAGE buffer system was developed for the analysis of membrane proteins. The method involves molecular weight‐based separations of fully denatured and solubilized proteins in two dimensions. This doubled SDS‐PAGE (dSDS‐PAGE) approach produced a diagonal arrangement of protein spots and successfully circumvented problems associated with membrane proteome analysis involving traditional gel‐based methods. Membrane proteins from the anaerobic bacterium Clostridium thermocellum were used for these investigations. Tricine‐dSDS‐PAGE and the newly developed Bicine‐dSDS‐PAGE were compared with the standard glycine‐dSDS‐PAGE (Laemmli protocol) in their suitability to separate C. thermocellum membrane proteins. Large‐format gel experiments using optimized gel preparation and running buffer conditions revealed a 112% increase in protein spot count for Tricine‐dSDS‐PAGE and a 151% increase for Bicine‐dSDS‐PAGE, compared to glycine‐dSDS‐PAGE. The data clearly indicated that Bicine‐dSDS‐PAGE is a superior method for the analysis of membrane proteins, providing enhanced resolution and protein representation.

A novel continuous high-pressure biphasic bioreactor was designed to investigate the toxicity of compressed and supercritical fluids on the thermophilic bacterium Clostridium thermocellum. Cultures were conducted at 1.8 and 7.0 MPa hydrostatic pressure and in the presence of compressed N(2) (7.0 MPa), gaseous (1.8 MPa) and supercritical ethane (7.0 MPa), and gaseous (1.8 MPa) and liquid (7.0 MPa) propane at a single dilution rate. No significant changes in metabolism or growth were observed in the presence of compressed N(2) relative to 7.0 MPa hydrostatic pressure, indicating that it acted as an inert fluid. However, dramatic inhibitions of growth and metabolism occurred in the presence of ethane and propane at 7.0 MPa. These inhibitions were reversed by depressurization from the supercritical (ethane) or liquid (propane) to gaseous state. Solvent toxicity by compressed and supercritical fluids was attributed to phase toxicity and was correlated with fluid density rather than conventional measures of toxicity (log P(o/w)). This biphasic reactor system facilitates investigations of solvent toxicity and dissolved gas effects on whole cells under elevated pressures.

A xylose ABC (ATP-binding cassette) transport operon, xylFGH, was cloned from Thermoanaerobacter ethanolicus, a thermophilic ethanol-producing eubacterium. The cistrons code for a periplasmic D-xylose-binding protein (XylF, partial sequence of 250 amino acids), ATP-binding protein (XylG, 505 amino acids), and integral membrane protein (XylH, 388 amino acids). These results, together with previous work, indicate that duplicate copies of both xylF and xylH are present in the T. ethanolicus chromosome, suggesting ancient gene duplication or lateral gene transfer events. XylG resembles other eubacterial monosaccharide ABC-ATPases in that its two nucleotide-binding domains (NBDs) are highly homologous, yet significantly different with respect to putative catalytic residues. Unlike most other integral membrane ABC transport proteins, XylH apparently contains 11 or 12 transmembrane segments (TMS) and is similar to a small group of ABC permeases that defy the “2 × 6” helix paradigm. This is the first report of a monosaccharide ABC transport operon in a thermophilic anaerobic eubacterium.

Dependence on foreign oil remains a serious issue for the U.S. economy. Additionally, automobile emissions related to petroleum-based, fossil fuel has been cited as one source of environmental problems, such as global warming and reduced air quality. Using agricultural and forest biomass as a source for the biofuel ethanol industry, provides a partial solution by displacing some fossil fuels. However, the use of high cost enzymes as an input is a significant limitation for ethanol production. Economic analyses of cellulase enzyme production costs using solid state cultivation (SSC) are performed and compared to the traditional submerged fermentation (SmF) method. Results from this study indicate that the unit costs for the cellulase enzyme production are $15.67 per kilogram ($/kg) and $40.36/kg, for the SSC and SmF methods, respectively, while the market price for the cellulase enzyme is $36.00/kg. Profitability analysis and sensitivity analysis also provide positive results. Since these results indicate that the SSC method is economical, ethanol production costs may be reduced, with the potential to make ethanol a viable supplemental fuel source in light of current political, economic and environmental issues.

