Wpływ immobilizacji komórek bakteryjnych na biodegradację naproksenu

Praca zawiera artykuły w języku angielskim.More and more researches are reporting the presence of pharmaceuticals, especially non-steroidal anti-inflammatory drugs in the natural environment. One of them, naproxen is not fully degraded in the human body. Additionally, wastewater treatment plants are not adapted to its utilization. In recent years, bacterial strains which are characterized by increased naproxen degradation potential have been isolated and described. Therefore, the aim of the doctoral dissertation was to immobilize bacterial strains capable to degrade naproxen. The characteristics of the carrier used and bacterial biofilms formed on its surface were made. The effect of immobilization on the course of drug degradation in monocultural conditions, as well as in the presence of autochthonous microflora of the trickling filter was determined. In addition, changes in the activity of enzymes involved in naproxen degradation as a result of immobilization were investigated. Studies on the effects of immobilization on the biodegradation of naproxen began with the optimization of the immobilization process. However, to correctly assess the physiological state of immobilized bacteria in the biofilm, the method based on the fluorescein diacetate hydrolysis was modified. The modification was to omit the detachment of the biofilm from the carrier and conducting the test on the intact biofilm together with the carrier. The developed procedure assumed shaking of samples in phosphate buffer with a pH in the range of 7.4-7.6 for 1 hour and the result was expressed as total metabolic activity (TEA). The sensitivity assay, during which changes in TEA as a result of starvation were measured, allowed to determine the minimal endogenous metabolism of the Bacillus thuringiensis B1(2015b) which was equal to 161-170 μg/g dry weight per hour. It was also observed that nutrient deficiency induced biofilm formation by B1(2015b) cells on the surface of polyurethane foam. As a result of optimization of the immobilization of the strain Planococcus sp. S5 on Loofah sponge, it was observed that the highest TEA values (1250.26 ± 87.61 μg/g dry weight per hour) were achieved during 72-hour incubation in mineral salt medium (MSM; pH 7.2) additionally supplemented with glucose, NaCl and MnSO₄, shaken at 90 rpm at 30°C and with high cell concentration. Strain Bacillus thuringiensis B1(2015b) immobilized on the Loofah sponge showed the highest TEA values (790.14 ± 40.60 μg/g dry weight per hour) after 48-hour incubation in HTC medium (pH 8), supplemented with glucose and MnSO4, shaken at 110 rpm at 20°C with low cell concentration. Analysis of naproxen degradation by Planococcus sp. S5 strain showed an inhibitory effect at a concentration higher than 12 mg/L on free S5 cells. It was observed that free S5 cells were able to completely degrade the drug in a concentration of 6, 9 and 12 mg/L in 38, 44 and 62 days, respectively. The degradation of the drug proceed in two phases. The first phase, lasting 29 days, was characterized by a slower naproxen degradation rate. During the second phase, drug degradation was twice as fast. Immobilized S5 cells on Loofah sponge were able to completely degrade the drug in all analyzed concentrations, and the degradation rate was constant, independent of the day of incubation and similar to degradation during phase II performed by free cells. Studies on the course of repeated cycles of naproxen degradation at the lowest analyzed concentration showed that as a result of immobilization, Planococcus sp. S5 cells maintained full degradation capacity for 55 days, during which degraded 3 doses of the drug. Additionally, during naproxen degradation, S5 cells secreted significant amounts of exopolysaccharides to increase the protective barrier against naproxen. Studies on the impact of the Planococcus sp. S5 immobilization on the activity of enzymes involved in naproxen degradation have shown that immobilization did not change the degradation pathway. However, significant changes were observed in the values of these activities. It was shown that the enzymatic activity in the first phase of drug degradation by free S5 cells was much lower than in the faster degradation phase. Despite the similar rate of drug degradation by free S5 cells in phase II and by immobilized S5 cells, the activity of the analyzed enzymes of immobilized cells was significantly higher than that of free cells. The naproxen biodegradation by Bacillus thuringiensis B1(2015b) immobilized on the Loofah sponge was monitored in a trickling filter augmented with autochthonous microflora from the Imhoff tank flow chamber in Krupski Młyn – Ziętek. Analysis showed that immobilized B1(2015b) cells degraded 70% of naproxen at a concentration of 1 mg/L in the trickling filter without autochthonous microflora. However, in the presence of indigenous microflora, immobilized B1(2015b) cells at the same time degraded 90% of the drug. Obtained results showed synergistic interactions between the autochthonous microflora of the trickling filter and introduced B1(2015b) cells, which resulted in acceleration of naproxen biodegradation. By analyzing the bacterial V3-V5 regions of the 16S rRNA gene using denaturing gradient gel electrophoresis (DGGE), it was confirmed that the introduced Bacillus thuringiensis B1(2015b) was able to survive and multiply in the trickling filter after the process of naproxen degradation. In addition, an analysis of the qualitative changes of bacterial and fungal communities of autochthonous microflora after exposure to naproxen, as well as after the introduction of immobilized B1(2015b) was performed. Naproxen has been shown to cause a significant reduction in bacterial microflora biodiversity. Fungal strains were less sensitive to the drug. However, as a result of the introduction of immobilized B1(2015b) cells, which were able to quickly eliminate the drug, an increase in the biodiversity of bacterial and fungal microflora was observed

