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Does fullerene C60 affect cell membranes integrity?
S146
Abstracts / New Biotechnology 33S (2016) S1–S213
Biological treatment of industrial wastewaters often requires implementation of specialized microbial biocenoses, resistant to toxic chemicals and capable of forming activated sludges organized in flocs. In biogas-producing plants, a waste effluent is generated bearing high environmental risk, especially of eutrophication. The so-called digester supernatant is difficult to treat conventionally since it has a relatively low organic carbon load and contains high amount of biogenic substances where nitrogen occurs in the reduced, ammonium form. The idea to employ microalgae, bacteria, yeast or complex microbial consortia may prove successful provided the proliferating biomass can be easily removed from bioreactors, preferentially by sedimentation, prior to clean water discharge. We tested the possibility of bioaugmenting microbial communities of eutrophic waters with zoogleal bacteria to favor floc formation. Ten bacterial strains were isolated from the activated sludge of an efficiently-operating (optimal process parameters, good sedimentation characteristics and floc structure morphology) municipal wastewater treatment plant. For isolation, selective media were used for culturing zoogleal bacteria, especially Zooglea ramigera. Four isolates, including Z. ramigera 115, showed extensive synthesis of zoogleal matrix. However, only one strain, Zooglea sp. ZB-IV/1, tolerated highly eutrophic synthetic media (NH4+ up to 6000 mg/l) and could grow in industrial effluents from sewage sludge fermentation, where it maintained the ability to produce zoogleal matrix. ZB-IV/1 was shown to promote flocs formation after inoculation to bacterial and yeast suspensions, and microbial consortia specialized at xenobiotic biodegradation kept their potential while acquiring morphology and sedimentation characteristics close to that of the mature activated sludge. http://dx.doi.org/10.1016/j.nbt.2016.06.1225
P15-9 Microbial communities in aerobic granules treating municipal wastewater containing bisphenol A (BPA) ´ Agnieszka Cydzik-Kwiatkowska ∗ , Magdalena Zielinska, Katarzyna Bernat, Katarzyna Bułkowska, Irena Wojnowska-Baryła University of Warmia and Mazury in Olsztyn, Poland Bisphenol A (BPA) is one of the most prevalent synthetic xenoestrogens in the environment with a global production rate of a million tons per year. In this study, aerobic granular sludge was used for BPA removal from municipal wastewater. The study determined how the concentration of BPA in wastewater influenced the microbial composition of granules. The study was conducted in four constantly aerated reactors with granules operated at an hydraulic retention time of 16 h. Influent to the control reactor did not contain BPA. BPA concentrations in the wastewater fed to the other three reactors were 2, 6 and 12 mg/L. The overall COD and ammonium concentrations in the influent were about 400 mg/L and 50 mg/L, respectively. Microbial composition of biomass was assessed on the basis of bacterial 16S rDNA gene using the MiSeq Illumina platform. The vast majority of sequences obtained belonged to proteobacteria. The predominant proteobacteria was betaproteobacteria, whose participation in aerobic granules ranged from 36.93% to 57.04%. The abundance of Sphingomonadales, Sphingobacteriales, Burkholderiales, Nitrosomonadales, Methylophilales and Methylococcales in granules increased with rising concentrations of BPA in the influent. Thauera sp., Azoarcus sp., and Aquimonas sp. were core bacterial genera in granules at all concentrations of BPA in the influent. Molecular data indicate that BPA was directly metabolized as the source of carbon and energy by Sphingomonas sp.
