Thermophilic anaerobic fermentation of hemicellulose and hemicellulose-derived aldose sugars by Thermoanaerobacter strain B6A

The thermophilic, anaerobic fermentation of hemicellulosic subtrates by Thermoanaerobacter strain B6A was investigated using a variety of commercially-available hemicelluloses which had been characterized by physical and chemical analysis. Products of hemicellulose fermentation included ethanol, acetic acid, lactic acid, H₂, and CO₂ in ratios which varied depending on the hemicellulose used. Both the rate and extent of substrate utilization (as estimated from product formation) varied in the order: unidentified mannan/xyloglucan > xylan > 4-O-methylglucuronoxylan > arabinoxylan > type II arabinogalactan=0. Rates of product formation were enhanced up to twofold by autoclaving of substrates,which partially depolymerized the substrates and in some cases altered their composition. Total extent of product formation, however, was similar in autoclaved and non-autoclaved substrates. Hemicellulose fermentations were mediated by one or more constitutive extracellular enzyme activities. Component monosaccharides of hemicelluloses in their natural isomeric configurations supported rapid growth (_max = 0.3–1.0 h^–1), while unnatural isomers were not utilized.The wide carbohydrate utilization spectrum of this strain apparently reflects its role as a versatile primary consumer in the hot spring bacterial-algal mat from which it was isolated.Abbreviations GC Gas chromatography - HPLC High-performance liquid chromatography - MS Mass spectroscopy - TLC Thinlayer chromatography - TMS Trimethylsilyl Contribution No. 3696 of the Central Research and Development Department     

Analysis of copia sequence variation within and between Drosophila species

The sequences of the 5' long-terminal repeat (LTR) and adjacent leader regions of 27 full-length copia elements isolated from natural populations of Drosophila melanogaster, D. simulans, and D. mauritiana are presented. Phylogenetic analyses indicate that although D. melanogaster copia elements are distinct from those of D. simulans and D. mauritiana, the elements of these latter two species are not distinguishable from one another. LTRs and adjacent 5' leader regions of elements isolated from D. simulans and D. mauritiana are structurally similar to one another and carry substantial deletional variation mapping to regions previously identified as being of potential importance for copia expression.      

Production of caproic acid by cocultures of ruminal cellulolytic bacteria and Clostridium kluyveri grown on cellulose and ethanol

Ruminal cellulolytic bacteria (Fibrobacter succinogenes S85 or Ruminococcus flavefaciens FD-1) were combined with the non-ruminal bacterium Clostridium kluyveri and grown together on cellulose and ethanol. Succinate and acetate produced by the cellulolytic organisms were converted to butyrate and caproate only when the culture medium was supplemented with ethanol. Ethanol (244 mM) and butyrate (30 mM at pH 6.8) did not inhibit cellulose digestion or product formation by S85 or FD-1; however caproate (30 mM at pH 6.8) was moderately inhibitory to FD-1. Succinate consumption and caproate production were sensitive to culture pH, with more caproic acid being produced when the culture was controlled at a pH near neutrality. In a representative experiment under conditions of controlled pH (at 6.8) 6.0 g cellulose 1^–1 and 4.4 g ethanol 1^–1 were converted to 2.6 g butyrate 1^–1 and 4.6 g caproate 1^–1. The results suggest that bacteria that efficiently produce low levels of ethanol and acetate or succinate from cellulose should be useful in cocultures for the production of caproic acid, a potentially useful industrial chemical and bio-fuel precursor.Mention of specific products is intended only to provide information and does not contitute an endorsement by the U.S. Department of Agriculture over other products not mentioned.     

Immunomagnetic detection of Bacillus stearothermophilus spores in food and environmental samples.

There are currently no methods for the rapid and sensitive detection of bacterial spores that could be used to direct raw materials containing high spore loads away from products that pose a food safety risk. Existing methods require an overnight incubation, cannot detect spores below 10(5) CFU/ml, or are not specific to particular species. This work describes a method to specifically detect < 10(4) CFU of bacterial spores per ml within 2 h. Polyclonal antibodies to Bacillus stearothermophilus spores were attached to 2.8-micron-diameter magnetic polystyrene beads by using a polythreonine cross-linker via the antibody carbohydrate moiety. A biotin-avidin-amplified sandwich enzyme-linked immunosorbent assay coupled to a fluorescent substrate was used to quantitate captured spores. The concentration of B. stearothermophilus spores in samples was linearly correlated to fluorescent activity (r2 = 0.99) with a lower detection limit of 8 x 10(3) CFU/ml and an upper detection limit of 8 x 10(5) CFU/ml. The detection limits are not fixed and can be changed by varying the immunomagnetic bead concentration. Several food and environmental samples were tested to demonstrate the versatility of the assay.     

Demography and blood genetics of Argentinian Mapuche Indians

The whole Mapuche Indian community of Blancura Centro was covered by a demographic census, with special attention given to variables of genetic interest. Afterwards a sample of it was investigated in relation to 22 genetic systems. The community can be characterized as a young group, with high fertility, but moderate mortality and endogamy. The index of opportunity for selection is relatively low (0.46). The presence of variation at the ABO and Lutheran loci suggests some non-Indian admixture, calculated as 7% in the sample studied. Unusual findings were the absence of L^*NS, low frequency (7%) of L^*MS and high frequency (37%) of L^*Ns. They also showed low frequencies of P^*1 (28%) and DI^*a (3%), but high of HP^*1 (74%). Similarities, but also differences, were noted with previous results obtained in this tribe in Argentina and Chile.     