Fluorescein diacetate hydrolysis using the whole biofilm as a sensitive tool to evaluate the physiological state of immobilized bacterial cells

Due to the increasing interest and the use of immobilized biocatalysts in bioremediation studies, there is a need for the development of an assay for quick and reliable measurements of their overall enzymatic activity. Fluorescein diacetate (FDA) hydrolysis is a widely used assay for measuring total enzymatic activity (TEA) in various environmental samples or in monoculture researches. However, standard FDA assays for TEA measurements in immobilized samples include performing an assay on cells detached from the carrier. This causes an error, because it is not possible to release all cells from the carrier without affecting their metabolic activity. In this study, we developed and optimized a procedure for TEA quantification in the whole biofilm formed on the carrier without disturbing it. The optimized method involves pre-incubation of immobilized carrier in phosphate buffer (pH 7.6) on the orbital shaker for 15 min, slow injection of FDA directly into the middle of the immobilized carrier, and incubation on the orbital shaker (130 rpm, 30◦C) for 1 h. Biofilm dry mass was obtained by comparing the dried weight of the immobilized carrier with that of the unimmobilized carrier. The improved protocol provides a simple, quick, and more reliable quantification of TEA during the development of immobilized biocatalysts compared to the original method

Enhanced Degradation of Naproxen by Immobilization of Bacillus thuringiensis B1(2015b) on Loofah Sponge

The naproxen-degrading bacterium Bacillus thuringiensis B1(2015b) was immobilised onto loofah sponge and introduced into lab-scale trickling filters. The trickling filters constructed for this study additionally contained stabilised microflora from a functioning wastewater treatment plant to assess the behavior of introduced immobilized biocatalyst in a fully functioning bioremediation system. The immobilised cells degraded naproxen (1 mg/L) faster in the presence of autochthonous microflora than in a monoculture trickling filter. There was also abundant colonization of the loofah sponges by the microorganisms from the system. Analysis of the influence of an acute, short-term naproxen exposure on the indigenous community revealed a significant drop in its diversity and qualitative composition. Bioaugmentation was also not neutral to the microflora. Introducing a new microorganism and increasing the removal of the pollutant caused changes in the microbial community structure and species composition. The incorporation of the immobilised B1(2015b) was successful and the introduced strain colonized the basic carrier in the trickling filter after the complete biodegradation of the naproxen. As a result, the bioremediation system could potentially be used to biodegrade naproxen in the future

Use of xanthan gum for whole cell immobilization and its impact in bioremediation - a review

