Enrichment of endophytic Actinobacteria in roots and rhizomes of Miscanthus × giganteus plants exposed to diclofenac and sulfamethoxazole.

This study investigates how wastewater containing 2 mg l-1 of sulfamethoxazole (SMX) and 2 mg l-1 of diclofenac (DCF) affects the composition of bacterial communities present in the roots and rhizomes of Miscanthus × giganteus plants grown in laboratory-scale constructed wetlands. Bacterial communities in plant roots and rhizomes were identified in treated and control samples by 16S rRNA amplicon sequencing. Moreover, bacterial endophytes were isolated in R2A and 1/10 869 media and screened for their ability to metabolize SMX and DCF in liquid medium by HPLC. Our results show significant changes in the abundance of main genera, namely Sphingobium and Streptomyces between control and treated plants. Around 70% of the strains isolated from exposed plants belonged to the phylum Actinobacteria and were classified as Streptomyces, Microbacterium, and Glycomyces. In non-exposed plants, Proteobacteria represented 43.5% to 63.6% of the total. We identified 17 strains able to remove SMX and DCF in vitro. From those, 76% were isolated from exposed plants. Classified mainly as Streptomyces, they showed the highest SMX (33%) and DCF (41%) removal efficiency. These isolates, alone or in combination, might be used as bio-inoculants in constructed wetlands to enhance the phytoremediation of SMX and DCF during wastewater treatment.

The treatment of wastewater containing pharmaceuticals in microcosm constructed wetlands: the occurrence of integrons (int1-2) and associated resistance genes (sul1-3, qacEΔ1).

The aim of this study was to analyze the occurrence of sulfonamide resistance genes (sul1-3) and other genetic elements as antiseptic resistance gene (qacEΔ1) and class 1 and class 2 integrons (int1-2) in the upper layer of substrate and in the effluent of microcosm constructed wetlands (CWs) treating artificial wastewater containing diclofenac and sulfamethoxazole (SMX), which is a sulfonamide antibiotic. The bacteria in the substrate and in the effluents were equipped with the sul1-2, int1, and qacEΔ1 resistance determinants, which were introduced into the CW system during inoculation with activated sludge and with the soil attached to the rhizosphere of potted seedlings of Phalaris arundinacea 'Picta' roots (int1). By comparing the occurrence of the resistance determinants in the upper substrate layer and the effluent, it can be stated that they neither were lost nor emerged along the flow path. The implications of the presence of antibiotic resistance genes in the effluent may entail a risk of antibiotic resistance being spread in the receiving environment. Additionally, transformation products of SMX were determined. According to the obtained results, four (potential) SMX transformation products were identified. Two major metabolites of SMX, 2,3,5-trihydroxy-SMX and 3,5-dihydroxy-SMX, indicated that SMX may be partly oxidized during the treatment. The remaining two SMX transformation products (hydroxy-glutathionyl-SMX and glutathionyl-SMX) are conjugates with glutathione, which suggests the ability of CW bacterial community to degrade SMX and resist antimicrobial stress.

Plant-derived pectin nanocoatings to prevent inflammatory cellular response of osteoblasts following Porphyromonas gingivalis infection.

BACKGROUND: Bioengineered plant-derived Rhamnogalacturonan-Is (RG-Is) from pectins are potential candidates for surface nanocoating of medical devices. It has recently been reported that RG-I nanocoatings may prevent bacterial infection and improve the biocompatibility of implants. The aim of the study was to evaluate in vitro impact of bioengineered RG-I nanocoatings on osteogenic capacity and proinflammatory cytokine response of murine osteoblasts following Porphyromonas gingivalis infection. METHODS: Murine MC3T3-E1 osteoblasts and isolated primary calvarial osteoblasts from C57BL/6J (B6J osteoblasts) mice were infected with P. gingivalis and incubated on tissue culture polystyrene plates with or without nanocoatings of unmodified RG-Is isolated from potato pulps (PU) or dearabinanated RG-Is (PA). To investigate a behavior of infected osteoblasts cultured on RG-Is cell morphology, proliferation, metabolic activity, mineralization and osteogenic and pro-inflammatory gene expression were examined. RESULTS: Following P. gingivalis infection, PA, but not PU, significantly promoted MC3T3-E1 and BJ6 osteoblasts proliferation, metabolic activity, and calcium deposition. Moreover, Il-1b, Il-6, TNF-α, and Rankl gene expressions were downregulated in cells cultured on PU and to a higher extent on PA as compared to the corresponding control, whereas Runx, Alpl, Col1a1, and Bglap gene expressions were upregulated vice versa. CONCLUSION: Our data clearly showed that pectin RG-Is nanocoating with high content of galactan (PA) reduces the osteoblastic response to P. gingivalis infection in vitro and may, therefore, reduce a risk of inflammation especially in immunocompromised patients with rheumatoid or periodontal disorders.

