Influence of oxygen and carbon dioxide on lignin degradation in solid state fermentation of wheat straw withStropharia rugosoannulata

Summary In solid state fermentation,Stropharia rugosoannulata degrades lignin of wheat straw slightly better in the presence of oxygen than that in air. The sub-atmospheric partial pressure of oxygen (0.05 atm.) inhibits lignin and organic matter degradation. the increasing partial pressure of carbon dioxide (0.1–0.3 atm.) along with 0.2 atm. of oxygen does not have any effect on lignin degradation, but slightly decreases organic matter loss and increases thein vitro digestibility of fermented wheat straw, thereby making the process more efficient.     

Oxygen poisoning in Drosophila

Fruit flies live longer at the partial pressure of oxygen found in air than at either larger or smaller partial pressures. Flies exposed to 1 atm of oxygen for 8 hr every day do not recover completely in the remaining 16 hr. In general, intermittent exposures to 1 atm of oxygen are better tolerated than continuous exposure to the same average oxygen concentration per day, but exposures to higher pressures of 2–5 atm of oxygen for as little as a half hour every two days markedly shorten the life-span. Older flies consume more oxygen per minute and are more sensitive to oxygen poisoning than young flies, and the rate of dying in 6 atm of O₂, or the reciprocal of the survival time, is a linear function of the age. The oxygen pressure-time curve can be well expressed by the general empirical equation (P_OO2)² x time = 120 where P is in atmosphere and survival time in hours. The progress of oxygen poisoning appears to be linear with time rather than exponential.     

Growth of Streptococcus faecalis under High Hydrostatic Pressure and High Partial Pressures of Inert Gases

Growth of Streptococcus faecalis in a complex medium was inhibited by xenon, nitrous oxide, argon, and nitrogen at gas pressures of 41 atm or less. The order of inhibitory potency was: xenon and nitrous oxide > argon > nitrogen. Helium appeared to be impotent. Oxygen also inhibited streptococcal growth and it acted synergistically with narcotic gases. Growth was slowed somewhat by 41 atm hydrostatic pressure in the absence of narcotic gases, but the gas effects were greater than those due to pressure. In relation to the sensitivity of this bacterium to pressure, we found that the volume of cultures increased during growth in a volumeter or dilatometer, and that this dilatation was due mainly to glycolysis. A volume increase of 20.3 ± 3.6 ml/mole of lactic acid produced was measured, and this value was close to one of 24 ml/mole lactic acid given for muscle glycolysis, and interestingly, close to the theoretic volume increase of activation calculated from the depression of growth rate by pressure.     

Improved Sugar Conversion and Ethanol Yield for Forage Sorghum (Sorghum bicolor L. Moench) Lines with Reduced Lignin Contents

Lignin is known to impede conversion of lignocellulose into ethanol. In this study, forage sorghum plants carrying brown midrib (bmr) mutations, which reduce lignin contents, were evaluated as bioenergy feedstocks. The near-isogenic lines evaluated were: wild type, bmr-6, bmr-12, and bmr-6 bmr-12 double mutant. The bmr-6 and bmr-12 mutations were equally efficient at reducing lignin contents (by 13% and 15%, respectively), and the effects were additive (27%) for the double mutant. Reducing lignin content was highly beneficial for improving biomass conversion yields. Sorghum biomass samples were pretreated with dilute acid and recovered solids washed and hydrolyzed with cellulase to liberate glucose. Glucose yields for the sorghum biomass were improved by 27%, 23%, and 34% for bmr-6, bmr-12, and the double mutant, respectively, compared to wild type. Sorghum biomass was also pretreated with dilute acid followed by co-treatment with cellulases and Saccharomyces cerevisiae for simultaneous saccharification and fermentation (SSF) into ethanol. Conversion of cellulose to ethanol for dilute-acid pretreated sorghum biomass was improved by 22%, 21%, and 43% for bmr-6, bmr-12, and the double mutant compared to wild type, respectively. Electron microscopy of dilute-acid treated samples showed an increased number of lignin globules in double-mutant tissues as compared to the wild-type, suggesting the lignin had become more pliable. The mutations were also effective for improving ethanol yields when the (degrained) sorghum was pretreated with dilute alkali instead of dilute acid. Following pretreatment with dilute ammonium hydroxide and SSF, ethanol conversion yields were 116 and 130 mg ethanol/g dry biomass for the double-mutant samples and 98 and 113 mg/g for the wild-type samples.     

