Analysis of the Bioaugmentation Potential of Pseudomonas putida OR45a and Pseudomonas putida KB3 in the Sequencing Batch Reactors Fed with the Phenolic Landfill Leachate

The treatment of landfill leachate could be challenging for the biological wastewater treatment systems due to its high toxicity and the presence of poorly biodegradable contaminants. In this study, the bioaugmentation technology was successfully applied in sequencing batch reactors (SBRs) fed with the phenolic landfill leachate by inoculation of the activated sludge (AS) with two phenol-degrading Pseudomonas putida OR45a and Pseudomonas putida KB3 strains. According to the results, the SBRs bioaugmented with Pseudomonas strains withstood the increasing concentrations of the leachate. This resulted in the higher removal efficiency of the chemical oxygen demand (COD) of 79–86%, ammonia nitrogen of 87–88% and phenolic compounds of 85–96% as compared to 45%, 64%, and 50% for the noninoculated SBR. Simultaneously, the bioaugmentation of the AS allowed to maintain the high enzymatic activity of dehydrogenases, nonspecific esterases, and catalase in this ecosystem, which contributed to the higher functional capacity of indigenous microorganisms than in the noninoculated AS. Herein, the stress level experienced by the microorganisms in the SBRs fed with the leachate computed based on the cellular ATP measurements showed that the abundance of exogenous Pseudomonas strains in the bioreactors contributed to the reduction in effluent toxicity, which was reflected by a decrease in the stress biomass index to 32–45% as compared to the nonbioaugmented AS (76%).

PROBLEMS SOLUTION OF SPECIAL IDENTIFICATION OF EISENIA CULTURAL POPULATIONS

Introduction. Intensification of various aspects of modern agricultural production, based on the use of a large number of mineral fertilizers and chemical means of plant and animal protection, actualizes the development and mastering of natural-like methods for restoring the quality of soils and bottom deposits of inland water bodies. The goal is their successful use in cultivating cultivated plants and conducting remediation measures. One such approach is vermiculturing – breeding of earthworms of the family Lumbricidae, for biotransformation of depleted soils and organic wastes in order to obtain biohumus. Relevance. Earthworms differ significantly in biological characteristics from animals, traditionally bred in agriculture. When working with them, a selection problem arises to assess their phenotypes – it is difficult to identify individuals and assess their performance. In this regard, the main task of effective selection becomes more complicated. Today in scientific sources on the study of the genetic structure of earthworms natural populations, their karyotypes and morphological features are not few data [3]. But the development of vermiculture, based on breeding and industrial use for biotransformation of earthworms, requires a clear species identification of cultural lines of invertebrate animals of the family Lumbricidae. Research objective. The aim of the study was to investigate the morphological and cytogenetic features of the Eisenia worms population that is being forms. Materials and methods. While forming of the new population, the worms that were purchased by the Department of Biotechnology of the UGHTU in the association "Bioconversion" and previously described as E. foetida were used. Of the array of these animals, 6 worms were selected that became the founders of the new population. After increasing the number of up to 300 animals, we formed groups of 20 individuals, each group irradiated with a laser of the LGN-208b type With a power of 1 mW, a wavelength of 633 nm, a beam diameter of 14 mm, various exposures in time from 5 to 30 min. The control was not irradiated. Irradiated animals were bred in separate groups and studied morphological, biochemical and cytogenetic features. Morphological study of the species affiliation was carried out that was determined and compared with the descriptions of these species given in the works of foreign taxonomy [5]. The intensity of the pigmentation of the integument of the body was determined in animals. All further morpho-metric studies were carried out on worms fixed in 75% ethanol. By the method of microscopy, parameters such as length and diameter of the body, total number of segments, location of the segments of the girdle, pubertal ridges and the first dorsal pore, type of setae were analyzed. Karyological analysis was performed with worms selected at the time of highest sexual activity. Preparations were prepared from the tissue of the seminal sacks by the method previously successfully used to study karyotypes of lumbricids [6]. The worms were injected with 0.1% solution of colchicine into the pre-lobe zone for 19 hours. 20 min to the autopsy. The animals were immobilized in 75% ethanol solution and digested along the median spinal line. Removed spermatic bags hypotone 50 min. in the distillate and fixed in three steps in a mixture of acetic acid and ethanol in a ratio of 1:3. Chromosome preparations were made by imprinting. Genetic markings were performed by electrophoresis in a 7.5% polyacrylamide gel Tris-EDTA • Na2-borate system with pH = 8.5 [7] 1 hour 20 minutes at a voltage of 200 V and a current strength of 140–mA. Extract of enzymes and proteins was obtained by grinding the final segments of the body with a size of 5–10 mm in the distillate in the ratio 1:1. After switching of the electric current, the gel was treated with a solution containing a special substrate that specifically reacts with the enzyme under study, forming spots corresponding to the spectra of the enzymes on the gels. The genotype of the individual according to the locus encoding the enzyme being studied is determined by the nature of the distribution of the spots on the gel [8]. Results of the research. The carried out researches have shown that the groups of animals under study belong to the species E. foetida, and by other attributes to the species E. venetta, which caused the problem of the species affiliation of the earthworm array when working to form a new population. Thus, the morphological indices studied have revealed that animals for all morphological features refer to the species Eisenia foetida. During the study of cytogenetic, it was found that the karyotype of the animals was 36 chromosomes but it should be 22. Just kind of Eisenia foetida is composed of 22 chromosomes and karyotypes of other species of the genus Eisenia have 36 chromosomes. Therefore, biochemical gene marking was carried out on enzyme systems, in particular nonspecific esterases. Nonspecific esterases of different species of the genus Eisenia differ in molecular weight. Esterаs of E. foetida have a lower mass than esterases of E. veneta. Our studies showed that in the place of the locus of spectra of nonspecific esterases, the individuals under study belong to the species E. veneta. Nonspecific esterases of different species of the genus Eisenia differ in molecular weight. Esterases E. foetida have a lower mass than E. veneta esterase. Our studies have shown that individuals at the locus of spectra of non-specific esterases belong to the species E. veneta. Conclusions The research may be a theoretical hypothesis for certain types of animal identification in vermiculture and creating biological diversity in its population. Despite the fact that it originates from 6 individuals obtained from a single array of animals, polymorphism by esterases showed a fairly high level of genetic variability in the forming line that indicates the reserve of its genetic variability and allows it to hope for its successful development in the future.

