Anaerobic degradation of soluble fractions of [C-lignin]lignocellulose

[C-lignin]lignocellulose was solubilized by alkaline heat treatment and separated into different molecular size fractions for use as the sole source of carbon in anaerobic enrichment cultures. This study is aimed at determining the fate of low-molecular-weight, polyaromatic lignin derivatives during anaerobic degradation. Gel permeation chromatography was used to preparatively separate the original C-lignin substrate into three component molecular size fractions, each of which was then fed to separate enrichment cultures. Biodegradability was assessed by monitoring total carbon dioxide and methane production, evolution of labeled gases, loss of C-activity from solution, and changes in gel permeation chromatographic elution patterns. Results indicated that the smaller the size of the molecular weight fraction, the more extensive the degradation to gaseous end products. In addition, up to 30% of the entire soluble lignin-derived carbon was anaerobically mineralized to carbon dioxide and methane.     

Determination of carbon dioxide evolution rate using on-line gas analysis during dynamic biodegradation experiments

Respirometry is a precious tool for determining the activity of microbial populations. The measurement of oxygen uptake rate is commonly used but cannot be applied in anoxic or anaerobic conditions or for insoluble substrate. Carbon dioxide production can be measured accurately by gas balance techniques, especially with an on-line infrared analyzer. Unfortunately, in dynamic systems, and hence in the case of short-term batch experiments, chemical and physical transfer limitations for carbon dioxide can be sufficient to make the observed carbon dioxide evolution rate (OCER) deduced from direct gas analysis very different from the biological carbon dioxide evolution rate (CER).To take these transfer phenomena into account and calculate the real CER, a mathematical model based on mass balance equations is proposed. In this work, the chemical equilibrium involving carbon dioxide and the measured pH evolution of the liquid medium are considered. The mass transfer from the liquid to the gas phase is described, and the response time of the analysis system is evaluated.Global mass transfer coefficients (K(L)a) for carbon dioxide and oxygen are determined and compared to one another, improving the choice of hydrodynamic hypotheses. The equations presented are found to give good predictions of the disturbance of gaseous responses during pH changes.Finally, the mathematical model developed associated with a laboratory-scale reactor, is used successfully to determine the CER in nonstationary conditions, during batch experiments performed with microorganisms coming from an activated sludge system. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 243-252, 1997.     

Selective desorption of carbon dioxide from sewage sludge for in situ methane enrichment--part I: pilot-plant experiments

In situ methane enrichment in anaerobic digestion of sewage sludge has been investigated by experiments and by modeling. In this first part, the experimental work on the desorption of carbon dioxide and methane from sewage sludge is reported. The bubble column, had a diameter of 0.3 m and a variable height up to 1.8 m. At operation the dispersion height in the column was between 1 and 1.3 m. Outdoor air was used. The column was placed close to a full-scale sewage sludge digester, at a municipal wastewater treatment plant. The digester was operated at mesophilic conditions with a hydraulic retention time of about 20 days. The bubble column was operated to steady-state, at which carbon dioxide concentration and alkalinity were determined on the liquid side, and the concentration of carbon dioxide and methane on the gas side. Thirty-eight experiments were performed at various liquid and gas flow rates. The experimental results show that the desorption rates achieved for carbon dioxide ranges from 0.07 to 0.25 m(3) CO(2)/m(3) sludge per day, which is comparable to the rate of generation by the anaerobic digestion. With increasing liquid flow rate and decreasing gas flow rate the amount of methane desorbed per amount of carbon dioxide desorbed increases. The lowest methane loss achieved is approximately 2% of the estimated methane production in the digestion process.     

