Prevention of formation of acid drainage from high-sulfur coal refuse by inhibition of iron- and sulfur-oxidizing microorganisms. I. Preliminary experiments in controlled shaken flasks

Changes of pH and sulfate concentration in high-sulfur coal refuse slurries are used as measurements of microbial pyrite oxidation in the laboratory. Sodium lauryl sulfate (SLS), alkylbenzene sulfonate (ABS), benzoic acid (BZ) and combinations of SLS plus BZ and ABS plus BZ effectively inhibited formation of sulfate and acid when added in concentrations greater than 50 mg/L to inoculated 20 or 30% coal refuse slurries. Here 25 mg/L concentrations of SLS, ABS, and ABS + BZ stimulated acid production. Formic, hexanoic, oxalic, propionic, and pyruvic acids at 0.1% concentrations were also effective inhibitors. Four different lignin sulfonates were only slightly effective inhibitors at 0.1% concentrations. It was concluded that acid formation resulting from microbial oxidation in high-sulfur coal refuse can be inhibited.     

PCR amplification of 16S rDNA sequences in Fe-rich sediment of coal refuse drainage

Acid drainage from coal mines or coal refuse, when treated in collection ponds, results in the formation of Fe-rich sediments. The Fe in these sediments inhibits the PCR and hinders identification of bacteria using non-culture 16S rDNA molecular methods. We describe a technique that employs EDTA to dissolve and remove Fe from these sediments, yielding DNA that can be amplified by PCR. The technique allows the identification of bacterial DNA sequences and increases our understanding of the bacterial community in Fe-rich sediments.     

Bacteria and Acidic Drainage from Coal Refuse: Inhibition by Sodium Lauryl Sulfate and Sodium Benzoate

The application of an aqueous solution of sodium lauryl sulfate and sodium benzoate to the surface of high-sulfur coal refuse resulted in the inhibition of iron-and sulfur-oxidizing chemoautotrophic bacteria and in the decrease of acidic drainage from the refuse, suggesting that acid drainage can be abated in the field by inhibiting iron- and sulfur-oxidizing bacteria.     

Anaerobic biodegradability of cellulose and hemicellulose in excavated refuse samples using a biochemical methane potential assay

Summary Improved techniques are needed to predict potential methane generation from refuse buried in landfills. The Biochemical Methane Potential (BMP) test was used to measure the methane potential of ten refuse samples excavated from a Berkeley, CA, landfill. The test was conducted in 125-ml serum bottles containing phosphate-buffered medium and inoculated with anaerobically digested sewage sludge. Comparison of the measured BMP to the theoretical BMP calculated from measured cellulose and hemicellulose concentrations indicated that cellulose plus hemicellulose is not well correlated with the measured BMP. The average of the measured to theoretical BMP was 19.1% (range 0–53%, s.d.=16.9%). Measured sulfate concentrations showed that sulfate was an insignificant electron sink in the samples tested. Once methane production from the refuse was complete, 0.072 g of Whatman no. 1 filter paper was added to two of the four serum bottles incubated for each sample. An average of 84.9% (s.d=2.5%) of the added filter paper was recovered as methane, suggesting that some cellulose and hemicellulose present in refuse is recalcitrant or otherwise not bioavailable.     

Preliminary investigation of the influence of pH on the solid-state refuse methanogenic fermentation

Laboratory refuse columns and bottle cultures were used to examine the responses of the interrelated metabolic processes of acid metabolism, solvent metabolism and methanogenesis of the solid-state refuse fermentation to single initial additions of exogenous buffering capacity. Refuse samples poised at neutral and alkaline pH values, by the addition of 0.2 mol/1 phosphate buffer, were found to promote acid-ogenesis to such an extent that methanogenesis was inhibited. The same inhibition was also apparent in the presence of acid pH regimes which were initially characterized by the accumulation of solvents. The possible implications of controlling pH by adding buffer to promote and exploit the refuse fermentation are discussed.     

Pyrite oxidation by thermophilic archaebacteria.

Three species of thermophilic archaebacteria of the genera Sulfolobus (Sulfolobus acidocaldarius and S. solfataricus) and Acidianus (Acidianus brierleyi) were tested for their ability to oxidize pyrite and to grow autotrophically on pyrite, to explore their potential for use in coal desulfurization. Only A. brierleyi was able to oxidize and grow autotrophically on pyrite. Jarosite was formed during the pyrite oxidation, resulting in the precipitation of sulfate and iron. The medium composition affected the extent of jarosite formation.     

Detection of Thiobacillus ferrooxidans in acid mine environments by indirect fluorescent antibody staining.