Enzyme histochemistry of monocytes/macrophages: a study in a murine model of immune complex-mediated glomerulonephritis.

In a model of immune complex glomerulonephritis in BALB/c mice, cells of monocyte/macrophage lineage (M phi), identifiable by electron microscopy, infiltrate the glomerulus. In spite of this, no unequivocal nonspecific esterase activity can be demonstrated histochemically in the glomeruli. On the other hand, many mononuclear cells with strong acid phosphatase activity are consistently present. This observation is in line with other studies that have demonstrated the heterogeneity of enzyme profiles in different M phi populations. Despite the wide acceptance of nonspecific esterases as markers for M phi, the present study indicates that exclusive reliance on a single marker in investigating the participation of M phi in a pathological process can lead to erroneous conclusions. Use of multiple markers and preferably multiple investigative modalities is recommended.

Nonspecific esterase isozymes that focus at identical isoelectric points are kinetically analogous.

Isoelectric focusing (IEF) of soluble nonspecific esterases of rat kidney and testis exhibits an identical array of organophosphate-resistant cathodal isozymes. To ascertain whether such isozymes that focus at the same pI are also kinetically analogous, two isozymes, both focused at pI 7.2, were isolated, one from each organ, by elution from cut-out, unstained gel segments. Although the esterases of the whole soluble fraction of kidney and testis exhibited different kinetic properties and organophosphate susceptibility, no differences were observed with regard to the isozymes. Therefore, because of similar electrophoretic and kinetic behavior, the two isozymes can be regarded as phenotypic expressions of similar genetic products.

Monocyte nonspecific esterase. Enzymologic characterization of a neutral serine esterase associated with myeloid cells.

Monocytes contain a characteristic, prominent set of membrane-bound nonspecific esterases with a slightly acid isoelectric point. These esterases are also detected at modest levels in some granulocyte preparations. They are not apparent in lymphocytes. Among 18 fresh myeloid leukemias and myeloid leukemia cell lines, those of subtypes M4 (myelomonocytic) and M5 (monocytic) were strongly positive; some of subtypes M1-M3 (granulocytic) were moderately positive. The esterases were not detected among 32 fresh lymphoid leukemias and lymphoid leukemia and lymphoblast cell lines. The membrane-bound monocyte esterases, solubilized by treatment of monocyte preparations with nonionic detergent, were resolved by ion-exchange chromatography. The monocyte species account for 80-95% of the total nonspecific esterase activity of monocytes. The resolved enzymes behave as neutral serine carboxyl esterases and are highly sensitive to inhibition by diisopropylfluorophosphate (DFP) and also by sodium fluoride. Similar analysis of a lymphocyte preparation yielded no detectable monocyte esterases, but yielded numerous other forms which were generally resistant to inhibition by DFP and NaF. These nonspecific esterases are also present at background levels in monocytes. The resolution and characterization of the membrane-bound serine esterases from monocytes demonstrates the basis for the well-known cytochemistry of monocytes. The results are also crucial to the development of an immunologic surface marker test for myeloid cells and the study of monocyte membrane physiology.