Anaerobic Degradation of Soluble Fractions of [14C-Lignin]Lignocellulose

[¹⁴C-lignin]lignocellulose was solubilized by alkaline heat treatment and separated into different molecular size fractions for use as the sole source of carbon in anaerobic enrichment cultures. This study is aimed at determining the fate of low-molecular-weight, polyaromatic lignin derivatives during anaerobic degradation. Gel permeation chromatography was used to preparatively separate the original ¹⁴C-lignin substrate into three component molecular size fractions, each of which was then fed to separate enrichment cultures. Biodegradability was assessed by monitoring total carbon dioxide and methane production, evolution of labeled gases, loss of ¹⁴C-activity from solution, and changes in gel permeation chromatographic elution patterns. Results indicated that the smaller the size of the molecular weight fraction, the more extensive the degradation to gaseous end products. In addition, up to 30% of the entire soluble lignin-derived carbon was anaerobically mineralized to carbon dioxide and methane.     

Modeling of anaerobic formate kinetics in mixed biofilm culture using dynamic membrane mass spectrometric measurement

The dynamics of the anaerobic conversion of formate in a microbial mixed culture taken from an anaerobic fluidized bed reactor was studied using a new stirred micro reactor equipped with a membrane mass spectrometer. The microreactor with a toroidally shaped bottom and pitched blade turbine and a cylindrical flow guide was thermostated and additionally equipped with a pH electrode and pH control. During fed-batch experiments using formate, the dissolved gases (methane, hydrogen, and carbon dioxide), as well as the acid consumption rates for pH control were monitored continuously. Initially and at the end of each experiment, organic acids were analyzed using ion chromatography (IC). It was found that about 50% of the formate was converted to methane via hydrogen and carbon dioxide, 40% gave methane either directly or via acetate. This was calculated from experiments using H(13)CO(3) (-) pulses and measurement of (12)CH(4) and (13)CH(4) production rates. About 10% of the formate was converted to lactate, acetate, and propionate, thereby increasing the measured CO(2)/CH(4) production ratio. The nondissociated formic acid was shown to be rate determining. From the relatively high K(s) value of 2.5 mmol m(-3), it was concluded that formate cannot play an important role in electron transfer. During dynamic feeding of formate, hydrogen concentration always increased to a maximum before decreasing again. This peak was found to be very discriminative during modeling. From the various models set up, only those with two-stage degradation and double Monod kinetics, both for CO(2) and hydrogen, were able to describe the experimental data adequately. Additional discrimination was possible with the IC measurement of organic acids. (c) 1995 John Wiley & Sons, Inc.     

Mass spectrometry as en ecological tool for in situ measurement of dissolved gases in sediment systems

Abstract The use of membrane-inlet quadrupole mass spectrometry, as a method for quantitative monitoring of dissolved gases in natural or semi-natural environments, is described. Its advantages over other methods lie in the fact that it provides an accurate, sensitive means for non-invasive, continuous analysis of several dissolved gases simultaneously. The potential of mass spectrometry as an ecological tool is illustrated by representative results from measurements made on undisturbed and experimentally amended estuarine and fresh-water sediments.
Dissolved gas profiles from the surface to a depth of 10 cm in the estuarine sediment showed that the dissolved oxygen decreased gradually until at 10 cm it was undetectable (< 0.25 μM); dinitrogen reached a maximum at 6 cm, where oxygen was 20 μM. In a fresh-water sediment, methane reached 1.5 mM at 10 cm depth. NO_x was also detected; quantitation of carbon dioxide necessitates a correction for the contribution of NO_x. Manipulation of conditions (gas phase, nitrogen and carbon sources) permitted ecological modelling.     

Spatial variation of dissolved gas concentrations in a willow vegetation filter treating landfill leachate