A Study of the Effects of Hydrostatic Pressure on Macromolecular Synthesis in Escherichia coli

In cultures of Escherichia coli 15 (thymine^-, leucine^-) which were incubated at high hydrostatic pressures, cell division occurred only at pressures below 430 atm but in a somewhat synchronous fashion at around 250 atm. The rate of leucine-¹⁴C incorporation into a macromolecular fraction of the cells diminished to a zero value at about 580 atm and that of uracil-¹⁴C incorporation to a zero value at about 770 atm. The rate of thymine-¹⁴C incorporation at pressures around 330 atm was that to be expected with a culture in which DNA synthesis is somewhat synchronous. At pressures above 500 atm, thymine-¹⁴C was incorporated only over the initial part of the pressure incubation and further incorporation under pressure was not observed no matter how long the duration of the incubation. We present evidence along several lines that the thymine incorporation kinetics reflect an effect of pressure on a locus at the origin (or termination) of a replication of the bacterial chromosome. The recovery of cell division and of the incorporation rates upon release of pressure were found to depend on the magnitude of the pressure and the duration of the pressure incubation.     

Evaluation of angiosperm and fern contributions to soil organic matter using two methods of pyrolysis-gas chromatography-mass spectrometry

Background

Ferns are an important plant group, and older phylogenies of non-polypod ferns contain relatively high concentrations of aliphatic leaf waxes, lignins, and tannins that could contribute to soil organic matter (SOM) biochemistry and stability.

Methods

Pyrolysis gas-chromatography mass-spectrometry (py-GC/MS) analyzes biochemical fragments which can be related to lignin, polysaccharide, lipid, nitrogen (N)-bearing, non-lignin aromatics, and phenol source compounds. Thermochemolysis using tetramethylammonium hydroxide (TMAH) combined with py-GC/MS improves detection of lignin, cutin, and suberin-derived compounds. To examine the advantages and disadvantages of both methods for characterizing plant and soil biochemistry, we characterized non-polypod and polypod fern and angiosperm live tissues, roots and soils from the Kohala Mountains, Hawaii.

Results

Py-GC/MS provided a broad biochemical overview of compound groups including lignin, polysaccharide, lipid, N-bearing, non-lignin aromatics and phenol groups while TMAH-py-GC/MS detailed lignin units and fatty acids at the expense of the other categories. TMAH-py-GC/MS provided more detailed data on lignin, cutin, suberin and tannin-derived compounds. Both methods detected differences in lignin units between species, although p-coumaric and ferulic acids, predominantly found in ferns, were only observed with TMAH-py-GC/MS.

Conclusions

Both py-GC/MS and TMAH-py-GC/MS are methods to detect compound-specific plant biomarkers, but are most useful when combined for their complimentary results.     

Growth of Geobacter sulfurreducens with Acetate in Syntrophic Cooperation with Hydrogen-Oxidizing Anaerobic Partners