Removal of diclofenac and sulfamethoxazole from synthetic municipal waste water in microcosm downflow constructed wetlands: Start-up results.

The objectives of this study were to investigate the start-up removal of pharmaceutical compounds diclofenac and sulfamethoxazole in microcosm downflow constructed wetlands and their effect on the performance of the studied constructed wetlands, and also to assess the effect of plants on the removal of these compounds. The experimental system that was used in this 86-day experiment consisted of 24 columns filled up to 70 cm with predominantly sandy material. Four types of columns were used (six replicates) depending on the presence of plants (Phalaris arundinacea L. var. picta L.) and the presence of pharmaceutical compounds in the influent. The influent was synthetic municipal waste water to which a mixture of 5 mg/L of diclofenac and 5 mg/L of sulfamethoxazole was added. The observed removal of diclofenac was moderate (approx. 50%) and the removal of sulfamethoxazole was relatively low (24-30%). It was found that the removal of diclofenac and sulfamethoxazole was not affected by the vegetation. The presence of diclofenac and sulfamethoxazole in the influent had significant effect on the effluent concentration of N-NO3 and the water loss in the columns, which in both cases were lower than in the control columns. The scope for further research was discussed.

Qualitative variability in microbial community of constructed wetlands used for purifying wastewater contaminated with pharmaceutical substances.

Pharmaceutical substances and their residues are increasingly present in the environment. Therefore, attempts at their removal are made by using different processes. Increasingly important among these processes are those modeled on natural phenomena which occur in wetland ecosystems, called technical scale constructed wetlands. Microbial degradation is an important process in these constructed wetlands. The biodegradation of chemicals often involves a complex series of biochemical reactions and usually varies with the microorganisms involved. The objectives of this study were to determine the impact of sulfamethoxazole and diclofenac on ammonia oxidizing bacteria and other parameters of wastewater in the microcosm of down-flow constructed wetlands. The Spearman correlation coefficient attained negative values in the case of comparison of the Shannon biodiversity index and the parameters of purified wastewater. This dependence was pronounced. In the case of pharmaceutical substances dosed with wastewater, the Spearman correlation coefficient assumed positive values. The highest value assumed by the Spearman correlation coefficient (0.9) was for the removal of diclofenac and Shannon index values for the planted columns, with a very high relationship. For unplanted columns, this value equaled 0.6. For sulfamethoxazole, the value for planted columns was 0.7, and for unplanted -0.7. The presence of plants did not have an impact on the Shannon biodiversity index.

R&D priorities in the field of sustainable remediation and purification of agro-industrial and municipal wastewater.

This article was presented as a position paper during the Environmental Biotechnology and Microbiology Conference in Bologna, Italy in April 2012. It indicates major and emerging environmental biotechnology research and development (R&D) priorities for EU members in the field of sustainable remediation and purification of agro-industrial and municipal wastewater. The identified priorities are: anaerobic/aerobic microbial treatment, combination of photochemical and biological treatment, phytoremediation and algae-based remediation, as well as innovative technologies currently investigated, such as enzyme-based treatment, bioelectrochemical treatment and recovery of nutrients and reuse of cleaned water. State of the art, research needs and prospective development in these domains are crucially discussed. As a result, goals of the future development of bioremediation and purification processes are defined and the way to achieve them is proposed.

Influence of calcium, magnesium, and iron ions on aerobic granulation.