Membrane inlet mass spectrometry (MIMS) was used to monitor continuously and simultaneously the concentrations of dissolved gases (O₂, CO₂, CH₄) within the treatment bed of a willow vegetation filter treating leachate at a landfill site in mid Wales. The distribution of dissolved gasses within the bed was shown to be highly heterogeneous at the small spatial scale with considerable variation between vertical profiles measured simultaneously at different locations. In general, aerobic conditions were observed above the water table with reduced levels of oxygen and increasing levels of carbon dioxide and methane below it. Distinct pockets of oxygen (up to 200 μM) were observed in anaerobic zones and pockets of reduced oxygen and elevated carbon dioxide were observed in the aerobic zone. Pockets of methane were observed in some profiles coexisting with up to 200 μM oxygen at 5 cm depth. These observations confirm the hypothesis that micro-sites exists within the soil/root matrix where aerobic organic matter decomposition and anaerobic processes such as methanogenesis can occur in relatively close proximity to each other. We hypothesise that the distribution of dissolved gases is determined by rapid diffusion of air maintaining aerobic conditions above the water table, removal of oxygen by microbial processes creating anaerobic conditions below the water table and the distribution of willow roots in the soil which create local aerobic zones by oxygen release.     

Hydrogenase activity and methanogenesis in anaerobic sewage sludge,in rumen liquid,and in freshwater sediments

Summary The applicability of hydrogenase determinations to the evaluation of hydrogen transfer reactions occurring within methanogenic environments was investigated. Enzymatic hydrogen production was determined in digester sludge, river sediments, and rumen liquid using reduced methyl viologen, formate, and pyruvate as hydrogen donors. Hydrogenase determinations turned out not to be inhibited by toxic compounds present in sediments of the polluted river Saar. Comparative kinetic studies of the conversion of acetate and of hydrogen to methane support the assumption that carbon dioxide reduction by hydrogen accounts for the major part of methane formed in river sediments. In rumen liquid and in river sediments similar enzyme patterns were observed which were different from that found in digester sludge. The rates of methanogenesis correlated well with hydrogenase activities in all ecosystems studied: Correlation coefficients ranged from 0.84 to 0.95. Rumen liquid and river sediments exhibited higher hydrogenase activities than digester sludge when compared at identical rates of methane production. According to these results, the hydrogenase determination is applicable to the evaluation of the hydrogen transfer, occurring within the microbial biomass of anaerobic ecosystems.     

Thermophilic anaerobic biodegradation of [C]lignin, [C]cellulose, and [C]lignocellulose preparations

Thermophilic (55 degrees C) anaerobic enrichment cultures were incubated with [C-lignin]lignocellulose, [C-polysaccharide]lignocellulose, and kraft [C]lignin prepared from slash pine, Pinus elliottii, and C-labeled preparations of synthetic lignin and purified cellulose. Significant but low percentages (2 to 4%) of synthetic and natural pine lignin were recovered as labeled methane and carbon dioxide during 60-day incubations, whereas much greater percentages (13 to 23%) of kraft lignin were recovered as gaseous end products. Percentages of label recovered from lignin-labeled substrates as dissolved degradation products were approximately equal to percentages recovered as gaseous end products. High-pressure liquid chromatographic analyses of CuO oxidation products of sound and degraded pine lignin indicated that no substantial chemical modifications of the remaining lignin polymer, such as demethoxylation and dearomatization, occurred during biodegradation. The polysaccharide components of pine lignocellulose and purified cellulose were relatively rapidly mineralized to methane and carbon dioxide; 31 to 37% of the pine polysaccharides and 56 to 63% of the purified cellulose were recovered as labeled gaseous end products. An additional 10 to 20% of the polysaccharide substrates was recovered as dissolved degradation products. Overall, these results indicate that elevated temperatures can greatly enhance rates of anaerobic degradation of lignin and lignified substrates to methane and low-molecular-weight aromatic compounds.     

Bioconversion of carbon dioxide to methane using hydrogen and hydrogenotrophic methanogens