Pure cultures of Geobacter sulfurreducens and other Fe(III)-reducing bacteria accumulated hydrogen to partial pressures of 5 to 70 Pa with acetate, butyrate, benzoate, ethanol, lactate, or glucose as the electron donor if electron release to an acceptor was limiting. G. sulfurreducens coupled acetate oxidation with electron transfer to an anaerobic partner bacterium in the absence of ferric iron or other electron acceptors. Cocultures of G. sulfurreducens and Wolinella succinogenes with nitrate as the electron acceptor degraded acetate efficiently and grew with doubling times of 6 to 8 h. The hydrogen partial pressures in these acetate-degrading cocultures were considerably lower, in the range of 0.02 to 0.04 Pa. From these values and the concentrations of the other reactants, it was calculated that in this cooperation the free energy change available to G. sulfurreducens should be about −53 kJ per mol of acetate oxidized, assuming complete conversion of acetate to CO₂ and H₂. However, growth yields (18.5 g of dry mass per mol of acetate for the coculture, about 14 g for G. sulfurreducens) indicated considerably higher energy gains. These yield data, measurement of hydrogen production rates, and calculation of the diffusive hydrogen flux indicated that electron transfer in these cocultures may not proceed exclusively via interspecies hydrogen transfer but may also proceed through an alternative carrier system with higher redox potential, e.g., a c-type cytochrome that was found to be excreted by G. sulfurreducens into the culture fluid. Syntrophic acetate degradation was also possible with G. sulfurreducens and Desulfovibrio desulfuricans CSN but only with nitrate as electron acceptor. These cultures produced cell yields of 4.5 g of dry mass per mol of acetate, to which both partners contributed at about equal rates. These results demonstrate that some Fe(III)-reducing bacteria can oxidize organic compounds under Fe(III) limitation with the production of hydrogen, and they provide the first example of rapid acetate oxidation via interspecies electron transfer at moderate temperature.     

Effects of Hyperbaric Pressure on a Deep-Sea Archaebacterium in Stainless Steel and Glass-Lined Vessels

The effects of hyperbaric helium pressures on the growth and metabolism of the deep-sea isolate ES4 were investigated. In a stainless steel reactor, cell growth was completely inhibited but metabolic gas production was observed. From 85 to 100°C, CO₂ production proceeded two to three times faster at 500 atm (1 atm = 101.29 kPa) than at 8 atm. At 105°C, no CO₂ was produced until the pressure was increased to 500 atm. Hydrogen and H₂S were also produced biotically but were not quantifiable at pressures above 8 atm because of the high concentration of helium. In a glass-lined vessel, growth occurred but the growth rate was not accelerated by pressure. In most cases at temperatures below 100°C, the growth rate was lower at elevated pressures; at 100°C, the growth rates at 8, 250, and 500 atm were nearly identical. Unlike in the stainless steel vessel, CO₂ production was exponential during growth and continued for only a short time after growth. In addition, relatively little H₂ was produced in the glass-lined vessel, and there was no growth or gas production at 105°C at any pressure. The behavior of ES4 as a function of temperature and pressure was thus very sensitive to the experimental conditions.     

Enhancing alfalfa conversion efficiencies for sugar recovery and ethanol production by altering lignin composition

Alfalfa (Medicago sativa L.) biomass was evaluated for biochemical conversion into ethanol using dilute-acid and ammonia pretreatments. The two alfalfa lines compared were a reduced S-lignin transgenic cultivar generated through down regulation of the caffeic acid O-methyltransferase gene and a wild-type control. Both were harvested at two maturities. All the samples had similar carbohydrate contents including a mean composition of 316 g glucan and 497 g total neutral carbohydrates per kg dry biomass, which corresponds to a theoretic ethanol yield of 382 l/ton. Ethanol yields for alfalfa stems pretreated with dilute-acid were significantly impacted by harvest maturity and lignin composition, whereas when pretreated with dilute-ammonia, yield was solely affected by lignin composition. Use of a recombinant xylose-fermenting Saccharomyces strain, for converting the ammonia pretreated alfalfa samples, further increased ethanol yields. Ethanol yields for the xylose-fermenting yeast were 232-278 l/ton and were significantly enhanced for the reduced S lignin cultivars.     