or unspecifically degraded by OH-producing oxidoreductases of ammonium-oxidizing and methylotrophic bacteria. http://dx.doi.org/10.1016/j.nbt.2016.06.1226
P15-10 Characteristics of selected environmental bacterial strains for their potential use in bioaugmentation of activated sludge laded with Kalina Pond leachate Justyna Michalska ∗ , Izabela Gren, ´ Danuta Wojcieszynska ´ University of Silesia, Poland Phenol and its derivatives are widely used in various industries, hence these compounds are most commonly present in the industrial wastewater. “Klimzowiec” Sewage Treatment Plant in Chorzów is a place of discharge not only municipal wastewater but also leachates from Kalina Pond, which are strongly loaded with many phenolic compounds. The Kalina Pond is a natural water reservoir situated in an interior basin in the southeastern part of the city Swietochlowice. The main source of the pond’s pollution has been the industrial waste tip of HAJDUKI S.A. chemical plant in Chorzów. Water in the pond is highly alkaline, deprived of oxygen and may be expected to contain phenols in concentration of a couple of hundreds to a couple of thousands mg dm3 . Discharged into Wastewater Treatment Plant, Kalina Pond leachate may negatively affect activated sludge microorganisms and reduce the effectiveness of wastewater treatment. One of the methods, that may protect sludge indigenous microorganisms from toxic effect due to the increase of wastewater contaminant load is bioaugmentation. The aim of this study was evaluation of selected environmental bacterial strains for their potential use in bioaugmentation of activated sludge exposed to high doses of phenolic compounds. Results show that some of tested microoorganisms are much more resistant to toxic effect of different Kalina Pond leachate concentration than activated sludge microorganisms. Due to the ability of coaggregation with activated sludge microorganisms and strong biofilm formation these bacteria are expected to incorporate into the structure of sludge flocs. http://dx.doi.org/10.1016/j.nbt.2016.06.1227
Nanobiotechnology P16-1 Does fullerene C60 affect cell membranes integrity? 1, ´ Agnieszka Borowik 1,∗ , Michał Rychłowski 1 , Grzegorz Gołunski Anna Woziwodzka 1 , Yuriy Prylutskyy 2 , Maxim Evstigneev 3 , Jacek Piosik 1 1 Intercollegiate Faculty of Biotechnology, University of Gda´ nsk & Medical University of Gda´ nsk, Poland 2 Taras Shevchenko National University of Kyiv, Ukraine 3 Belgorod State University, Russia
C60 fullerene (FC60 ) is reported to directly interact with biomacromolecules, such as aromatic mutagens or anticancer drugs, and penetrate into cells. Therefore, it is extensively studied for its potential application as a drug carrier in novel chemotherapy systems. However, there are some indications suggesting FC60 toxicity (e.g. induced by interactions with cell walls), but these studies
Abstracts / New Biotechnology 33S (2016) S1–S213
remain inconclusive. A question arises how FC60 can damage cells and increase treated organisms lifespan at the same time. To shed a light on this problem we employed several biophysical and biological techniques. We visualized FC60 adhesion to Salmonella typhimurium cells wall with atomic force microscopy (AFM). Subsequently, using mutagenicity assay (Ames test) based on the same bacteria species, the influence of FC60 on biological activity of the chosen anticancer drugs was analyzed. Additionally, we performed combined DAPI and propidium iodide (PI) staining on either bacterial or eukaryotic cells to define FC60 uptake and its possible impact on the cell wall continuity and cells viability. In this approach DAPI selectively stains living cells, in contrary to PI, which stains only dead cells or cells possessing destabilized membranes. Fluorescence signals were detected using confocal microscopy. Obtained results provide practical insight into FC60 impact on diverse cells membranes, what is essential for FC60 pharmaceutical application. What is more, such knowledge may be exploited in future cancer prevention or treatment. http://dx.doi.org/10.1016/j.nbt.2016.06.1228
P16-2 Evaluation of multi-walled carbon nanotubes on the neuronal dopamine receptor in Helix aspersa Erasmo Orrantia 1,∗ , Estefanía Eileen Mendoza 2 , Juan Bernal-Martínez 1 1 2
Centro de Investigación en Materiales Avanzados, Mexico S. C, Mexico
S147