Biogas produced from organic wastes contains energetically usable methane and unavoidable amount of carbon dioxide. The exploitation of whole biogas energy is locally limited and utilization of the natural gas transport system requires CO₂ removal or its conversion to methane. The biological conversion of CO₂ and hydrogen to methane is well known reaction without the demand of high pressure and temperature and is carried out by hydrogenotrophic methanogens. Reducing equivalents to the biotransformation of carbon dioxide from biogas or other resources to biomethane can be supplied by external hydrogen. Discontinuous electricity production from wind and solar energy combined with fluctuating utilization cause serious storage problems that can be solved by power-to-gas strategy representing the production of storable hydrogen via the electrolysis of water. The possibility of subsequent repowering of the energy of hydrogen to the easily utilizable and transportable form is a biological conversion with CO₂ to biomethane. Biomethanization of CO₂ can take place directly in anaerobic digesters fed with organic substrates or in separate bioreactors. The major bottleneck in the process is gas-liquid mass transfer of H₂ and the method of the effective input of hydrogen into the system. There are many studies with different bioreactors arrangements and a way of enrichment of hydrogenotrophic methanogens, but the system still has to be optimized for a higher efficiency. The aim of the paper is to gather and critically assess the state of a research and experience from laboratory, pilot and operational applications of carbon dioxide bioconversion and highlight further perspective fields of research.     

The use of disposable gaas generating kits for the growth of hydrogen-oxidizing bacteria and the determination of hydrogen autotrophy

A simplified procedure for the determination of autotropic growth of hydrogen-oxidizing bacteria has been developed. The method uses commercially available disposable hydrogen and carbon dioxide kits, commonly used in anaerobic bacteriology, to produce a gaseous atmosphre containing by volume approximately 41% hydrogen, 6% carbon dioxide, 11% oxygen and 42% nitrogen. The atmosphere was suitable for the growth of strains assigned to the species Alcaligenes eutrophus, Alcaligenes paradoxus, Paracoccus denitrificans, Pseudomonas facilis, Pseudomonas flava, Pseudomonas palleronii, Pseudomonas saccahrophilia and Rhodococcus sp. (‘Nocardia opaca’). The method can also be used for the screening of hydrogen-oxidizing ability in bacterial isolates, thus eliminating the need for complex gas mixing devices or expensive gas mixtures.     

Continuous measurement of dissolved H2 in an anaerobic reactor using a new hydrogen/air fuel cell detector

A miniature fuel cell, using a hydrophobic Teflon(R) membrane, designed to continuously measure dissolved H(2) in nonbiological media, was tested for use in anaerobic digestion conditions. In water, this detector responds quickly and efficiently to variation of hydrogen concentration in the range from 80 to 770 nM The media used, and the metabolites or products found in anaerobic digestion media, i. e. inorganic carbon and phosphate buffers, formate, acetate, and dissolved methane, did not interfere with the signal of the detector cell. Dissolved hydrogen sulfide did not poison the cell but was detected. In spite of the detector's high sensitivity to hydrogen (about 21,000 times higher for hydrogen than for hydrogen sulfide), interferences can occur in media containing high sulfide levels.In a methanogenic reactor, the detector cell response to dissolved hydrogen was fast and reliable with time. The observed values ranged values ranged from 2 to 3.5muM. Dissolved hydrogen concentrations were 40 to 70 times higher than values calculated from measured hydrogen partial pressures and Henry's coefficient, suggesting a limitation of the process in the hydrogen transfer from the liquid to the gaseous phase.     

Thermophilic Anaerobic Biodegradation of [14C]Lignin, [14C]Cellulose, and [14C]Lignocellulose Preparations

Thermophilic (55°C) anaerobic enrichment cultures were incubated with [¹⁴C-lignin]lignocellulose, [¹⁴C-polysaccharide]lignocellulose, and kraft [¹⁴C]lignin prepared from slash pine, Pinus elliottii, and ¹⁴C-labeled preparations of synthetic lignin and purified cellulose. Significant but low percentages (2 to 4%) of synthetic and natural pine lignin were recovered as labeled methane and carbon dioxide during 60-day incubations, whereas much greater percentages (13 to 23%) of kraft lignin were recovered as gaseous end products. Percentages of label recovered from lignin-labeled substrates as dissolved degradation products were approximately equal to percentages recovered as gaseous end products. High-pressure liquid chromatographic analyses of CuO oxidation products of sound and degraded pine lignin indicated that no substantial chemical modifications of the remaining lignin polymer, such as demethoxylation and dearomatization, occurred during biodegradation. The polysaccharide components of pine lignocellulose and purified cellulose were relatively rapidly mineralized to methane and carbon dioxide; 31 to 37% of the pine polysaccharides and 56 to 63% of the purified cellulose were recovered as labeled gaseous end products. An additional 10 to 20% of the polysaccharide substrates was recovered as dissolved degradation products. Overall, these results indicate that elevated temperatures can greatly enhance rates of anaerobic degradation of lignin and lignified substrates to methane and low-molecular-weight aromatic compounds.     