The influence of CO2 on the bilayer fluidity of liposomes, which are representative of model cellular membranes, was examined for the first time at the elevated pressures (up to 13.9 MPa) associated with CO2-based processing of liposomes and microbial sterilization. Fluidization and melting point depression of aqueous dipalmitoylphosphatidylcholine (DPPC) liposomes by pressurized CO2 (present as an excess phase) were studied by steady-state fluorescence anisotropy using the membrane probe 1,6-diphenyl-1,3,5-hexatriene (DPH). Isothermal experiments revealed reversible, pressure-dependent fluidization of DPPC bilayers at temperatures corresponding to near-gel (295 K) and fluid (333 K) phases at atmospheric pressure, where the gel-to-fluid phase transition (Tm) occurs at approximately 315 K. Isobaric measurements (PCO2 =1.8, 7.0, and 13.9 MPa) of DPH anisotropy demonstrate substantial melting point depression (DeltaTm = -4.8 to -18.5 K) and a large broadening of the gel-fluid phase transition region, which were interpreted using conventional theories of melting point depression. Liposome fluidity is influenced by CO2 accumulation in the hydrocarbon core and polar headgroup region, as well as the formation of carbonic acid and/or the presence of buffering species under elevated CO2 pressure.

The continuous culture of Clostridium thermocellum, a thermophilic bacterium capable of producing ethanol from cellulosic material, is demonstrated at elevated hydrostatic pressure (7.0 MPa, 17.3 MPa) and compared with cultures at atmospheric pressure. A commercial limitation of ethanol production by C. thermocellum is low ethanol yield due to the formation of organic acids (acetate, lactate). At elevated hydrostatic pressure, ethanol:acetate (E/A) ratios increased >10(2) relative to atmospheric pressure. Cell growth was inhibited by approximately 40% and 60% for incubations at 7.0 MPa and 17.3 MPa, respectively, relative to continuous culture at atmospheric pressure. A decrease in the theoretical maximum growth yield and an increase in the maintenance coefficient indicated that more cellobiose and ATP are channeled towards maintaining cellular function in pressurized cultures. Shifts in product selectivity toward ethanol are consistent with previous observations of hydrostatic pressure effects in batch cultures. The results are partially attributed to the increasing concentration of dissolved product gases (H2, CO2) with increasing pressure; and they highlight the utility of continuous culture experiments for the quantification of the complex role of dissolved gas and pressure effects on metabolic activity.

Ethanol and acetone were extracted from aqueous feed streams (10wt.% solute) at ambient temperature using compressed propane (34.5bar) and CO2 (69bar) in a polypropylene hollow fiber membrane contactor (HFC). Overall mass transfer coefficients were determined and related to tubeside, membrane, and shellside mass transfer by varying the aqueous velocity (0.6–11.8cm/s) and molar solvent to feed ratio (1 to 3) in countercurrent flow. The overall mass transfer coefficient for ethanol and acetone extraction with compressed propane ranged from 3.8×10−4 to 1.1×10−2 and 1.1×10−4 to 1.7×10−3cm/s, respectively. For compressed CO2, the overall mass transfer coefficient ranged from 5.1×10−4 to 1.4×10−3cm/s for ethanol extraction and 4.1×10−4 to 9.5×10−4cm/s for acetone extraction. Experimentally determined shellside mass transfer coefficients were compared to values predicted by existing correlations for HFC extraction with non-pressurized solvents. Enhanced HFC mass transfer is observed with compressed solvents relative to both conventional liquid HFC extraction and to column extraction with compressed CO2. Our results demonstrate the applicability of compressed hydrocarbons to HFC-based processes, and the ability to analyze compressed solvent-based extraction using conventional HFC techniques.

The feasibility of extracting aqueous ethanol and acetone within a hollow fiber membrane contactor (HFC) has been examined using compressed CO2 (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 CO2 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 CO2 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.