Xanthan gum is one of the exo-polysaccharides produced by bacteria and is characterized by unique non-Newtonian properties. Its structure and conformation strongly depend on the fermentation conditions and such factors as temperature and ions concentration. The properties of the xanthan gum were appreciated in the controlled drug delivery but in the crosslinked form. Due to its ability to enhance the survival rate of immobilized bacteria, the potential of a crosslinked form is promising. Unfortunately, xanthan gum crosslinking procedures often require toxic substances or harsh environmental conditions, which cannot be used in the entrapment of living cells. In this study, we summarised a crosslinking method that could potentially be modified to reduce its toxicity to living cells. Moreover, this review also includes using xanthan gum in bioremediation studies and possible utilization methods to avoid carrier accumulation in the environment

Biotransformation of naproxen by immobilized Bacillus thuringiensis B1 (2015b)

tekst w j. pol. i ang.Bezpiecznymi i stosunkowo tanimi metodami usuwania NLPZ są metody biologicznej degradacji. Dotychczas wyizolowano jednak tylko nieliczne szczepy bakteryjne zdolne do biotransformacji/biodegradacji naproksenu. Znaczącym potencjałem biodegradacyjnym, charakteryzuje się wyizolowany ze środowiska naturalnego Bacillus thuringiensis B1(2015b), zdolny do całkowitej biotransformacji naproksenu w stężeniu 6 mg/L w ciągu 5 tygodni, w obecności dodatkowego źródła węgla (Marchlewicz i in. 2016). Celem przeprowadzonych badań było zwiększenie wydajności kometabolicznej biodegradacji naproksenu przez badany szczep, poprzez jego immobilizację na naturalnej gąbce pochodzącej z rośliny Luffa aegyptiaca. W trakcie eksperymentu monitorowano proces biotransformacji naproksenu przez wolne oraz unieruchomione komórki bakteryjne. Sprawdzono również, jak zmienia się aktywność metaboliczna uformowanego na powierzchni nośnika biofilmu w trakcie rozkładu leku

Immobilization of Planococcus sp. S5 strain on the loofah sponge and its application in naproxen removal

Planococcus sp. S5, a Gram-positive bacterium isolated from the activated sludge is known to degrade naproxen in the presence of an additional carbon source. Due to the possible toxicity of naproxen and intermediates of its degradation, the whole cells of S5 strain were immobilized onto loofah sponge. The immobilized cells degraded 6, 9, 12 or 15 mg/L of naproxen faster than the free cells. Planococcus sp. cells immobilized onto the loofah sponge were able to degrade naproxen efficiently for 55 days without significant damage and disintegration of the carrier. Analysis of the activity of enzymes involved in naproxen degradation showed that stabilization of S5 cells in exopolysaccharide (EPS) resulted in a significant increase of their activity. Changes in the structure of biofilm formed on the loofah sponge cubes during degradation of naproxen were observed. Developed biocatalyst system showed high resistance to naproxen and its intermediates and degraded higher concentrations of the drug in comparison to the free cells

TEFM (c17orf42) is necessary for transcription of human mtDNA

Here we show that c17orf42, hereafter TEFM (transcription elongation factor of mitochondria), makes a critical contribution to mitochondrial transcription. Inactivation of TEFM in cells by RNA interference results in respiratory incompetence owing to decreased levels of H- and L-strand promoter-distal mitochondrial transcripts. Affinity purification of TEFM from human mitochondria yielded a complex comprising mitochondrial transcripts, mitochondrial RNA polymerase (POLRMT), pentatricopeptide repeat domain 3 protein (PTCD3), and a putative DEAD-box RNA helicase, DHX30. After RNase treatment only POLRMT remained associated with TEFM, and in human cultured cells TEFM formed foci coincident with newly synthesized mitochondrial RNA. Based on deletion mutants, TEFM interacts with the catalytic region of POLRMT, and in vitro TEFM enhanced POLRMT processivity on ss- and dsDNA templates. TEFM contains two HhH motifs and a Ribonuclease H fold, similar to the nuclear transcription elongation regulator Spt6. These findings lead us to propose that TEFM is a mitochondrial transcription elongation factor