In this study, we investigated the effect of different multivalent cations on granule formation. Previous experiments showed that formation of matrix EPS and their structure depend of the presence of divalent cations. This study indicates that trivalent cations are also playing an important role. However, the more compact granules were formed in the presence of all cations. The authors tried also to determine changes in proteomic profile of slime and tightly bound EPS. These results showed that matrix EPS is composed of a variety of large and complex proteins, but there are also small proteins, like for example, lectins. These small proteins have a role in the interaction between cells and exopolysaccharides and in granules formation.

Synergetic toxic effect of an explosive material mixture in soil.

Explosives materials are stable in soil and recalcitrant to biodegradation. Different authors report that TNT (2,4,6-trinitrotoluene), RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) and HMX (octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine) are toxic, but most investigations have been performed in artificial soil with individual substances. The aim of the presented research was to assess the toxicity of forest soil contaminated with these substances both individually as well in combinations of these substances. TNT was the most toxic substance. Although RDX and HMX did not have adverse effects on plants, these compounds did cause earthworm mortality, which has not been reported in earlier research. Synergistic effects of explosives mixture were observed.

Phytoremediation of explosives (TNT, RDX, HMX) by wild-type and transgenic plants.

The large-scale production and processing of munitions has led to vast environmental pollution by the compounds TNT(2,4,6-trinitrotoluene), RDX(hexahydro-1,3,5-trinitro-1,3,5-triazine) and HMX(octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine). Explosives contain these toxic and mutagenic xenobiotics, which are stable in the environment and recalcitrant to remediation. Certain technologies used thus far (incineration, adsorption, advanced oxidations processes, chemical reduction etc.) have not only been very expensive but also caused additional environmental problems. During recent decades, the most popular technologies have been biotechnological methods, such as phytoremediation, which is relatively cheap, environmentally friendly, and a highly accepted solution by society. The most promising of these technologies is the usage of genetically modified plants, which combines the ability of bacterial genes to detoxify compounds with the phytoremediation benefits of plants. This paper is a review related to the latest and most important achievements in the field of phytoremediation of water and soil contaminated with TNT, RDX and HMX.

The population dynamics of nitrifiers in ammonium-rich systems.

Non-optimal pH, dissolved oxygen concentration, the presence of toxic substances, or the influence of grazers are known to cause disturbances in nitrification. Because activated sludge is a mixture of different organisms, bacteria, and higher organisms, the stability of processes such as carbon removal, nitrification, denitrification, and dephosphatation depends on a range of interactions. These interactions occur both between and within trophic levels. Understanding of the ecology of microorganisms involved in bioprocesses is essential for effective control of startup and operation of a particular process. The aim of the study was to gain further insight into the dynamics of nitrifiers in activated sludge at various sludge ages while treating higher concentrations of ammonium. The results confirmed the importance of Nitrosococcus mobilis and Nitrobacter sp. as the dominant nitrifiers responsible for nitritation and nitratation, respectively, in the presence of unlimited ammonium. The size of the dominant bacteria colony was larger compared to the other species present and reached 25 microm. Problems with nitrification occurred in all high-ammonium loaded reactors. The dynamics of nitrifier population was monitored by oxygen uptake rate (OUR) using a test enabling the OUR measurement separately for ammonium-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB). The results reveal the hypersensitivity of nitrifiers to the substrate and products of incomplete nitrification.

Ammonia oxidizing bacteria community in activated sludge monitored by denaturing gradient gel electrophoresis (DGGE).

Biological treatment based on activated sludge is efficient in municipal wastewater treatment. The removal of nitrogen compounds is crucial for sewage purification. Due to the fact that ammonia is toxic for a water environment and causes eutrophication, ammonia oxidation is of the utmost importance in wastewater treatment. Observing the changeability of ammonia oxidizing bacteria (AOB) and identifying their most abundant species can be helpful in the optimization of wastewater treatment. In this study we used denaturing gradient gel electrophoresis (DGGE), combined with cloning and sequencing of 16S rRNA and AmoA gene fragments in order to estimate AOB biodiversity and temporal community changes. Activated sludge samples were collected from the municipal WWTP in Gliwice (Poland) at 2-week intervals. Ammonia concentration in the influent during the experiment was 30.2-57.6 mg N-NH4+/L. The research revealed a high diversity of uncultured bacteria. It is suspected that these bacteria could be involved in the nitrification, which points to the fact that these bacteria might be efficient in the process. However such a situation is not confirmed and it requires further research. The appearance of Ferribacterium-like bacteria together with Nitrosomonas sp. as the most abundant bacteria was found.