Nanomaterials play crucial role in modern civilisation, and may be found in food industry, medicine, engineering and agriculture. Novel nanomaterials are being described every year. Nevertheless, they may enter natural environment and for that reason they should be examined in terms of action against living organisms. Bacteria and fungi are likely to come into contact with nanostructures before any other organisms. The latest studies show that nanomaterials may affect biodiversity of environmental microbiota. On the other hand, they may induce various activities in microorganisms such as idiolites secretion. Therefore, the aim of the study was an assessment of interaction between chosen microorganisms and novel carbon–cobalt nanocomposite. Nanocomposite of cobalt nanoparticles and graphene was obtained from the carbonized metal organic frameworks. Studied carbon–cobalt nanocomposite was analysed regarding the influence of the different nanocomposite’s cobalt oxide crystallographic phases on bactericidal properties. The reaction to the nanomaterials was tested on four reference microorganisms – three bacterial (Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus) and one yeast (Candida albicans). The tested microorganisms were cultured with the addition of nanostructures in a liquid Tryptic Soy Broth medium. Viability (Koch’s plate tests, Optical Density measures, Transmission Electron Microscopy), metabolic activity of cells (AlamarBlue® ) and biofilm formation (Crystal Violet Staining) were tested. All results were analysed statistically (ANOVA). Results indicate that chosen microorganisms react differently to the used nanomaterial. Surprisingly, the nanocomposite caused an increase in the bacterial quantity during incubation, but was neutral to the growth of C. albicans. Moreover, nanostructures significantly altered an ability to form biofilm in selected bacteria. http://dx.doi.org/10.1016/j.nbt.2016.06.1230
Carbon nanotubes (CNTs) are well known for their unique mechanical, electrical, chemical and structural properties. However, their use in biological systems is more recent. CNTs have demonstrated the ability to transport a wide range of molecules across membranes and into living cells; therefore, they have attracted great interest in biomedical applications such as tissue regeneration, advanced imaging and drug or gene delivery. On the other hand, Helix aspersa’s neurons have been used in various investigations as biological models due to their easy morphological and functional identification by the size they present (∼150 m), spontaneous electrical activity, among other characteristics. This research aims to present an electrophysiological study of the effects of multi-walled carbon nanotubes (MWCNTs) on the dopamine ligand-dependent receptor in neurons of Helix aspersa. Different agonist and antagonist of dopamine receptor drugs were also used to evaluate the response to distinct stimulations. Here, we describe the non-modification of the electrophysiological properties of dopamine receptor in neurons. Conversely, using small concentrations of functionalized MWCNTs dissolved in Ringer’s solution potentiated the expected response. http://dx.doi.org/10.1016/j.nbt.2016.06.1229
P16-3 Reaction of different microorganisms to novel graphene-based nanomaterials Adrian Augustyniak 1,∗ , Krzysztof Cendrowski 2 , Martyna Barylak 1 , Ewa Mijowska 2 , Paweł Nawrotek 1 1 2
West Pomeranian University of Technology, Poland Szczecin, Poland
P16-5 Proteomics analysis of Desulfovibrio alaskensis in response to platinum and palladium Michael Capeness ∗ , Virginia Echavarri-Bravo, Thierry Le Bihan, Louise Horsfall The University of Edinburgh, United Kingdom Desulfovibrio alaskensis is able to form nanoparticles from a variety of heavy, often toxic metals, and can do so from impure feed stocks. Here we have used them to form nanoparticles of Pt and Pd, which are highly catalytic and very desirable for industrial purposes. To further understand the pathways involved in their formation we have carried out whole cell shotgun-proteomics to elucidate the response of D. alaskensis to Pt and Pd. We found there was a shared set of proteins with increased expression in both the Pt and Pd datasets, as well as proteins unique to each, underpinning a shared potential pathway of 13 proteins with 7 proteins specific to each metal. From this we took one