Poplar Lignin Decomposition by Gram-Negative Aerobic Bacteria

Eleven gram-negative aerobic bacteria (Pseudomonadaceae and Neisseriaceae) out of 122 soil isolates were selected for their ability to assimilate poplar dioxane lignin without a cosubstrate. Dioxane lignin and milled wood lignin degradation rates ranged between 20 and 40% of initial content after 7 days in mineral medium, as determined by a loss of absorbance at 280 nm; 10 strains could degrade in situ lignin, as evidenced by the decrease of the acetyl bromide lignin content of microtome wood sections. No degradation of wood polysaccharides was detected. Lignin biodegradation by Pseudomonas 106 was confirmed by ¹⁴CO₂ release from labeled poplar wood, although in lower yields compared with results obtained through chemical analysis based on acetyl bromide residual lignin determination.     

Bacteriology of Manganese Nodules: V. Effect of Hydrostatic Pressure on Bacterial Oxidation of MnII and Reduction of MnO2

It was experimentally demonstrated that two strains of Arthrobacter 37, one growing at 25 C and the other at 5 C, could catalyze Mn^II oxidation at hydrostatic pressures well in excess of the pressure encountered by the parent culture in its original habitat in the ocean (80 atm). The strain grown at 5 C showed an increase in temperature optimum for manganese oxidation with increase in pressure. It was like-wise experimentally shown that induced Bacillus 29 without added ferricyanide and uninduced Bacillus 29 with added ferricyanide could catalyze MnO₂ reduction at hydrostatic pressures in excess of the pressure encountered by this organism in its original habitat (187 atm). The uninduced Bacillus 29, in the presence of ferricyanide, was active over a wider range of pressures (1 to 1,000 atm) than the induced Bacillus 29 in the absence of ferricyanide (1 to 467 atm). At corresponding pressures, the uninduced culture was also considerably more active than the induced culture. Special techniques were developed for measuring Mn^II-oxidizing and MnO₂-reducing activity under pressure.     

Ethanol Production by Thermophilic Bacteria: Relationship Between Fermentation Product Yields of and Catabolic Enzyme Activities in Clostridium thermocellum and Thermoanaerobium brockii

Significant quantitative differences in end-product yields by two strains of Clostridium thermocellum and one strain of Thermoanaerobium brockii were observed during cellobiose fermentation. Most notably, the ethanol/H₂ and lactate/acetate ratios were drastically higher for T. brockii as compared with C. thermocellum strains LQRI and AS39. Exogenous H₂ addition (0.4 to 1.0 atm) during culture growth increased the ethanol/acetate ratio of both T. brockii and AS39 but had no effect on LQRI. All strains had an operative Embden-Meyerhof glycolytic pathway and displayed catabolic activities of fructose-1,6-diphosphate–activated lactate dehydrogenase, coenzyme A acetylating pyruvate and acetaldehyde dehydrogenase, hydrogenase, ethanol dehydrogenase, and acetate kinase. Enzyme kinetic properties (apparent K_m, V_max, and Q₁₀ values) and the specificity of electron donors/acceptors for different oxidoreductases involved in pyruvate conversion to fermentation products were compared in the three strains. Both species contained ferredoxin-linked pyruvate dehydrogenase and pyridine nucleotide oxidoreductases. Ferredoxin-nicotinamide adenine dinucleotide (NAD) reductase activity was significantly higher in T. brockii than in AS39 and was not detectable in LQRI. H₂ production and hydrogenase activity were inversely related to ferredoxin-NAD reductase activity in the three strains. Ferredoxin-NAD phosphate reductase activity was present in cell extracts of both species. Alcohol dehydrogenase activity in C. thermocellum was NAD dependent, unidirectional, and inhibited by low concentrations of NAD and ethanol. Ethanol dehydrogenase activity of T. brockii was both NAD and NADP linked, reversible, and not inhibited by low levels of reaction products. The high lactate yield of T. brockii correlated with increased fructose-1,6-diphosphate. The relation of catabolic enzyme activity and quantitative differences in intracellular electron flow and fermentation product yields of these thermophilic bacteria is discussed.     

