Shifts in Methanogenic Subpopulations Measured with Antibody Probes in a Fixed-Bed Loop Anaerobic Bioreactor Treating Sulfite Evaporator Condensate

A fixed-bed loop, high-rate anaerobic bioreactor treating sulfite evaporator condensate was sampled when it reached steady state and afterwards following perturbations during a 14-month period. By using immunotechnology, it was observed that shifts in methanogenic subpopulations occurred in association with perturbations, such as restarting and relocating the biomass into a different tank. Methanogens related to Methanobacterium bryantii MoHG and Methanobrevibacter smithii ALI were numerous throughout the observation period, while Methanosarcina mazei S6 and Methanosarcina thermophila TM1 were found in the early and late samples, respectively. Also, Methanobacterium formicicum was more numerous at the top portion of the bioreactor, while Methanobrevibacter arboriphilus AZ and DC were at the bottom. Sample formalinization required for prolonged storage proved suitable for antigen preservation.     

Quantitative Immunologic Analysis of the Methanogenic Flora of Digestors Reveals a Considerable Diversity

To determine which methanogens occur in digestors, we performed a quantitative immunologic analysis of a variety of samples. A comprehensive panel of calibrated polyclonal antibody probes of predefined specificity spectra was used. This allowed precise identification of bacteria by antigenic fingerprinting. A considerable diversity of methanogens was uncovered, much larger than previously reported, encompassing at least 14 strains of 11 species. Strategies were developed to measure the load of any given methanogen in a sample and to compare samples quantitatively. Two methanogens were found to predominate which were antigenically closely related with either Methanobacterium formicicum MF or Methanobrevibacter arboriphilus AZ. Fundamental data, probes, and methods are now available to monitor methanogenic subpopulations during digestor operation and thus learn about their respective roles and predictive significance.     

Isolation and Characterization of Methanogenic Bacteria from Landfills

Methanogenic bacteria were isolated from landfill sites in the United Kingdom. Strains of Methanobacterium formicicum, Methanosarcina barkeri, several different immunotypes of Methanobacterium bryantii, and a coccoid methanogen distinct from the reference immunotypes were identified.     

Antigenic determinants distinctive of Methanospirillum hungatei and Methanogenium cariaci identified by monoclonal antibodies

Monoclonal antibodies were prepared against two species of Methanomicrobiaceae. Antibody 1A is specific for Methanospirillum hungatei strain JF1 and the determinant it recognizes is expressed on the surface of JF1 cells, where it is exposed and accessible to antibody. The determinant is found in a polypeptide (MW<12,000) in the sheath that covers the bacterial cell; it is not present in Methanospirillum hungatei strain GP1; and it is not expressed on the surface of whole cells of the other 24 methanogenic bacteria tested. It is therefore a marker of strain JF1, consequently, antibody 1A is potentially useful for tracking JF1 and fragments thereof in a variety of samples. Antibody 7A is specific for Methanogenium cariaci JR1c. It did not react with any other methanogen tested, not even with Mg. marisnigri or Ms. hungatei JF1, although these cross-react with Mg. cariaci if tested with polyclonal antisera. Therefore antibody 7A recognizes specifically a marker of Mg. cariaci JR1c.Abbreviations SIA slide immunoenzymatic assay - SDS-PAGE sodium dodecylsulfate polyacrylamide gel electrophoresis     

Effect of magnesium on methanogenic subpopulations in a thermophilic acetate-degrading granular consortium.

The effects of Mg2+ on thermophilic (55 degrees C) granules grown on acetate in 0.2-liter upflow anaerobic sludge blanket reactors were studied. The methanogens in the granules were identified and counted by using antibody probes and the antigenic fingerprinting method. Packets of large coccoidal cells antigenically related to Methanosarcina thermophila TM-1 were scarce in the absence of Mg2+ but increased with increasing Mg2+ concentrations up to 30 mM; Methanosarcina packets immunologically related to Methanosarcina barkeri R1M3 showed a similar trend, and their numbers increased up to 100 mM Mg2+. The number of single cells antigenically related to TM-1, R1M3, and Methanosarcina mazei S-6 were scarce at low Mg2+ concentrations but increased drastically at 30 and 100 mM Mg2+. The number of rod-shaped bacteria antigenically related to Methanobacterium thermoautotrophicum GC1 and delta H was highest with no Mg2+ present, and their numbers decreased with increasing concentrations of the cation. These quantitative data, obtained by counting cells in suspensions made from disrupted granules, were confirmed by microscopic observation of the methanogenic subpopulations in thin histologic sections of the granules.     