of the corresponding genes, and re-introduced it into D. alaskensis under increased, constitutive expression to observe its effect on the morphology of the Pt nanoparticles produced. We found there was a significant increase in the size of the resultant nanoparticles, demonstrating that we can effectively change the morphology of the nanoparticles and thus their catalytic characteristics by changing the underlying genetic components of the production pathway. This will form the basis for further work to finely tailor the characteristics of desirable nanoparticles for specific industrial roles and through strain enhancement. http://dx.doi.org/10.1016/j.nbt.2016.06.1231
Abstracts / New Biotechnology 33S (2016) S1–S213
Biological treatment of industrial wastewaters often requires implementation of specialized microbial biocenoses, resistant to toxic chemicals and capable of forming activated sludges organized in flocs. In biogas-producing plants, a waste effluent is generated bearing high environmental risk, especially of eutrophication. The so-called digester supernatant is difficult to treat conventionally since it has a relatively low organic carbon load and contains high amount of biogenic substances where nitrogen occurs in the reduced, ammonium form. The idea to employ microalgae, bacteria, yeast or complex microbial consortia may prove successful provided the proliferating biomass can be easily removed from bioreactors, preferentially by sedimentation, prior to clean water discharge. We tested the possibility of bioaugmenting microbial communities of eutrophic waters with zoogleal bacteria to favor floc formation. Ten bacterial strains were isolated from the activated sludge of an efficiently-operating (optimal process parameters, good sedimentation characteristics and floc structure morphology) municipal wastewater treatment plant. For isolation, selective media were used for culturing zoogleal bacteria, especially Zooglea ramigera. Four isolates, including Z. ramigera 115, showed extensive synthesis of zoogleal matrix. However, only one strain, Zooglea sp. ZB-IV/1, tolerated highly eutrophic synthetic media (NH4+ up to 6000 mg/l) and could grow in industrial effluents from sewage sludge fermentation, where it maintained the ability to produce zoogleal matrix. ZB-IV/1 was shown to promote flocs formation after inoculation to bacterial and yeast suspensions, and microbial consortia specialized at xenobiotic biodegradation kept their potential while acquiring morphology and sedimentation characteristics close to that of the mature activated sludge. http://dx.doi.org/10.1016/j.nbt.2016.06.1225
P15-9 Microbial communities in aerobic granules treating municipal wastewater containing bisphenol A (BPA) ´ Agnieszka Cydzik-Kwiatkowska ∗ , Magdalena Zielinska, Katarzyna Bernat, Katarzyna Bułkowska, Irena Wojnowska-Baryła University of Warmia and Mazury in Olsztyn, Poland Bisphenol A (BPA) is one of the most prevalent synthetic xenoestrogens in the environment with a global production rate of a million tons per year. In this study, aerobic granular sludge was used for BPA removal from municipal wastewater. The study determined how the concentration of BPA in wastewater influenced the microbial composition of granules. The study was conducted in four constantly aerated reactors with granules operated at an hydraulic retention time of 16 h. Influent to the control reactor did not contain BPA. BPA concentrations in the wastewater fed to the other three reactors were 2, 6 and 12 mg/L. The overall COD and ammonium concentrations in the influent were about 400 mg/L and 50 mg/L, respectively. Microbial composition of biomass was assessed on the basis of bacterial 16S rDNA gene using the MiSeq Illumina platform. The vast majority of sequences obtained belonged to proteobacteria. The predominant proteobacteria was betaproteobacteria, whose participation in aerobic granules ranged from 36.93% to 57.04%. The abundance of Sphingomonadales, Sphingobacteriales, Burkholderiales, Nitrosomonadales, Methylophilales and Methylococcales in granules increased with rising concentrations of BPA in the influent. Thauera sp., Azoarcus sp., and Aquimonas sp. were core bacterial genera in granules at all concentrations of BPA in the influent. Molecular data indicate that BPA was directly metabolized as the source of carbon and energy by Sphingomonas sp.