Mathematical modelling of the anaerobic digestion process: Application of dynamic mass-energy balance

The method of mass and energy balance was used in the design of a dynamic model of anaerobic digestion of complex organic substrates with production of methane. Distribution of mass flow, represented by the most abundant elements (C, H, N, O), and energy flow, represented by redoxons (available electrons), into gas and liquid output streams is influenced by environmental conditions in a continuous flow digester. Two pathways of methane generation,via cleavage of acetate andvia carbon dioxide reduction by hydrogen, are described in the model. The model was compared with experimental data from laboratory and pilot-plant experiments     

Oxygen absorption in stirred tanks: A correlation for ionic strength effects

A new correlation is given for the prediction of the volumetric coefficient for mass transfer (K_La) in stirred tanks from dispersed gas bubbles to basal salt solutions of ionic strengths representative of fermentation media. The correlation includes the effects of both the operating parameters (agitation power per unit volume and gas superficial velocity) and the physicochemical properties of the system: interfacial tension, viscosity, density, diffusion, coefficient and, in particular, ionic strength. The effect of the latter was found to be most significant in the Newtonian systems of water-like viscosity investigated; no previous correlations have included the effect of ionic strength. K_La values were determined by using a dissolved oxygen probe to monitor the steady-state oxygen tension in continuous flow experiments, and/or the rate of change of oxygen tension in unsteady-state semibatch experiments. In the latter cases, results were computed by a nonlinear, least squares computer program which fitted the experimental data to a model of probe transient response characteristics. The general applicability of the model and the computational procedure was verified by comparing the results to those obtained with the same electrolyte solution in the steady-state mode. The experiments were run over a wide range of agitation power inputs, including those typical of both soluble- and insoluble-substrate fermentations. The correlation appears to be valid for both oxygen mass transfer with and without homogeneous chemical reaction in the liquid phase; in the former case, for example, sulfite oxidation, knowledge of the chemical reaction enhancement factor is required. In addition to predicting oxygen transfer capabilities, the correlation may be used for other sparingly soluble gases of interest in fermentation systems, such as methane, hydrogen, and carbon dioxide.     

Cellulose Fermentation by a Rumen Anaerobic Fungus in Both the Absence and the Presence of Rumen Methanogens

The fermentation of cellulose by an ovine rumen anaerobic fungus in the absence and presence of rumen methanogens is described. In the monoculture, moles of product as a percentage of the moles of hexose fermented were: acetate, 72.7; carbon dioxide, 37.6; formate, 83.1; ethanol, 37.4; lactate, 67.0; and hydrogen, 35.3. In the coculture, acetate was the major product (134.7%), and carbon dioxide increased (88.7%). Lactate and ethanol production decreased to 2.9 and 19%, respectively, little formate was detected (1%), and hydrogen did not accumulate. Substantial amounts of methane were produced in the coculture (58.7%). Studies with [2-¹⁴C]acetate indicated that acetate was not a precursor of methane. The demonstration of cellulose fermentation by a fungus extends the range of known rumen organisms capable of participating in cellulose digestion and provides further support for a role of anaerobic fungi in rumen fiber digestion. The effect of the methanogens on the pattern of fermentation is interpreted as a shift in flow of electrons away from electron sink products to methane via hydrogen. The study provides a new example of intermicrobial hydrogen transfer and the first demonstration of hydrogen formation by a fungus.     

