I. B. Kotova

Utilization of aminoaromatic acids by a methanogenic enrichment culture and by a novel Citrobacter freundii strain

Following incubation of mesophilic methanogenic floccular sludge from a lab-scale upflow anaerobic sludge bed reactor used to treat cattle manure wastewater, a stable 5-aminosalicylate-degrading enrichment culture was obtained. Subsequently, a Citrobacter freundii strain, WA1, was isolated from the 5-aminosalicylate-degrading methanogenic consortium. The methanogenic enrichment culture degraded 5-aminosalicylate completely to CH4, CO2 and NH4+, while C. freundii strain WA1 reduced 5-aminosalicylate with simultaneous deamination to 2-hydroxybenzyl alcohol during anaerobic growth with electron donors such as pyruvate, glucose or serine. When grown on pyruvate, C. freundii WA1 converted 3-aminobenzoate to benzyl alcohol and also reduced benzaldehyde to benzyl alcohol. Pyruvate was fermented to acetate, CO2, H2 and small amounts of lactate, succinate and formate. Less lactate (30%) was produced from pyruvate when C. freundii WA1 grew with 5-aminosalicylate as co-substrate.

Decolorization and partial degradation of selected azo dyes by methanogenic sludge

The toxicity potential and decolorization of three acid azo dyes (Acid Orange 6, Acid Orange 7, and Acid Orange 52) by methanogenic granular sludge from an anaerobic expanded granular sludge bed reactor was assayed. Complete bioreduction was found for all three azo dyes. Sulfanilic acid and 4-aminoresorcinol were detected from the decolorization of Acid Orange 6, sulfanilic acid and 1-amino-2-naphtol were detected from the reduction of Acid Orange 7, and sulfanilic acid and N,N-dimethyl-1,4-phenylenediamine (DMP) were found to be intermediates of Acid Orange 52 degradation. Sulfanilic acid and 1-amino-2-naphtol were persistent in the anaerobic conditions, whereas 4-aminoresorcinol was completely mineralized by anaerobic sludge and DMP was transformed into 1,4-phenylenediamine. Enrichment cultures obtained via consecutive passages on basal medium with only azo dye as a carbon and an energy source seemed to be morphologically heterogeneous. Baculiform and coccus cells were found when viewed under a light microscope. Cocci were joined in chains. Because anaerobic sludge contains sulfate-reducing bacteria and therefore may generate sulfide, azo dyes were tested for chemical decolorization by sulfide to compare rates of chemical and biologic reduction.

Isolation of strain Pseudomonas sp. ASA2 from a methanogenic community degrading aminobenzoate and aminosalicylate

Aminoaromatic compounds are the products of anaerobic degradation of azo dyes and microbial and abiotic reduction of homocyclic nitroaromatics. In recent decades, such xenobiotics, due to their mutagenic and carcinogenic properties, have caused severe environmental problems. These compounds accumulate in bottom sediments of water bodies and soils near varnish-and-dye, textile, and pharmaceutical plants and crucially affect the trophic chains and microbial diversity in these environments. Anaerobic microbial treatment is the main method of eliminating these pollutants, since, under aerobic conditions, certain toxic intermediates may form, and aminoaromatic compounds may polymerize in the presence of oxygen to persistent macromolecules. The behavior of microbial populations in waste treatment plants, as well as in natural environments, is poorly understood. In this connection, it was of interest to study anaerobic microbial communities degrading aminoaromatic substrates and to isolate bacteria responsible for the first stages of this process. The degradation of isomers of aminobenzoic (ABA) and aminosalicylic (ASA) acids was shown in methanogenic sludges and enrichment cultures [1–3]. Anaerobic degradation of ABA by pure bacterial cultures has been investigated [2, 4–6], whereas anaerobic degradation of ASA is still insufficiently studied [7].

Special traits of decomposition of azo dyes by anaerobic microbial communities

We present the results of an investigation into the special traits of conversion of azo dyes Acid Orange 6, Acid Orange 7, Methyl Orange, and Methyl Red under anaerobic conditions in comparison to aerobic conditions. In the presence of oxygen, only Methyl Red underwent decomposition, while under oxygen-free conditions, all remaining substances were fully decolourised under the action of a methanogenous consortium of microorganisms. The products of reduction of the azo bond are determined in the case of each dye. Introduction of additional acceptors of electrons (sulfate and nitrate) had a negative influence on the discoloration of azo dyes. Addition of ethanol as an available organic cosubstrate accelerated decomposition of azo dyes both under methanogenous and sulfate- and nitrate-reducing conditions. There is no direct correlation between the rates of conversion of azo dyes under anaerobic conditions or their toxicity to acetoclastic methanogens. Changes in the morphological composition of the community decolouring an azo dye depended on the duration of its impact on microorganisms. The mechanism of the reduction of the azo bond under the action of substances acting as mediators is explained. These substances are products of the metabolism of the microbial community in anaerobic conditions. It is shown that the supposed mediators NADH and sulfide efficiently decolourise azo dyes in a cell-free system, while riboflavin significantly increased the rate of conversion of substrates in recurrent cycles of discoloration only in the presence of an anaerobic microbial consortium.