Removal of analgesic drugs from the aquatic environment using photochemical methods.

The occurrence of analgesics in the environment can be explained by the fact that they are very popular and in common use, for example: to treat the symptoms of colds, aches and pains or for the treatment of painful diseases of rheumatic and non-rheumatic origin. Analgesic drugs are only partly removed from wastewater using the biological wastewater treatment processes. The photochemical methods are mentioned as a useful tool for the removal of analgesic medicines from the aquatic environment. The elimination of three analgesic drugs: diclofenac, naproxen and ibuprofen from the aquatic environment using UV- and UV/H(2)O(2)-processes was the aim of the study. All experiments were performed in the water with the presence and the absence of the urea as the main urine component. With the presence of urea the values of photo-oxidation rate constants (in the UV/H(2)O(2)-process) varied from 0.22 min(-1) (ibuprofen) to 0.39 min(-1) (diclofenac). The values of the photodegradation rate constants in the solution without urea (in the UV/H(2)O(2)-process) varied from 0.25 min(-1) (ibuprofen) to 0.45 min(-1) (diclofenac). The study showed that naproxen, ibuprofen and diclofenac may be effectively removed from the aquatic environment (e.g. from the urine) by means of photochemical methods.

Molecular analysis of temporal changes of a bacterial community structure in activated sludge using denaturing gradient gel electrophoresis (DGGE) and fluorescent in situ hybridization (FISH).

Wastewater treatment based on activated sludge is known to be one of the most effective and popular wastewater purification methods. An estimation of microbial community variability in activated sludge allows us to observe the correlation between a particular bacterial group's appearance and the effectiveness of the removal of chemical substances. This research is focused on microbial community temporal changes in membrane bioreactors treating wastes containing a high level of ammonia nitrogen. Samples for this study were collected from two membrane bioreactors with an activated sludge age of 12 and 32 days, respectively. The activated sludge microbial community was adapted for the removal of ammonia nitrogen up to a level of 0.3 g NH4(+) - N g/VSS/d (VSS - volatile suspended solids). The methods - denaturing gradient gel electrophoresis (DGGE) based on 16S rRNA gene PCR products and fluorescent in situ hybridization (FISH) with 16S rRNA gene probes - revealed significant differences in the microbial community structure in the two bioreactors, caused mainly by a difference in sludge age. According to the results obtained in this study, a bioreactor with a sludge age of 12 days is characterized by a much higher microbial community diversity than a bioreactor with a sludge age of 32 days. Interestingly, the appearance of particular species of nitrifying bacteria was constant throughout the experiment in both bioreactors. Changes occured only in the case of the Nitrosomonas oligotropha lineage bacteria. This study demonstrates that the bacterial community of bioreactors operating with different sludge ages differs in total community structure. Nevertheless, the changeability of the bacterial community structure did not have any influence on the efficiency of nitrification.

Acute toxicity and genotoxicity of five selected anionic and nonionic surfactants.

The results of four toxicity bioassays of selected anionic and nonionic surface active agents were presented. Three widely used anionic surfactants that belong to alkyl sulphates (AS), alkylbenzene sulphonates (LAS) and alkylpolyoxyethylene sulphates (AES) as well as nonionic surfactants: polyoxyethylene alkyl ethers (AE) and polyoxylethylene alkylphenyl ethers (APE) were tested. Three different toxicity assays to aquatic organisms: Physa acuta Draparnaud, Artemia salina and Raphidocelis subcapitata were applied. Additionally, the genotoxicity test with Bacillus subtilis M45 Rec- and H17 Rec+ strains was performed. The obtained results showed that none of the surfactants studied was genotoxic at the concentration 1000 mg l(-1). On the basis of toxicity tests to aquatic organisms all tested anionic surfactants were harmful (LC50 between 10 and 100 mg l(-1)), whereas nonionic ones were toxic (LC50 between 1 and 10 mg l(-1)) or even highly toxic (LC50 below 1 mg l(-1)). Moreover, the bigger was the molecular weight of the tested compound, the higher toxicity was observed.