高效产氢菌株Enterococcus sp. LG1的分离及产氢特性

采用Hungate厌氧培养技术分别从厌氧污泥、好氧污泥及河底泥中分离出12株厌氧产氢细菌,并对其中的Enterococcus sp.LG1(注册号:EU258743)进行了研究.结果表明,该株细菌为专性厌氧菌,经革兰氏染色结果为阴性.通过16S rDNA碱基测序和比对证实,该菌株是目前尚未报道过的1个新菌种,初步确定其细菌学上的分类地位.同时,以灭菌预处理的污泥为底物培养基,对该菌的产氢能力及污泥发酵过程中底物性质变化(SCOD、可溶性蛋白质、总糖和pH值等)进行了探讨.实验结果显示,产氢茵Enterococcus sp.LG1的发酵过程中只有H2和CO2产生,无CH4产生.产气量最高为36.48 mL/g TCOD,氢气含量高达73.5%,为已报道文献中以污泥为底物发酵制氢中之最高.根据污泥发酵产物分析得知,该菌的发酵类行为典型的丁酸型发酵.     

Wildfire reduces carbon dioxide efflux and increases methane uptake in ponderosa pine forest soils of the southwestern USA

Severe wildfire may cause long-term changes in the soil-atmosphere exchange of carbon dioxide and methane, two gases known to force atmospheric warming. We examined the effect of a severe wildfire 10?years after burning to determine decadal-scale changes in soil gas fluxes following fire, and explored mechanisms responsible for these dynamics. We compared soil carbon dioxide efflux, methane uptake, soil temperature, soil water content, soil O horizon mass, fine root mass, and microbial biomass between a burned site and an unburned site that had similar stand conditions to the burned site before the fire. Compared to the unburned site, soil carbon dioxide efflux was 40% lower and methane uptake was 49% higher at the burned site over the 427-day measurement period. Soil O horizon mass, microbial biomass, fine root mass, and surface soil water content were lower at the burned site than the unburned site, but soil temperature was higher. A regression model showed soil carbon dioxide efflux was more sensitive to changes in soil temperature at the burned site than the unburned site. The relative importance of methane uptake to carbon dioxide efflux was higher at the burned site than the unburned site, but methane uptake compensated for only 1.5% of the warming potential of soil carbon dioxide efflux at the burned site. Our results suggest there was less carbon available at the burned site for respiration by plants and microbes, and the loss of the soil O horizon increased methane uptake in soil at the burned site.     

Carbon cycling within the sulfate-methane-transition-zone in marine sediments from the Ulleung Basin

The significance of the various carbon cycling pathways in driving the sharp sulfate methane transition zone (SMTZ) observed at many locations along continental margins is still a topic of debate. Unraveling these processes is important to our understanding of the carbon cycle in general and to evaluate whether the location of this front can be used to infer present and past methane fluxes from deep reservoirs (e.g., gas hydrate). Here we report the pore water data from the second Ulleung Basin Gas Hydrate Expedition and on the results of a box model that balances solute fluxes among different carbon pools and satisfies the observed isotopic signatures. Our analysis identifies a secondary methanogenesis pathway within the SMTZ, whereby 25–35 % of the dissolved inorganic carbon (DIC) produced by the anaerobic oxidation of methane (AOM) is consumed by CO₂ reduction (CR). To balance this DIC consumption, a comparable rate of organic matter degradation becomes necessary, which in turn consumes a significant amount of sulfate. The fraction of sulfate consumed by AOM ranges from 70 to 90 %. Whereas a simple mass balance would suggest a one to one relationship between sulfate and methane fluxes; our isotopic considerations show that methane flux estimates based solely on sulfate data may be in error by as much as 30 %. Furthermore, the carbon cycling within the SMTZ is fueled by a significant contribution (10–40 %) of methane produced by CR just below the SMTZ. Therefore sulfate gradient cannot necessarily be used to infer methane contributions from gas hydrate reservoirs that may lay tens to hundreds of meters below the SMTZ.