Characterization of human DNGR-1+ BDCA3+ leukocytes as putative equivalents of mouse CD8α+ dendritic cells

In mouse, a subset of dendritic cells (DCs) known as CD8α+ DCs has emerged as an important player in the regulation of T cell responses and a promising target in vaccination strategies. However, translation into clinical protocols has been hampered by the failure to identify CD8α+ DCs in humans. Here, we characterize a population of human DCs that expresses DNGR-1 (CLEC9A) and high levels of BDCA3 and resembles mouse CD8α+ DCs in phenotype and function. We describe the presence of such cells in the spleens of humans and humanized mice and report on a protocol to generate them in vitro. Like mouse CD8α+ DCs, human DNGR-1+ BDCA3hi DCs express Necl2, CD207, BATF3, IRF8, and TLR3, but not CD11b, IRF4, TLR7, or (unlike CD8α+ DCs) TLR9. DNGR-1+ BDCA3hi DCs respond to poly I:C and agonists of TLR8, but not of TLR7, and produce interleukin (IL)-12 when given innate and T cell–derived signals. Notably, DNGR-1+ BDCA3+ DCs from in vitro cultures efficiently internalize material from dead cells and can cross-present exogenous antigens to CD8+ T cells upon treatment with poly I:C. The characterization of human DNGR-1+ BDCA3hi DCs and the ability to grow them in vitro opens the door for exploiting this subset in immunotherapy

Natural carriers in bioremediation: a review

Bioremediation of contaminated groundwater or soil is currently the cheapest and the least harmful method of removing xenobiotics from the environment. Immobilization of microorganisms capable of degrading specific contaminants significantly promotes bioremediation processes, reduces their costs, and also allows for the multiple use of biocatalysts. Among the developed methods of immobilization, adsorption on the surface is the most common method in bioremediation, due to the simplicity of the procedure and its non-toxicity. The choice of carrier is an essential element for successful bioremediation. It is also important to consider the type of process (in situ or ex situ), type of pollution, and properties of immobilized microorganisms. For these reasons, the article summarizes recent scientific reports about the use of natural carriers in bioremediation, including efficiency, the impact of the carrier on microorganisms and contamination, and the nature of the conducted research

Antigen presenting cell-selective drug delivery by glycan-decorated nanocarriers.

Targeted drug delivery systems hold promise for selective provision of active compounds to distinct tissues or cell subsets. Thus, locally enhanced drug concentrations are obtained that would confer improved efficacy. As a consequence adverse effects should be diminished, as innocent bystander cells are less affected. Currently, several controlled drug delivery systems based on diverse materials are being developed. Some systems exhibit material-associated toxic effects and/or show low drug loading capacity. In contrast, liposomal nanocarriers are particularly favorable because they are well tolerated, poorly immunogenic, can be produced in defined sizes, and offer a reasonable payload capacity. Compared with other immune cells, professional antigen-presenting cells (APCs) demonstrate enhanced liposome uptake mediated by macropinocytosis, phagocytosis and presumably also by clathrin- and caveolae-mediated endocytosis. In order to further enhance the targeting efficacy toward APCs, receptor-mediated uptake appears advisable. Since APC subsets generally do not express single linage-specific receptors, members of the C-type lectin receptor (CLR) family are compelling targets. Examples of CLR expressed by APCs include DEC-205 (CD205) expressed by myeloid dendritic cells (DC) and monocytes, the mannose receptor C type 1 (MR, CD206) expressed by DC, monocytes and macrophages, DC-SIGN (CD209) expressed by DC, and several others. These receptors bind glycans, which are typically displayed by pathogens and thus support pathogen uptake and endocytosis. Further research will elucidate whether glycan-decorated liposomes will not only enhance APCs targeting but also enable preferential delivery of their payload to discrete subcellular compartments