Ethane production by Methanosarcina barkeri during growth in ethanol supplemented medium

Methanosarcina barkeri strain 227 produced ethane during growth on H₂/CO₂ when ethanol was added to the medium in concentrations of 89–974 mM; ethane production varied from 14 to 38 nmoles per tube (20 ml gas phase, 5.7 ml liquid) with increasing ethanol concentrations. Cells grown to mid-logarithmic phase (A₆₀₀ 0.46, protein = 64 g/ml) on H₂/CO₂, thoroughly flushed with H₂/CO₂, then exposed to ethanol, produced maximal ethane levels (at 585 and 974 mM ethanol) of about 215 nmoles per tube, with an ethane/methane ratio of 1×10^-3. Mid-logarithmic-phase cultures of Methanosarcina barkeri strain Fusaro also produced ethane (up to 20 nmoles per tube) when exposed to ethanol. Cultures of strain 227 growing on methanol in the absence of H₂ produced 6 nmoles per tube of ethane when supplemented with ethanol whereas those lacking ethanol but containing H₂ and/or methanol produced 1.6 nmoles per tube. Cultures of Methanococcus deltae strains LH and RC, Methanospirillum hungatei or Methanobacterium thermoautotrophicum produced 5 nmoles ethane per tube when grown in medium containing ethanol. Ethanol concentrations of 177–886 mM were inhibitory to growth of all methanogens examined. Production of ethane by Methanosarcina was inhibited by >62 mM methanol, and both methanogenic inhibitors tested, CCl₄ and Br–CH₂–CH₂–SO _inf3 ^sup- , inhibited ethane and methane production concurrently. The data suggest that ethanol is converted to ethane by Methanosarcina species using the terminal portion of the methanol-to-methane pathway.     

Assimilatory reduction of sulfate and sulfite by methanogenic bacteria.

A variety of sulfur-containing compounds were investigated for use as medium reductants and sulfur sources for growth of four methanogenic bacteria. Sulfide (1 to 2 mM) served all methanogens investigated well. Methanococcus thermolithotrophicus and Methanobacterium thermoautotrophicum Marburg and delta H grew well with S0, SO3(2-), or thiosulfate as the sole sulfur source. Only Methanococcus thermolithotrophicus was able to grow with SO4(2-) as the sole sulfur source. 2-Mercaptoethanol at 20 mM was greatly inhibitory to growth of Methanococcus thermolithotrophicus on SO4(2-) or SO2(2-) and Methanobacterium thermoautotrophicum Marburg on SO3(2-) but not to growth of strain delta H on SO3(2-). Sulfite was metabolized during growth by Methanococcus thermolithotrophicus. Sulfide was produced in cultures of Methanococcus thermolithotrophicus growing on SO4(2-), SO3(2-), thiosulfate, and S0. Methanobacterium thermoautotrophicum Marburg was successfully grown in a 10-liter fermentor with S0, SO3(2-), or thiosulfate as the sole sulfur source.     

Relationship of formate to growth and methanogenesis by Methanococcus thermolithotrophicus

Methanococcus thermolithotrophicus is a methanogenic archaebacterium that can use either H2 or formate as its source of electrons for reduction of CO2 to methane. Growth and suspended-whole-cell experiments show that H2 plus CO2 methanogenesis was constitutive, while formate methanogenesis required adaptation time; selenium was necessary for formate utilization. Cells grown on formate had 20 to 100 times higher methanogenesis rates on formate than cells grown on H2-CO2 and transferred into formate medium. Enzyme assays with crude extracts and with F420 or methyl viologen as the electron acceptor revealed that hydrogenase was constitutive, while formate dehydrogenase was regulated. Cells grown on formate had 10 to 70 times higher formate dehydrogenase activity than cells grown on H2-CO2 with Se present in the medium; when no Se was added to H2-CO2 cultures, even lower activities were observed. Adaptation to and growth on formate were pH dependent, with an optimal pH for both about one pH unit above that optimal for H2-CO2 (pH 5.8 to 6.5). When cells were grown on H2-CO2 in the presence of formate, formate (greater than or equal to 50 mM) inhibited both growth and methanogenesis at pH 5.8 to 6.2, but not at pH greater than 6.6. Both acetate and propionate produced similar inhibition. Formate inhibition was also observed in Methanospirillum hungatei.     