Patterns of sequence variation in the mitochondrial D-loop region of shrews

Direct sequencing of the mitochondrial displacement loop (D-loop) of shrews (genus Sorex) for the region between the tRNA(Pro) and the conserved sequence block-F revealed variable numbers of 79-bp tandem repeats. These repeats were found in all 19 individuals sequenced, representing three subspecies and one closely related species of the masked shrew group (Sorex cinereus cinereus, S. c. miscix, S. c. acadicus, and S. haydeni) and an outgroup, the pygmy shrew (S. hoyi). Each specimen also possessed an adjacent 76-bp imperfect copy of the tandem repeats. One individual was heteroplasmic for length variants consisting of five and seven copies of the 79-bp tandem repeat. The sequence of the repeats is conducive to the formation of secondary structure. A termination-associated sequence is present in each of the repeats and in a unique sequence region 5' to the tandem array as well. Mean genetic distance between the masked shrew taxa and the pygmy shrew was calculated separately for the unique sequence region, one of the tandem repeats, the imperfect repeat, and these three regions combined. The unique sequence region evolved more rapidly than the tandem repeats or the imperfect repeat. The small genetic distance between pairs of tandem repeats within an individual is consistent with a model of concerted evolution. Repeats are apparently duplicated and lost at a high rate, which tends to homogenize the tandem array. The rate of D- loop sequence divergence between the masked and pygmy shrews is estimated to be 15%-20%/Myr, the highest rate observed in D-loops of mammals. Rapid sequence evolution in shrews may be due either to their high metabolic rate and short generation time or to the presence of variable numbers of tandem repeats.      

大鼠胼胝体内神经肽Y免疫反应阳性纤维的发育

本实验用免疫组织化学ＡＢＣ法研究了大鼠胼胝体内神经肽Ｙ免疫反应阳性（ＮＰＹ－ＩＲ）纤维的生后发育。结果发现,许多ＮＰＹ－ＩＲ纤维在大鼠出生时便存在于胼胝体内。ＮＰＹ－ＩＲ胼胝体纤维的密度在生后１周内继续逐渐增高,在第２周内达到最高峰。之后,ＮＰＹ－ＩＲ胼胝体纤维的密度逐渐下降,至第３周末时接近成年时的水平,即仅有少量ＮＰＹ－ＩＲ纤维存在于胼胝体内。这些结果提示在大鼠早期生后发育过程中许多ＮＰＹ－ＩＲ胼胝体纤维是暂时性的,其作用可能与大脑皮质的机能发育有关。     

Start-up of a thermophilic upflow anaerobic sludge bed (UASB) reactor with mesophilic granular sludge

Summary Fast start-up of thermophilic upflow anaerobic sludge bed (UASB) reactors was achieved at process temperatures of 46, 55 and 64° C, using mesophilic granular sludge as inoculum and fatty acid mixtures as feed. The start-up was brought about by increasing the temperature of mesophilic UASB reactors in a single step, which initially led to a sharp drop in the methane production rate. Thereafter, stable thermophilic methanogenesis was achieved within a period of 1 or 2 weeks depending on the temperature of operation. Mesophilic granules functioned initially as effective carrier material for thermophilic organisms. However, long-term operation led to disintegration of the granules, resulting in wash-out of thermophilic biomass. The temperature optima for acetotrophic methanogenic activity of the sludges cultivated at 46, 55 and 64° C, were similar, but differed significantly from the temperature optimum of the mesophilic inoculum. All the sludges examined were dominated by Methanothrix-like rods. These could be distinguished by antigenic fingerprinting into two subpopulations, one predominant at 36° C and the other predominant at 46° C and above. Offprint requests to: J. B. van Lier