Genetic structure of two urban afro-brazilian populations

A total of 218 individuals living in the Brazilian cities of Porto Alegre (in the South) and Salvador (in the Northeast) were variously studied in relation to nine erythrocyte and four plasma protein systems. The results were compared with previous studies in some of these systems, and estimates of interethnic admixture obtained in subsamples according to morphological appearance. As a whole, Afro-Brazilians from Salvador show 42% of non-African genes, the corresponding figure for Porto Alegre being 59%. The Amerindian contribution to these individuals was estimated as null or negligible. Average heterozygosities are similar to those obtained for African groups, but the gene differentiation coefficient (GST') is small. The phylogenetic tree indicates a closer relationship of Salvador with the African subcluster, as would be expected by the admixture and istorical data. Analyses such as this one are important for the unraveling of the complex networks responsible for the present variability of human populations, and for the dispelling of racist concepts.     

The Coagulation Factor XIIa Inhibitor rHA-Infestin-4 Improves Outcome after Cerebral Ischemia/Reperfusion Injury in Rats

Background and Purpose

Ischemic stroke provokes severe brain damage and remains a predominant disease in industrialized countries. The coagulation factor XII (FXII)-driven contact activation system plays a central, but not yet fully defined pathogenic role in stroke development. Here, we investigated the efficacy of the FXIIa inhibitor rHA-Infestin-4 in a rat model of ischemic stroke using both a prophylactic and a therapeutic approach.

Methods

For prophylactic treatment, animals were treated intravenously with 100 mg/kg rHA-Infestin-4 or an equal volume of saline 15 min prior to transient middle cerebral artery occlusion (tMCAO) of 90 min. For therapeutic treatment, 100 mg/kg rHA-Infestin-4, or an equal volume of saline, was administered directly after the start of reperfusion. At 24 h after tMCAO, rats were tested for neurological deficits and blood was drawn for coagulation assays. Finally, brains were removed and analyzed for infarct area and edema formation.

Results

Within prophylactic rHA-Infestin-4 treatment, infarct areas and brain edema formation were reduced accompanied by better neurological scores and survival compared to controls. Following therapeutic treatment, neurological outcome and survival were still improved although overall effects were less pronounced compared to prophylaxis.

Conclusions

With regard to the central role of the FXII-driven contact activation system in ischemic stroke, inhibition of FXIIa may represent a new and promising treatment approach to prevent cerebral ischemia/reperfusion injury.     

Response surface analysis of the effects of pH and dilution rate on Ruminococcus flavefaciens FD-1 in cellulose-fed continuous culture.

The ruminal cellulolytic bacterium Ruminococcus flavefaciens FD-1 was grown in cellulose-fed continuous culture with 20 different combinations of pH and dilution rate (D); the combinations were selected according to the physiological pH range of the organism (6.0 to 7.1) and growth rate of the organism on cellulose (0.017 to 0.10 h-1). A response surface analysis was used to characterize the effects of pH and D on the extent of cellulose consumption, growth yield, soluble sugar concentration, and yields of fermentation products. The response surfaces indicate that pH and D coordinately affect cellulose digestion and growth yield in this organism. As expected, the net cellulose consumption increased with increasing D while the fraction of added cellulose that was utilized decreased with increasing D. The effect of changes in pH within the physiological range on cellulose consumption was smaller than that of changes in D. Cellulose degradation was less sensitive to low pH than to high pH. At low Ds (longer retention times), cellulose degradation did not follow first-order kinetics. This decreased rate of cellulose digestion was not due to poor mixing, limitation by other medium components, or preferential utilization of the more amorphous fraction of the cellulose. The cell yield increased from 0.13 to 0.18 mg of cells per mg of cellulose with increasing Ds from 0.02 to 0.06 h-1 and decreased when the pH was shifted from the optimum of 6.5 to 6.8. The effect of pH on cell yield increased with increasing D. The reduced cell yield at low pH appears to be due to both an increase in maintenance energy requirements and a decrease in true growth yield.     

A segmented gas/liquid delivery system for continuous culture of microorganisms on insoluble substrates and its use for growth of Ruminococcus flavefaciens on cellulose

Summary An anaerobic continuous culture device was constructed that permits accurate delivery of media containing insoluble substrates, even at very low volumetric flow rates (<3 ml/h). The system consisted of: (1) a reservoir in which the medium slurry was mixed well by the combined use of stirring and diffusive gas sparging to suspend a cellulose substrate of small (< 45 m) particle size; (2) a delivery system that segmented the slurry into small (~ 20l) discrete liquid segments separated by intervening bubbles of CO₂ gas; and (3) a stirred, temperature-controlled 2-l fermentation vessel. The device was used to examine substrate consumption, product formation, and cell yield by the anaerobic ruminal cellulolytic bacterium Ruminococcus flavefaciens FD-1 under steady-state, cellulose-limited conditions at six different dilution rates (D) ranging from 0.017 to 0.101 h^–1 (pH 6.4–6.6). Cellulose consumption decreased from 4.00 g/1 (at D=0.017 h^–1) to 2.56 g/1 (at D=0.101 h^–1). Increases in D resulted in a progressive shift toward production of more acetate and formate, and less succinate. Redox balance calculations revealed a deficiency in reduced products, probably due to the production of H₂, which was not directly measured. Reducing sugar values remained low (0.05–0.10 g/1, as glucose) at all D values. The cellulose fermentation was characterized by a low maintenance coefficient (0.07 g cellulose/g cells per hour) and a high true growth yield (Y_G = 0.24 g cells/g cellulose, corrected for maintenance). Comparison of the data with literature values suggests that the fermentation of cellulose by this organism gives cell yields at least as great as the yields obtained from the fermentation of soluble sugars.Mention of specific products is for the benefit of the reader and does not constitute an endorsement of said products over other products not mentionedOffprint requests to: P. J. Weimer