[Surviving Forms in Antibiotic-Treated Pseudomonas aeruginosa].

Survival of bacterial populations treated with lethal doses of antibiotics is ensured by very small numbers of persister cells. Unlike antibiotic-resistant cells, antibiotic tolerance of persisters is not inheritable and reversible. The present work provides evidence supporting the hypothesis on transformation (maturation) of persisters of an opportunistic pathogen Pseudomonas aeruginosa, revealed by ciprofloxacin (CF) treatment (25–100 μg/mL), into dormant cystlike cells (CLC) and nonculturable cells (NC), as was described previously for a number of non-spore-forming bacteria. Subpopulations of type 1 and type 2 persisters, which survived antibiotic treatment and developed into dormant forms, were heterogeneous in their capacity to form colonies or microcolonies upon germination as resistance to heating at 70°C and in cell morphology. Type 1 persisters, which were formed after 1-month incubation of the stationary-phase cultures grown in the medium with decreased C and N concentrations, developed in several types of surviving cells, including those similar to CLC in cell morphology. In the course of 1-month incubation of type 2 persisters, which were formed in exponentially growing cultures, other types of surviving cells developed: immature CLC and L-forms. Unlike P. aeruginosa CLC formed in the control post-stationary phase cultures without antibiotic treatment, most of 1-month persisters, especially type 2 ones, were characterized by the loss of colony-forming capacity, probably due to transition into an nonculturable state with relatively high numbers of live intact cells (Live/Dead test). Another survival strategy of P. aeruginosa populations was ensured by a minor subpopulation of CF-tolerant and CF-resistant cells able to grow in the form of microcolonies or regular colonies of decreased size in the presence of the antibiotic. The described P. aeruginosa dormant forms may be responsible for persistent forms in bacteria carriers and latent infections and, together with antibiotic-resistant cells, are important as components of test systems to assay the efficiency of potential pharmaceuticals against resistant infections.

Anaerobic Microbial Associations Degrading Aminoaromatic Acids

Anaerobic microbial associations have been isolated that degrade aminoaromatic acids to methane and carbon dioxide at high rates. Significant differences between the morphological, cytological, and physiological traits of cultures isolated from samples of adapted and unadapted sludge are shown. The effects of cultivation temperature, illumination, and presence of mineral nitrogen and bicarbonate in the medium upon adaptation of enrichment cultures to substrates and subsequent behavior of the anaerobic associations have been studied. Intermediate and final products of degradation of aminoaromatic compounds and the sequence of their formation in the cultures have been determined. We have also studied the effects of exogenous electron acceptors and additional carbon sources on the degradation of aminoaromatic compounds.

The Isolation, Identification and Analyses of Lactobacillus Genus Bacteria with Probiotic Potential.

103 strains of lactic acid bacteria of Lactobacillus genus were isolated from natural sources and identified for genus and species level with API tests and 16S rRNA sequencing. However, only 27 strains from isolated cultures demonstrated a high stability to gastric stress and from that - only 15 strains were highly resistant to intestinal stress. Results indicated that only some isolated cultures of lactobacilli possessed potential probiotical properties and could serve as new probiotics for dairy industry with high resistance to gastro-intestinal stresses.

Methanogenic degradation of (amino)aromatic compounds by anaerobic microbial communities

Abstractdegradation of a range of aromatic substrates by anaerobic microbial communities was studied. Active methanogenic microbial communities decomposing aminoaromatic acids and azo dyes into CH4 and CO2 were isolated. Products of primary conversion were found to be 2-hydroxybenzyl and benzyl alcohols gradually transforming into benzoate. It was shown that isolated microbial communities are capable of converting the initial substrates—benzyl alcohol, benzoate, salicylic acid, and azo dye Acid Orange 6—into biogas without a lag-phase but with different velocities. Aromatic and linear intermediates of biodegradation of aromatic amines by obtained enrichment cultures were determined for the first time. Selective effect of aromatic substrates on a microbial community that was expressed in decrease in diversity and gradual change of dominant morphotypes was revealed.

Production of Proteinase with Plasmin-Like and Prekallikrein Activating Activity by the Micromycete Aspergillus terreus

The effect of nitrogen sources in the fermentation medium and the cultivation conditions on the production of proteinases with plasmin-like and prekallikrein activation activity by micromycete Aspergillus terreus 2 was investigated. The highest secretion of proteinases was achieved when the micromycete was cultivated on a medium containing both amine- and mineral nitrogen sources at an initial pH of 5.5 and at 28°С. It was established that the extracellular micromycete proteinases are equally capable of hydrolyzing fibrin and fibrinogen.