Relationships among tetraploid wheat (Triticum turgidum L.) landrace populations revealed by isozyme markers and agronomic traits

Diversity and relationships among ten tetraploid wheat landrace populations, collected from different localities in the central highlands of Ethiopia, were studied using isozyme markers and agronomic traits. This type of analysis in crop species is fundamental for designing optimal germ plasm collection, management practices and for developing an index for parental selection. The populations differed in allelic frequencies. Gene-diversity estimates showed that the populations encompass an appreciable amount of variation. However, differentiation between them was low, as was also confirmed by the presence of gene flow. Much of the diversity (85%), was attributable to the within-population level. The genetic distances were mostly small with the exception of those between a few pairs of populations. Thus, the relationships discerned among the populations were more of a similarity nature which could be ascribed to sharing a common ancestral population and/or adaptation to similar climatic conditions. The pattern of genetic divergence appeared to be independent of geographic distance. Considerable divergence in the agronomic traits was observed for certain populations. Cluster analyses of the isozyme and agronomic data produced different patterns and memberships of groupings. This lack of agreement could be ascribed to the different forces of evolution acting on isozyme markers and agronomic traits since agronomic traits, are the prime target of artificial selection. The clustering based on agronomic traits resulted in grouping together populations with similar agronomic performance. The results of this study suggest that taking more samples within a locality or population would be a better approach to capture the range of variation in the landrace populations of the central highlands of Ethiopia.     

Halotolerance of Methanobacterium thermoautotrophicum delta H and Marburg.

Methanobacterium thermoautotrophicum delta H and Marburg were adapted to grow in medium containing up to 0.65 M NaCl. From 0.01 to 0.5 M NaCl, there was a lag before cell growth which increased with increasing external NaCl. The effect of NaCl on methane production was not significant once the cells began to grow. Intracellular solutes were monitored by nuclear magnetic resonance (NMR) spectroscopy as a function of osmotic stress. In the delta H strain, the major intracellular small organic solutes, cyclic-2,3-diphosphoglycerate and glutamate, increased at most twofold between 0.01 and 0.4 M NaCl and decreased when the external NaCl was 0.5 M. M. thermoautotrophicum Marburg similarly showed a decrease in solute (cyclic-2,3-diphosphoglycerate, 1,3,4,6-tetracarboxyhexane, and L-alpha-glutamate) concentrations for cells grown in medium containing > 0.5 M NaCl. At 0.65 M NaCl, a new organic solute, which was visible in only trace amounts at the lower NaCl concentrations, became the dominant solute. Intracellular potassium in the delta H strain, detected by atomic absorption and 39K NMR, was roughly constant between 0.01 and 0.4 M and then decreased as the external NaCl increased further. The high intracellular K+ was balanced by the negative charges of the organic osmolytes. At the higher external salt concentrations, it is suggested that Na+ and possibly Cl- ions are internalized to provide osmotic balance. A striking difference of strain Marburg from strain delta H was that yeast extract facilitated growth in high-NaCl-containing medium. The yeast extract supplied only trace NMR-detectable solutes (e.g., betaine) but had a large effect on endogenous glutamate levels, which were significantly decreased. Exogenous choline and glycine, instead of yeast extract, also aided growth in NaCl-containing media. Both solutes were internalized with the choline converted to betaine; the contribution to osmotic balance of these species was 20 to 25% of the total small-molecule pool. These results indicate that M. thermoautotrophicum shows little changes in its internal solutes over a wide range of external NaCl. Furthermore, they illustrate the considerable differences in physiology in the delta H and Marburg strains of this organism.     

Development of an immunoassay to detect insect contamination of microalgal products

While the insect fragment count is currently the primary test used for assessing insect contamination of food products, this technique is very problematical for assaying microalgal materials. An account is given of a new immunoassay technique,which is based on an enzyme-linked immunosorbent assay(ELISA) detection of insect myosin and which provides a rapid and convenient means of quantitatively determining the amount of insect contamination in algal product samples with a high degree of replicability. Up to 30 samples can be tested in duplicate in 2.5–3 h. Experiments were carried out with a variety of common contaminant insects of algal products, using various life stages, including Corixidae, Ephydridaeand Chironomidae using both Spirulina (Arthrospira) and Chlorella as typical algal materials. As little as one insect per 50 g sample can readily be detected, with excellent correlation (r² = 0.99) between the number of insects present and the color produced. A matrix analysis to determine the ruggedness of the immunoassay was carried out following the protocols of the AOAC International and established that minor departures in seven variables from the standard assay resulted in no substantial differences. The insect myosin assay offers a quantitative and reliable means for assessing insect contamination of algal materials and should be considered for adoption as a standard method for this type of product. This revised version was published online in August 2006 with corrections to the Cover Date.     