An indirect fluorescent antibody (FA) staining technique was developed for the rapid detection of Thiobacillus ferrooxidans. The specificity of the FA stain for T. ferrooxidans was demonstrated with both laboratory and environmental samples. Coal refuse examined by scanning electron microscopy exhibited a rough, porous surface, which was characteristically covered by water-soluble crystals. Significant numbers of T. ferrooxidans were detected in the refuse pores. A positive correlation between numbers of T. ferrooxidans and acid production in coal refuse in the laboratory was demonstrated with the FA technique.     

Detection of Thiobacillus ferrooxidans in acid mine environments by indirect fluorescent antibody staining.

An indirect fluorescent antibody (FA) staining technique was developed for the rapid detection of Thiobacillus ferrooxidans. The specificity of the FA stain for T. ferrooxidans was demonstrated with both laboratory and environmental samples. Coal refuse examined by scanning electron microscopy exhibited a rough, porous surface, which was characteristically covered by water-soluble crystals. Significant numbers of T. ferrooxidans were detected in the refuse pores. A positive correlation between numbers of T. ferrooxidans and acid production in coal refuse in the laboratory was demonstrated with the FA technique.     

Effect of Water Potential on Growth and Iron Oxidation by Thiobacillus ferrooxidans

The effect of water potential on the growth of two strains of Thiobacillus ferroxidans was determined by adding defined amounts of sodium chloride or glycerol to the culture medium. The two strains differed slightly, and the most tolerant strain had a minimum water potential for growth of -15 to -32 bars when sodium chloride was used and -6 bars when glycerol was used. In another approach, the limiting water potential was determined by equilibrating small amounts of culture medium with atmospheres of relative humidities equivalent to specific water potentials, and the ability of the organism to grow and oxidize ferrous iron was determined. Under these conditions, which are analogous to those which might control water potential in a coal refuse pile or copper leaching dump, the lower limit at which iron oxidation occurred was -23 bars. The water potential of some coal refuse materials in which T. ferrooxidans was present were determined, and it was found that the water potentials at which the organism was active in these habitats were similar to those at which it was able to grow in culture. However, marked variation in water potential of coal refuse materials was found, presumably due to differences in clays and organic materials, and some coal refuse materials would probably never have water potentials at which the organism could grow. Some literature on the water potentials in copper leach dumps is reviewed, and it is concluded that control of water potential is essential to maximize the success of leaching operations. Because adequate drainage is necessary in a leach dump to ensure sufficient aeration, in many cases water availability in leach dumps may restrict the development of the bacterium necessary for the process.     

The co-disposal of a model brewery wastewater with domestic refuse

By use of refuse columns, it was shown that, at appropriate organic loading rates, the co-disposal of a synthetic brewery wastewater with refuse stimulated methane production and did not reduce leachate quality, in terms of pH or volatile fatty acid concentrations. When the application rate was doubled, from imposed dilution rate ( D ) 0·025 to 0·056 h⁻¹, there was no breakthrough of volatile fatty acids and methane production was promoted. However, when the same total organic load was achieved by doubling the organic strength of the wastewater, but maintaining D = 0·025 h⁻¹, leachate quality was temporarily reduced, with elevated concentrations of acetate and propionate.     

In vitro loading of apoferritin.

This study compared the effect of loading apoferritin either with ferrous ammonium sulfate in various buffers or with ceruloplasmin and chelated ferrous iron. It was shown that loading of apoferritin with ferrous ammonium sulfate was dependent on buffer and pH, and was directly related to the rate of iron autoxidation. The ceruloplasmin-dependent loading of apoferritin, however, was unaffected by these factors. Isoelectric focusing and amino acid analysis of the differently loaded ferritins showed that ferrous ammonium sulfate loading of apoferritin resulted in the depletion of the basic amino acids, lysine and histidine, probably as a result of protein oxidation. No significant differences in amino acid composition was noted for ceruloplasmin-loaded ferritin. Furthermore, ferritin loaded with ferrous ammonium sulfate released more iron than either native or ceruloplasmin-loaded ferritin when either paraquat or EDTA was used as an iron mobilizing agent. We suggest that the loading of apoferritin with ferrous ammonium sulfate occurred as a result of iron autoxidation and may result in oxidation of amino acids and loss of integrity of the protein, and that ceruloplasmin may act as a catalyst for the incorporation of iron into apoferritin in a manner more closely related to that occurring in vivo.     

