Fed-batch cultivation of methanobacterium formicicum and its fluorometric monitoring

Fed-batch cultivation of Methanobacterium formicicum Z-281 was performed under substrate limiting conditions. Specific rate of culture growth amounted to 0.041 h^?1, cell yield related to substrate was 0.6 mg proteine/mM formate. Significant increase of coenzyme F₄₂₀ content in cells depending on time of cultivation was observed. Increase of the fluorescence intensity of the medium was also observed with the accumulation of biomass. These parameters were suggested for tracking culture growth. The possibility of application of fluorescent factors for methanogenic population control in the anaerobic digester is discussed.     

Methanocalculus natronophilus sp. nov., a new alkaliphilic hydrogenotrophic methanogenic archaeon from a soda lake,and proposal of the new family Methanocalculaceae

A mesophilic hydrogenotrophic methanogenic archaeon, strain Z-7105^T, was isolated from the bottom sediments of a collector in the vicinity of a soda lake Tanatar II (Altai, Russia). The cells were motile, irregular cocci 0.2–1.2 μm in diameter. The organism was an obligate alkaliphile, growing within a pH range from 8.0 to 10.2, with the optimum at pH 9.0–9.5. It was obligately dependent on carbonates, growing at 0.5 to 1.6 M total carbonates with the optimum at 0.7–0.9 M. Sodium ions were also obligately required at concentrations from 0.9 to 3.3 M Na⁺ (optimum at 1.4–1.9 M). The organism was halotolerant, but Clions were not required. Hydrogen and formate were used as electron donors. Acetate was required for anabolism. The DNA G+C content was 50.2 mol %. According to the results of its 16S rRNA gene sequence analysis, the isolate belonged to the genus Methanocalculus, being the first known alkaliphilic member of this genus. Its similarity to the neutrophilic and halotolerant Methanocalculus species (M. halotolerans, M. taiwanensis, M. pumilus, and M. chunghsingensis) was 98.2–97.1%, which is within the interspecific range for this genus. The level of DNA-DNA hybridization between strain Z-7105^T and the Methanocalculus type species M. halotolerans DSM 14092^T was 32%. The genus Methanocalculus, including the new isolate and the previously described species, is distant from other genera of methanogens (<90% 16S rRNA gene similarity). Based on significant phenotypic differences and the results of phylogenetic analysis, including DNA-DNA hybridization, it is proposed to assign strain Z-7105^T (=DSM 25006^T, =VKM B-2765^T) to the new species Methanocalculus natronophilus sp. nov., and to incorporate the genus into the new family Methanocalculaceae fam. nov.     

Peptidase Activity of Proteinivorax Bacteria and Their Possible Ecological Role in the Microbial Communities of Tanatar Soda Lakes (Altai Krai,Russia)

Microbiology - Peptidase activity of alkaliphilic anaerobic proteolytic Proteinivorax bacteria, P. tanatarense Z-910T and P. hydrogeniformans Z-710T, isolated from the Tanatar system of hypersaline...     

Cellulase activity of a haloalkaliphilic anaerobic bacterium, strain Z-7026

Summary The cellulolytic activity of an alkaliphilic obligate anaerobic bacterium, Z-7026, which was isolated from the microbial community of soda-lake sediments and belongs to the cluster III of Clostridia with low G+C content, was studied. The bacterium was capable of growing in media with cellulose or cellobiose as the sole energy sources. Its maximal growth rate on cellobiose (0.042–0.046 h^–1) was observed at an initial pH value of 8.5–9.0, whereas the maximal rate of cellulase synthesis, assayed by using a novel fluorimetric approach, was found to be 0.1 h^–1 at pH 8–8.5. Secreted proteins revealed high affinity for cellulose and were represented by two major forms of molecular masses of 75 and 84 kDa, whereas the general protein composition of the precipitated and cellulose-bound preparations was similar to cellulosome subunits of Clostridium thermocellum. The optimum pH of the partially purified enzyme preparation towards both amorphous and crystalline cellulose was in the range 6–9, with more than 70% and less than 50% of maximal activity being retained at pH 9.2 and 5.0, respectively.     