Isolation and characterization of plasma membrane-associated cortical granules from sea urchin eggs

Cortical granules, which are specialized secretory organelles found in ova of many organisms, have been isolated from the eggs of the sea urchins Arbacia punctulata and Strongylocentrtus pupuratus by a simple, rapid procedure. Electron micropscope examination of cortical granules prepared by this procedure reveals that they are tightly attached to large segments of the plasma membrane and its associated vitelline layer. Further evidence that he cortical granules were associated with these cell surface layers was obtained by (125)I-labeling techniques. The cortical granule preparations were found to be rich in proteoesterase, which was purified 32-fold over that detected in a crude homogenate. Similarly, the specific radioactivity of a (125)I-labeled, surface glycoprotein was increased 40-fold. These facts, coupled with electron microscope observations, indicate the isolation procedure yields a preparation in which both the cortical granules and the plasma membrane-vitelline layer are purified to the same extent. Gel electrophoresis of the membrane-associated cortical granule preparation reveals the presence of at least eight polypeptides. The major polypeptide, which is a glycotprotein of apparent mol wt of 100,000, contains most of the radioactivity introduced by (125)I-labeling of the intact eggs. Lysis of the cortical granules is observed under hypotonic conditions, or under isotonic conditions if Ca(2+) ion is present. When lysis is under isotonic conditions is induced by addition of Ca(2+) ion, the electron-dense contents of the granules remain insoluble. In contrast, hypotonic lysis results in release of the contents of the granule in a soluble form. However, in both cases the (125)I-labeled glycoprotein remains insoluble, presumably because it is a component of either the plasma membrane or the vitelline layer. All these findings indicate that, using this purified preparation, it should be possible to carry out in vitro studies to better define some of the initial, surface-related events observed in vivo upon fertilization.     

Ecosystem responses to woody plant encroachment in a semiarid savanna rangeland

Woody plant encroachment alters the structure and function of rangeland ecosystems. The objective of this study was to explore the association between woody plant encroachment and various ecosystem properties (i.e. vascular plant species diversity, richness, evenness, soil organic matter, herbaceous biomass, leaf litter and bare ground cover) in a semiarid savanna rangeland, and also to test whether the relationships were influenced by woody species composition, elevation and site. We carried out a vegetation survey in four rangeland sites in the lower Omo region of southwestern Ethiopia, and regressed each one of the ecosystem properties, separately, against woody plant density, elevation and site using multiple linear regressions. We found that vascular plant species diversity, richness and evenness increased with woody plant density, most likely due to increased spatial heterogeneity and soil microclimate improvement. Bare ground cover increased significantly, whereas herbaceous biomass and soil organic matter did not respond to woody encroachment. In a subsequent investigation, we used a redundancy analysis to assess whether ecosystem properties were influenced by the identity of encroaching woody plant species. Species diversity and richness responded positively to Lannea triphylla, whereas leaf litter responded positively to Grewia tenax and G. villosa. Our findings suggest that woody plant encroachment in a semiarid rangeland does alter ecosystem properties. However, its impact is highly variable, influenced by a set of factors including the level of encroachment and identity of encroaching woody species.     

Programmatic Cost Evaluation of Nontargeted Opt-Out Rapid HIV Screening in the Emergency Department

Background

The Centers for Disease Control and Prevention recommends nontargeted opt-out HIV screening in healthcare settings. Cost effectiveness is critical when considering potential screening methods. Our goal was to compare programmatic costs of nontargeted opt-out rapid HIV screening with physician-directed diagnostic rapid HIV testing in an urban emergency department (ED) as part of the Denver ED HIV Opt-Out Trial.

Methods

This was a prospective cohort study nested in a larger quasi-experiment. Over 16 months, nontargeted rapid HIV screening (intervention) and diagnostic rapid HIV testing (control) were alternated in 4-month time blocks. During the intervention phase, patients were offered HIV testing using an opt-out approach during registration; during the control phase, physicians used a diagnostic approach to offer HIV testing to patients. Each method was fully integrated into ED operations. Direct program costs were determined using the perspective of the ED. Time-motion methodology was used to estimate personnel activity costs. Costs per patient newly-diagnosed with HIV infection by intervention phase, and incremental cost effectiveness ratios were calculated.

Results

During the intervention phase, 28,043 eligible patients were included, 6,933 (25%) completed testing, and 15 (0.2%, 95% CI: 0.1%–0.4%) were newly-diagnosed with HIV infection. During the control phase, 29,925 eligible patients were included, 243 (0.8%) completed testing, and 4 (1.7%, 95% CI: 0.4%–4.2%) were newly-diagnosed with HIV infection. Total annualized costs for nontargeted screening were $148,997, whereas total annualized costs for diagnostic HIV testing were $31,355. The average costs per HIV diagnosis were $9,932 and $7,839, respectively. Nontargeted HIV screening identified 11 more HIV infections at an incremental cost of $10,693 per additional infection.

Conclusions

Compared to diagnostic testing, nontargeted HIV screening was more costly but identified more HIV infections. More effective and less costly testing strategies may be required to improve the identification of patients with undiagnosed HIV infection in the ED.