五年填埋龄准好氧和厌氧填埋体及陈垃圾的理化特性

研究了5年填埋龄准好氧和厌氧填埋体的沉降量、温度和填埋气产生特性,并分析了填埋处理陈腐垃圾的理化性质.结果表明：5年填埋龄准好氧填埋体的表面沉降量和减容率均显著高于厌氧填埋体,同时渗滤液回灌比清水回灌更容易引起垃圾填埋体表面的不均匀沉降.厌氧填埋体的平均温度（25.6 ℃）略高于准好氧填埋体（24.8 ℃）,但差异不显著.准好氧填埋体不同时期的O₂浓度分别高于厌氧填埋体,而CH₄浓度则显著低于厌氧填埋体.5年填埋龄准好氧和厌氧填埋体陈垃圾中厨余物和纸类等易降解有机物含量显著降低,塑料、玻璃、砖瓦和竹木等含量则升高.其中,陈垃圾中有机质与营养元素含量均高于典型南方土壤;除厌氧填埋体陈垃圾中Cr含量超标外,其余重金属含量均未超出《土壤环境质量标准（GB 15618—1995）》的三级标准要求.     

Iron (II) Activated Persulfate Oxidation of MGP Contaminated Soil

The persulfate anion (S₂O₈ ^2?) is a strong oxidant with a redox potential of 2.01 V. However, when mixed with iron (II), it is capable of forming the sulfate radical (SO₄ ^?.) that has an even higher redox potential (E ^o = 2.6 V). In these studies the sulfate radical was investigated to determine if it was a feasible oxidant for the destruction of BTEX and PAH compounds found in MGP contaminated soil. The sulfate radical was generated by either the sequential addition of iron (II) solutions or by a single addition of a citric acid chelated iron (II) solution. The sequentially added iron destroyed 86% of the total BTEX concentration and 56% of the total PAH concentration in the soil. The citric acid chelated iron destroyed 95% of the total BTEX concentration and 85% of the total PAH concentration. A second dose of persulfate and citric acid chelated iron (II) resulted in the destruction of 99% of the total BTEX concentration and 92% of the total PAH concentration. In both the sequential and chelated iron studies the lower molecular weight BTEX compounds were oxidized to a greater extent than the higher molecular weight BTEX compounds, whereas the oxidation of PAH compounds showed no preference to molecular weight.     

Microbial production of methane from municipal refuse

A heat and caustic pretreatment process has been tested to determine the increase in gas yield that can be obtained from fermentation of organic municipal refuse. A treatment temperature of 130°C and a NaOH concentration of 3 g/100 g of dry solids resulted in the highest gas yield for the conditions tested. The probable increase in gas yield was 20% for the best conditions tested. The treatment procedure also substantially increased the rates of gas production. A high conversion efficiency is possible at much shorter reactor retention times with pretreatment than without pretreatment.     

Molecular phylogenetic analysis of dominant microbial populations in aged refuse

The phylogenetic analysis of dominant microbial populations in 8-year-old refuse samples was done in terms of the whole Bacterial and Archaeal domains. The results indicated that the Bacterial 16S rRNA genes sequences from the aged refuse were largely affiliated with the genus Bacillus, and that more than 60 % of the Archaeal sequences were closely related to the methanogenic archaeon. Some inferentially identified extremophilic organisms, particularly alkaliphiles and/or halophiles, were noted to be present in the aged refuse. Moreover, molecular evidence for the occurrence of ammonia-oxidizing Archaea in aged refuse was reported, which opens up avenues for elucidating its role in ammonia transformation in landfill systems. It seems reasonable to assume that the highly complex environment within the landfill systems may select for microbial populations with versatile metabolism and strong adaptation. These findings underline the need for further biochemical and ecological study of these organisms in aged refuse.     

泥炭沼泽湿地关键元素地球化学特征及其对碳排放的影响机制

硫、铁是泥炭沼泽湿地（泥炭地）中重要的生源要素,其参与下的生物地球化学过程对泥炭地碳循环意义重大。选取德国中部两处典型的雨养型泥炭地高海拔样点（TBP）和低海拔样点（TSP）,通过原位采集泥炭剖面孔隙水和可溶性气体等,研究了硫、铁元素等地球化学变化规律,结合DOC、甲烷（CH₄）和二氧化碳（CO₂）浓度分布,探讨其对泥炭地碳排放的影响。研究结果表明：（1） TBP中总还原无机硫（TRIS）浓度随深度先增后减,且上部0-87 cm平均浓度远高于87 cm深度以下,上部硫酸盐还原作用强烈。结合上部亚铁、硫化氢（H₂S）浓度分布,得知该范围内H₂S主要是通过微生物硫酸盐还原作用（BSR）生成,同时H₂S在孔隙水扩散过程中易与亚铁结合为硫化亚铁,进而生成稳定的黄铁矿,这一反应过程在约60 cm处减缓。（2） TBP、TSP两处采样点中DOC与亚铁、硫酸盐均有较强相关性,是由于地下水位的波动影响氧化还原程度以及微生物活性。两处采样点DOC均与亚铁呈显著正相关关系,表明铁氧化物在厌氧环境中被还原溶解产生亚铁,与其结合的有机碳被释放到溶液中从而导致DOC浓度的升高。TBP中DOC与硫酸盐呈显著负相关关系,表明硫酸盐作为电子受体被还原的过程中消耗酸度使pH值升高,增强了其中微生物的活性,DOC浓度由此增加。（3） CH₄与硫酸盐、TRIS浓度在剖面上均呈现相反变化趋势,表明硫酸盐输入的增加以及硫酸盐还原活动均会抑制CH₄生成。CO₂/CH₄均大于4,表明硫酸盐作为替代电子受体会使厌氧条件下碳矿化转向多CO₂和少CH₄生成。此外,亚铁对于CH₄生成一定程度上会起到低促高抑的效果,而对于CO₂的生成的影响较弱。表明硫酸盐对于CH₄和CO₂生成的影响高于亚铁。研究着重探究硫、铁等关键元素地下部生物地球化学过程对碳排放的影响机制,研究结果可为泥炭地碳排放核算提供理论支撑。     