or unspecifically degraded by OH-producing oxidoreductases of ammonium-oxidizing and methylotrophic bacteria. http://dx.doi.org/10.1016/j.nbt.2016.06.1226
P15-10 Characteristics of selected environmental bacterial strains for their potential use in bioaugmentation of activated sludge laded with Kalina Pond leachate Justyna Michalska ∗ , Izabela Gren, ´ Danuta Wojcieszynska ´ University of Silesia, Poland Phenol and its derivatives are widely used in various industries, hence these compounds are most commonly present in the industrial wastewater. “Klimzowiec” Sewage Treatment Plant in Chorzów is a place of discharge not only municipal wastewater but also leachates from Kalina Pond, which are strongly loaded with many phenolic compounds. The Kalina Pond is a natural water reservoir situated in an interior basin in the southeastern part of the city Swietochlowice. The main source of the pond’s pollution has been the industrial waste tip of HAJDUKI S.A. chemical plant in Chorzów. Water in the pond is highly alkaline, deprived of oxygen and may be expected to contain phenols in concentration of a couple of hundreds to a couple of thousands mg dm3 . Discharged into Wastewater Treatment Plant, Kalina Pond leachate may negatively affect activated sludge microorganisms and reduce the effectiveness of wastewater treatment. One of the methods, that may protect sludge indigenous microorganisms from toxic effect due to the increase of wastewater contaminant load is bioaugmentation. The aim of this study was evaluation of selected environmental bacterial strains for their potential use in bioaugmentation of activated sludge exposed to high doses of phenolic compounds. Results show that some of tested microoorganisms are much more resistant to toxic effect of different Kalina Pond leachate concentration than activated sludge microorganisms. Due to the ability of coaggregation with activated sludge microorganisms and strong biofilm formation these bacteria are expected to incorporate into the structure of sludge flocs. http://dx.doi.org/10.1016/j.nbt.2016.06.1227
Nanobiotechnology P16-1 Does fullerene C60 affect cell membranes integrity? 1, ´ Agnieszka Borowik 1,∗ , Michał Rychłowski 1 , Grzegorz Gołunski Anna Woziwodzka 1 , Yuriy Prylutskyy 2 , Maxim Evstigneev 3 , Jacek Piosik 1 1 Intercollegiate Faculty of Biotechnology, University of Gda´ nsk & Medical University of Gda´ nsk, Poland 2 Taras Shevchenko National University of Kyiv, Ukraine 3 Belgorod State University, Russia
C60 fullerene (FC60 ) is reported to directly interact with biomacromolecules, such as aromatic mutagens or anticancer drugs, and penetrate into cells. Therefore, it is extensively studied for its potential application as a drug carrier in novel chemotherapy systems. However, there are some indications suggesting FC60 toxicity (e.g. induced by interactions with cell walls), but these studies
Abstracts / New Biotechnology 33S (2016) S1–S213
remain inconclusive. A question arises how FC60 can damage cells and increase treated organisms lifespan at the same time. To shed a light on this problem we employed several biophysical and biological techniques. We visualized FC60 adhesion to Salmonella typhimurium cells wall with atomic force microscopy (AFM). Subsequently, using mutagenicity assay (Ames test) based on the same bacteria species, the influence of FC60 on biological activity of the chosen anticancer drugs was analyzed. Additionally, we performed combined DAPI and propidium iodide (PI) staining on either bacterial or eukaryotic cells to define FC60 uptake and its possible impact on the cell wall continuity and cells viability. In this approach DAPI selectively stains living cells, in contrary to PI, which stains only dead cells or cells possessing destabilized membranes. Fluorescence signals were detected using confocal microscopy. Obtained results provide practical insight into FC60 impact on diverse cells membranes, what is essential for FC60 pharmaceutical application. What is more, such knowledge may be exploited in future cancer prevention or treatment. http://dx.doi.org/10.1016/j.nbt.2016.06.1228
P16-2 Evaluation of multi-walled carbon nanotubes on the neuronal dopamine receptor in Helix aspersa Erasmo Orrantia 1,∗ , Estefanía Eileen Mendoza 2 , Juan Bernal-Martínez 1 1 2
Centro de Investigación en Materiales Avanzados, Mexico S. C, Mexico
S147