<Emphasis Type="Italic">Proteinivorax hydrogeniformans</Emphasis> sp. nov., an anaerobic,haloalkaliphilic bacterium fermenting proteinaceous compounds with high hydrogen production

A search for the organisms responsible for the degradation of biomass of primary producers in Tanatar lakes resulted in the isolation of a novel anaerobic, haloalkaliphilic microorganism, strain Z-710^T. The strain grows on proteinaceous substrates (peptides) but not on proteins. A rather limited range of substances of other classes can be utilised together with tryptone but not individually. An interesting physiological feature of the novel strain is a high capacity for hydrogen production (up to 30% v/v) during proteolytic fermentation. Phylogenetic analysis based on the 16S rRNA gene sequence similarity revealed that the organism can be assigned to the previously described genus Proteinivorax. According to its physiological features and the low DNA–DNA hybridisation level of the strain with the type strain of the only previously described Proteinivorax species—Proteinivorax tanatarense Z-910^T—strain Z-710^T is described here as representing a novel species with the name Proteinivorax hydrogeniformans sp. nov. The type strain is Z-710^T (= DSM 102085^T = VKM B-3042^T).     

Reduction of synthetic ferrihydrite by a binary anaerobic culture of <Emphasis Type="Italic">Anaerobacillus alkalilacustris</Emphasis> and <Emphasis Type="Italic">Geoalkalibacter ferrihydriticus</Emphasis> grown on mannitol at pH 9.5

In the course of an investigation of alkaliphilic iron reduction, metabiotic interactions in a binary culture reducing synthetic ferrihydrite (SF) have been studied. The binary culture contained two anaerobic bacteria: the alkaliphilic organotrophic bacillus Anaerobacillus alkalilacustris, which ferments sugars and sugar alcohols and is incapable of iron reduction, and the dissimilatory iron-reducing bacterium Geoalkalibacter ferrihydriticus, which is able to grow on acetate at the expense of anaerobic respiration. The experiments were performed under conditions of SF excess and deficiency. It was expected that G. ferrihydriticus would oxidize the acetate formed in the course of mannitol fermentation by A. alkalilacustris. The results were different from the expected ones: in the binary culture, fermentation products other than acetate were used for iron reduction; these were primarily formate and ethanol, which led to acetate accumulation rather than consumption. The reduction of SF to magnetite and/or siderite followed the earlier established regularities. The preferential order of donor utilization by G. ferrihydriticus did not conform to the energy yields of the corresponding reactions. Thus, it has been shown that there may be interactions in microbial communities that cannot be predicted from the characteristics of pure cultures. The degradation pathways of organic matter in communities may differ considerably from those observed in pure cultures, even in pure cultures of highly specialized organisms.     

Prediction of the three-dimensional structure of aflatoxin of Aspergillus flavus by homology modelling

Aflatoxins are polyketide-derived secondary metabolites produced by Aspergillus spp. The toxic effects of aflatoxins have adverse consequences for human health and agricultural economics. The aflR gene, a regulatory gene for aflatoxin biosynthesis, encodes a protein containing a zinc-finger DNA-binding motif. AFLR-Protein three-dimensional model was generated using Robetta server. The modeled AFLR-Protein was further optimization and validation using Rampage. In the simulations, we monitored the backbone atoms and the C-α-helix of the modeled protein. The low RMSD and the simulation time indicate that, as expected, the 3D structural model of AFLR-protein represents a stable folding conformation. This study paves the way for generating computer molecular models for proteins whose crystal structures are not available and which would aid in detailed molecular mechanism of inhibition of aflatoxin.     

Physiological and biochemical properties of the alkaliphilic anaerobic hydrolytic bacterium <Emphasis Type="Italic">Alkaliflexus imshenetskii</Emphasis>

The growth of Alkaliflexus imshenetskii and concentrations of metabolites produced by this microorganism during growth on various organic substrates were studied. It was shown that, although the composition and quantitative ratios of the fermentation products depended on the substrates utilized, acetate and succinate were always the major metabolites, while only minor amounts of formate were produced. During growth on xylan and starch, diauxy was observed caused by the successive decomposition of oligosaccharides and monosaccharides. It was demonstrated that, when grown on cellobiose, A. imshenetskii is capable of succinate fermentation mediated by phosphoenolpyruvate carboxykinase, pyruvate kinase, fumarate reductase, pyruvate ferredoxin oxidoreductase, malate dehydrogenase, and methylmalonyl-CoA decarboxylase. Succinate may be both the intermediate and final product of the A. imshenetskii metabolism, being fermented to propionate by methylmalonyl-CoA decarboxylase.