Temperature effects on anaerobic fermentation of domestic refuse

Anaerobic fermentation of organic solid waste can provide a significant source of fuel gas (methane). Application of this process requires a better understanding of the kinetics of the biological system. The literature is replete with kinetic studies of this process as applied to waste solids from water pollution control systems. Much of this work has been conducted in the mesophilic temperature range. Increased temperatures yield higher reaction rates that will improve the economics of the process. The rate limiting step in the fermentation of refuse is the hydrolysis of the complex organic solids, in particular cellulose. Cellulose is a major component of the refuse. A laboratory study employing domestic refuse has shown the effect of temperature on the rate of methane fermentation. The optimum mesophilic temperature was found to be 42°C, while the optimum thermophilic temperature was at least 60°C. No data was obtained beyond the 60°C temperature. Reaction rate constants are presented for anaerobic fermentation of domestic refuse. Because of the characteristics of the substrate it^?was not possible to obtain the necessary measurements for evaluation of constants in the Monod model. An overall system constant was developed.     

Formation and secretion of glycolithocholate-3-sulfate in primary hepatocyte cultures

Bile acid sulfation was studied in primary hepatocyte cultures. The primary hepatocyte cultures formed glycolithocholate-3-sulfate (GLC-S) when glycolithocholate (GLC) was added to the medium. The relative percentage of GLC-S formation increased when the GLC concentration was increased from 10 microM to 100 microM. GLC-S formation was linear to 60 min. GLC-S secretion into the medium was detectable at 75 min and linear to 8 hr. In contrast to the effect of GLC concentration, there was no difference in GLC-S formation or secretion when inorganic sulfate in the medium was increased 16-fold (100 microM-1600 microM). We conclude that the rate of bile acid sulfate formation in cultured primary hepatocytes is primarily controlled by bile acid, but not inorganic sulfate, concentration.     

Linking sedimentary sulfur and iron biogeochemistry to growth patterns of a cold‐water coral mound in the Porcupine Basin,S.W. Ireland (IODP Expedition 307)

Challenger Mound, a 150‐m‐high cold‐water coral mound on the eastern flank of the Porcupine Seabight off SW Ireland, was drilled during Expedition 307 of the Integrated Ocean Drilling Program (IODP). Retrieved cores offer unique insight into an archive of Quaternary paleo‐environmental change, long‐term coral mound development, and the diagenetic alteration of these carbonate fabrics over time. To characterize biogeochemical carbon–iron–sulfur transformations in the mound sediments, the contents of dithionite‐ and HCl‐extractable iron phases, iron monosulfide and pyrite, and acid‐extractable calcium, magnesium, manganese, and strontium were determined. Additionally, the stable isotopic compositions of pore‐water sulfate and solid‐phase reduced sulfur compounds were analyzed. Sulfate penetrated through the mound sequence and into the underlying Miocene sediments, where a sulfate–methane transition zone was identified. Small sulfate concentration decreases (<7 mm ) within the top 40 m of the mound suggested slow net rates of present‐day organoclastic sulfate reduction. Increasing δ³⁴S‐sulfate values due to microbial sulfate reduction mirrored the decrease in sulfate concentrations. This process was accompanied by oxygen isotope exchange with water that was indicated by increasing δ¹⁸O‐sulfate values, reaching equilibrium with pore‐water at depth. Below 50 mbsf, sediment intervals with strong ³⁴S‐enriched imprints on chromium‐reducible sulfur (pyrite S), high degree‐of‐pyritization values, and semi‐lithified diagenetic carbonate‐rich layers characterized by poor coral preservation, were observed. These layers provided evidence for the occurrence of enhanced microbial sulfate‐reducing activity in the mound in the past during periods of rapid mound aggradation and subsequent intervals of non‐deposition or erosion when geochemical fronts remained stationary. During these periods, especially during the Early Pleistocene, elevated sulfate reduction rates facilitated the consumption of reducible iron oxide phases, coral dissolution, and the subsequent formation of carbonate cements.