Nanomaterials play crucial role in modern civilisation, and may be found in food industry, medicine, engineering and agriculture. Novel nanomaterials are being described every year. Nevertheless, they may enter natural environment and for that reason they should be examined in terms of action against living organisms. Bacteria and fungi are likely to come into contact with nanostructures before any other organisms. The latest studies show that nanomaterials may affect biodiversity of environmental microbiota. On the other hand, they may induce various activities in microorganisms such as idiolites secretion. Therefore, the aim of the study was an assessment of interaction between chosen microorganisms and novel carbon–cobalt nanocomposite. Nanocomposite of cobalt nanoparticles and graphene was obtained from the carbonized metal organic frameworks. Studied carbon–cobalt nanocomposite was analysed regarding the influence of the different nanocomposite’s cobalt oxide crystallographic phases on bactericidal properties. The reaction to the nanomaterials was tested on four reference microorganisms – three bacterial (Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus) and one yeast (Candida albicans). The tested microorganisms were cultured with the addition of nanostructures in a liquid Tryptic Soy Broth medium. Viability (Koch’s plate tests, Optical Density measures, Transmission Electron Microscopy), metabolic activity of cells (AlamarBlue® ) and biofilm formation (Crystal Violet Staining) were tested. All results were analysed statistically (ANOVA). Results indicate that chosen microorganisms react differently to the used nanomaterial. Surprisingly, the nanocomposite caused an increase in the bacterial quantity during incubation, but was neutral to the growth of C. albicans. Moreover, nanostructures significantly altered an ability to form biofilm in selected bacteria. http://dx.doi.org/10.1016/j.nbt.2016.06.1230
Carbon nanotubes (CNTs) are well known for their unique mechanical, electrical, chemical and structural properties. However, their use in biological systems is more recent. CNTs have demonstrated the ability to transport a wide range of molecules across membranes and into living cells; therefore, they have attracted great interest in biomedical applications such as tissue regeneration, advanced imaging and drug or gene delivery. On the other hand, Helix aspersa’s neurons have been used in various investigations as biological models due to their easy morphological and functional identification by the size they present (∼150 m), spontaneous electrical activity, among other characteristics. This research aims to present an electrophysiological study of the effects of multi-walled carbon nanotubes (MWCNTs) on the dopamine ligand-dependent receptor in neurons of Helix aspersa. Different agonist and antagonist of dopamine receptor drugs were also used to evaluate the response to distinct stimulations. Here, we describe the non-modification of the electrophysiological properties of dopamine receptor in neurons. Conversely, using small concentrations of functionalized MWCNTs dissolved in Ringer’s solution potentiated the expected response. http://dx.doi.org/10.1016/j.nbt.2016.06.1229
P16-3 Reaction of different microorganisms to novel graphene-based nanomaterials Adrian Augustyniak 1,∗ , Krzysztof Cendrowski 2 , Martyna Barylak 1 , Ewa Mijowska 2 , Paweł Nawrotek 1 1 2
West Pomeranian University of Technology, Poland Szczecin, Poland
P16-5 Proteomics analysis of Desulfovibrio alaskensis in response to platinum and palladium Michael Capeness ∗ , Virginia Echavarri-Bravo, Thierry Le Bihan, Louise Horsfall The University of Edinburgh, United Kingdom Desulfovibrio alaskensis is able to form nanoparticles from a variety of heavy, often toxic metals, and can do so from impure feed stocks. Here we have used them to form nanoparticles of Pt and Pd, which are highly catalytic and very desirable for industrial purposes. To further understand the pathways involved in their formation we have carried out whole cell shotgun-proteomics to elucidate the response of D. alaskensis to Pt and Pd. We found there was a shared set of proteins with increased expression in both the Pt and Pd datasets, as well as proteins unique to each, underpinning a shared potential pathway of 13 proteins with 7 proteins specific to each metal. From this we took one of the corresponding genes, and re-introduced it into D. alaskensis under increased, constitutive expression to observe its effect on the morphology of the Pt nanoparticles produced. We found there was a significant increase in the size of the resultant nanoparticles, demonstrating that we can effectively change the morphology of the nanoparticles and thus their catalytic characteristics by changing the underlying genetic components of the production pathway. This will form the basis for further work to finely tailor the characteristics of desirable nanoparticles for specific industrial roles and through strain enhancement. http://dx.doi.org/10.1016/j.nbt.2016.06.1231