Effects of hyperoxia on composition and rate of synthesis of fatty acids in Escherichia coli

Growth of and fatty acid synthesis in Escherichia coli were inhibited by oxygen at partial pressures above 1 atm and were prevented by exposure to oxygen at 4.2 atm on membranes incubated on a minimal medium. Growth and fatty acid synthesis returned to control rates when cells were removed from hyperoxia to air. The spectrum of fatty acids produced was unchanged by oxygen at pressures which reduced the rate of synthesis. In situ fatty acids were stable to oxygen at pressures which prevented growth and synthesis. Reinitiation of synthesis after complete inhibition in hyperoxia occurred without production of aberrant fatty acids. Fatty acid synthetase specific activity was virtually unchanged, compared with air controls, in cells exposed either to 3.2 or to 15.2 atm of oxygen. The spectrum of fatty acids synthesized by cell-free extracts during incubation in 4.2 atm of oxygen was not different from air-incubated controls. Synthetase assays included added NADPH, acyl carrier protein, mercaptoethanol, and malonyl coenzyme A; hence, damage, other than reversible sulfhydryl oxidation, to the apoenzymes of synthetase was ruled out.     

Further Experiments on the Inhibition of Respiration of Peas Induced by Oxygen at High Pressures

Turner and Quartley (1956) concluded that the tricarboxylicacid cycle was a major respiratory pathway in green shelledpeas subjected to high pressures of oxygen. Additional supportfor this view has been obtained by demonstrating the depletionin content of oxaloacetic acid during treatment with oxygenand by the complete reversal of all the functions determinedfollowing a return of the samples to air after treatment forshort periods with oxygen at high pressures. Oxygen at pressures of either 5 or 3.5 atmospheres caused asimilar pattern in the behaviour of the carbon dioxide production,pH, citric and keto-acids, whilst oxygen at a pressure of 2atm. affected mainly the carbon dioxide production. This suggeststhat the respiration process can be inhibited by oxygen at highpressures in at least two different stages.     

Pressure and Temperature Dependence of Growth and Morphology of Escherichia coli: Experiments and Stochastic Model

We have investigated the growth of Escherichia coli, a mesophilic bacterium, as a function of pressure (P) and temperature (T). Escherichia coli can grow and divide in a wide range of pressure (1–400 atm) and temperature (23–40°C). For T > 30°C, the doubling time of E. coli increases exponentially with pressure and exhibits a departure from exponential behavior at pressures between 250 and 400 atm for all the temperatures studied in our experiments. The sharp change in doubling time is followed by a sharp change in phenotypic transition of E. coli at high pressures where bacterial cells switch to an elongating cell type. We propose a model that this phenotypic change in bacteria at high pressures is an irreversible stochastic process, whereas the switching probability to elongating cell type increases with increasing pressure. The model fits well the experimental data. We discuss our experimental results in the light of structural and thus functional changes in proteins and membranes.     

Ethanol and acetate synthesis from waste gas using batch culture of Clostridium ljungdahlii

Single inorganic carbon source was used for production of chemicals and fuels via fermentation processes. Clostridium ljungdahlii, a strictly anaerobic autotrophic bacterium, was grown on synthesis gas to produce acetate and ethanol from gaseous substrates. C. ljungdahlii was grown on a various concentrations of carbon monoxide with synthesis gas total pressures of 0.8–1.8 atm with an interval of 0.2 atm. The cell and product yields were 0.015 g cell/g CO and 0.41 g acetate/g CO, respectively. Formation of acetate was steady and the production trend was about the same for all of the gases initial pressure and at constant cell density. The ethanol concentration was enhanced by the initial presence of hydrogen and carbon dioxide in the liquid phase. There was no substrate inhibition while C. ljungdahlii was grown in the batch fermentation, even at high system pressure of 1.6 and 1.8 atm. A desired product molar ratio of ethanol:acetate (5:1) was achieved with total gas pressure of 1.6 and 1.8 atm.     

Mechanism of lignin inhibition of enzymatic biomass deconstruction

Background

The conversion of plant biomass to ethanol via enzymatic cellulose hydrolysis offers a potentially sustainable route to biofuel production. However, the inhibition of enzymatic activity in pretreated biomass by lignin severely limits the efficiency of this process.

Results

By performing atomic-detail molecular dynamics simulation of a biomass model containing cellulose, lignin, and cellulases (TrCel7A), we elucidate detailed lignin inhibition mechanisms. We find that lignin binds preferentially both to the elements of cellulose to which the cellulases also preferentially bind (the hydrophobic faces) and also to the specific residues on the cellulose-binding module of the cellulase that are critical for cellulose binding of TrCel7A (Y466, Y492, and Y493).

Conclusions

Lignin thus binds exactly where for industrial purposes it is least desired, providing a simple explanation of why hydrolysis yields increase with lignin removal.

     

Characterization of lignin-rich residues remaining after continuous super-critical water hydrolysis of poplar wood (Populus albaglandulosa) for conversion to fermentable sugars

Poplar wood flour (Populous albaglandulosa) was treated with sub- and super-critical water (subcritical: 325, 350 °C; super-critical: 380, 400, 425 °C) for 60 s at 220 ± 10 atm. Hydrochloric acid (0.05% v/v) was added to samples as acidic catalyst. The final products were separated into water soluble fraction and undegraded solids. The yields of undegraded solids were thoroughly dependent on temperature severity and mainly composed of lignin fragments. Average molecular weights of the lignins were between 1500 and 4400 Da, which was only 1/3-1/8-fold of poplar milled wood lignin (13,250 Da). DFRC (Derivatization Followed by Reductive Cleavage) analysis revealed that C6C3 phenols (coniferyl and sinapyl alcohol) were rarely detected in the lignins, indicating occurrence of two probable lignin reactions during SCW hydrolysis: lignin fragmentation via splitting of β-O-4 linkage and loss of propane side chains. These results were also confirmed by ¹H and ¹³C NMR spectroscopic analysis.     

Inhibition of plasma membrane Ca2+-atpase pump activity in cultured c6 glioma cells by halothane and xenon

We have compared the effect of two inhalational anesthetics, halothane and xenon, on Ca²⁺-ATPase (PMCA) pumping activity in plasma membrane vesicles prepared from cultured rat C6 glioma cells. Halothane, at concentrations ranging from 0.5 to 1.75 vol% (equivalent to 0.5 to 1.6 MAC), significantly inhibited Ca²⁺ uptake (transport) by plasma membrane vesicles in a dose-related fashion. Xenon, at partial pressures ranging from 0.5 to 1.5 atm (equivalent to 0.5 to 1.6 MAC), similarly inhibited PMCA pumping activity. Additive effects on suppression of PMCA pump activity were observed when C6 cell plasma membrane vesicles were exposed to increasing partial pressures of xenon in the presence of halothane (1 vol%). Halothane also inhibited PMCA pumping in cells from two other lines of neural origin, B104 (rat neuroblastoma) and PC12 (rat pheochromocytoma). Studies described in this report support the thesis that PMCA in cells of neural origin is inhibited by quite different inhalational anesthetics at clinically relevant concentrations.     

Pressure and Temperature Dependence of Growth and Morphology of?Escherichia coli: Experiments and Stochastic Model

We have investigated the growth of Escherichia coli, a mesophilic bacterium, as a function of pressure (P) and temperature (T). Escherichia coli can grow and divide in a wide range of pressure (1–400 atm) and temperature (23–40°C). For T > 30°C, the doubling time of E. coli increases exponentially with pressure and exhibits a departure from exponential behavior at pressures between 250 and 400 atm for all the temperatures studied in our experiments. The sharp change in doubling time is followed by a sharp change in phenotypic transition of E. coli at high pressures where bacterial cells switch to an elongating cell type. We propose a model that this phenotypic change in bacteria at high pressures is an irreversible stochastic process, whereas the switching probability to elongating cell type increases with increasing pressure. The model fits well the experimental data. We discuss our experimental results in the light of structural and thus functional changes in proteins and membranes.