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Degradation of endocrine disruptor compounds in wastewater by Pleurotus ostreatus fungal batch bioreactor
Abstracts / New Biotechnology 33S (2016) S1–S213
P14-20 Rhodococcus equi protects selected plant species from arsenic toxicity
S139
strate seems to be obligatory for decay of repeated doses of 4-chlorophenol. http://dx.doi.org/10.1016/j.nbt.2016.06.1203
Anna Kowalczyk 1,∗ , Dorota Tabor 2 , Dariusz Latowski 1 1 2
Jagiellonian University, Poland AGH University of Science and Technology, Poland
The new strain of arsenic resistant bacteria was isolated from soil contaminated with arsenic and identified as Rhodococcus equi. Isolated strain is capable to grow under laboratory conditions on LB medium supplemented with 2% (w/v) of pure As. Winter wheat as a starch-accumulating species; rapeseed (oil plant) and field pea (crude protein-rich species) were R. equi treated in order to test the bacteria influence on the plants tolerance to As. The final concentration of arsenic in culture medium was 0.25 mg/100 ml. The effect of R. equi on germination, average root and above ground parts length and biomass production were tested. The most tolerant to arsenic was winter wheat, whereas field pea was the least resistant. The supplementation with As in case of wheat and rapeseed slightly increased the number of germinated seeds, but inhibited pea germination. Average root and above ground parts length were reduced for all tested species. The inoculation of culture medium with R. equi increased the tolerance of plants to As, as the values of the controlled parameters increased. The treatment of wheat, rapeseed and pea with R. equi without arsenic demonstrated positive effect on measured parameters or the effect was not observed. The effect of R. equi on tolerance index and toxicity index of selected species to arsenic were tested. The presence of isolated strain in culture medium improved wheat and rapeseed tolerance to arsenic. The toxicity index demonstrated the increase of investigated plants tolerance, what indicates beneficial effect of R. equi on plants. http://dx.doi.org/10.1016/j.nbt.2016.06.1202
P14-21 Vanillic acid as the growth substrate in cometabolic biodegradation of 4-chlorophenol Izabela Gren´ 1,∗ , Justyna Michalska 2 , Klaudia Krzykała 3 , 1 ˛ Agnieszka Gaszczak
P14-22 Groundwater passive sampling and fungal biodegradation of psychopharmaceuticals Zdena Kresinova 1,∗ , Lucie Krejcova 2 , Klára Petru 3 , Tomas Cajthaml 1 1
Institute for Environmental Studies, Czech Republic Faculty of Science, Czech Republic 3 Charles University of Prague, Czech Republic 2
Passive sampling is a rapidly developing technology which offers significant advantages over traditional grab sampling and it is being widely used for the monitoring of pollutants in different environments. In the present study, an analytical methodology for passive sampling of psychopharmaceuticals was evaluated and applied on contaminated locality in Czech Republic. Four pharmaceutical drugs (amitriptyline, dosulepin, chlorprothixene, butamirate) and five compounds which are either used during drug manufacturing or are created as by-products were detected in groundwater in the range from nanograms up to hundreds of milligrams per litter. A biodegrading potential of ligninolytic fungus Pleurotus ostreatus was tested towards a mixture of all detected contaminants. The initial concentration of individual compounds in the mixture was 2 mg L−1 . Five compounds were totally or partially degraded (>52%) during the in vivo cultivation of P. ostreatus on complex liquid medium. Applied acute toxicity tests with Vibrio fischeri indicated the potential formation of metabolites that exhibited higher toxicity than the one or more original substances. EC50 values in the acute toxicity of V. fischeri for individual substances ranged from 5.45 mg L−1 to 131.98 mg L−1 . The metabolites are studied using MS techniques. Acknowledgements: The research leading to these results has received funding from the Norwegian Financial Mechanism 2009–2014 and the Ministry of Education, Youth and Sports under Project Contract no. MSMT-23681/2015-1. http://dx.doi.org/10.1016/j.nbt.2016.06.1204
1
Faculty of Biology and Environmental Protection, Poland University of Silesia in Katowice, Poland 3 Silesian University of Katowice, Poland 2
Vanillic acid belongs to the group of phenolic compounds of plant origin which can serve as the source of carbon and energy for the cells of Stenotrophomonas maltophilia KB2. But simultaneously its concentration of 4 mM and more causes 50% inhibition of KB2 cells growth. Biodegradation of vanillic acid engages enzymes of protocatechuate degradation pathway and contributes to increase of KB2 cell biomass. While vanillic acid was introduced together with 4-chlorophenol as the additional source of carbon and energy time needed for its biodegradation was prolonged, but complete removal of 4-chlorophenol was observed in the presence of vanillic acid in the concentration of 3 mM. While 4-chlorophenol concentration was halved, its removal took place even in the presence of eight times more concentration of vanillic acid. Because this phenolic compound does not induce enzymes of 4-chlorophenol biodegradation, presence of phenol as the second growth sub-
P14-23 Degradation of endocrine disruptor compounds in wastewater by Pleurotus ostreatus fungal batch bioreactor Lucie Linhartová 1,∗ , Zdena Kˇresinová 2 , Alena Filipová 1 , Martin Ezechiáˇs 2 , Tomáˇs Cajthaml 1 1 Institute of Microbiology, Academy of Sciences of the Czech Republic, Czech Republic 2 v.v.i, Czech Republic
It has been previously reported that the ligninolytic fungus Pleurotus ostreatus is able to degrade selected endocrine disruptor compounds (EDCs) under both in vivo and in vitro conditions. In this work we demonstrate a convenient fungal batch reactor arrangement for the degradation of EDCs in real contaminated, as well as EDCs fortified wastewater. Concentration of EDCs in real contaminated wastewater from locality A was found to be in the range from 0.117 g L−1 (triclosan, TCR) to 0.288 g L−1 (bisphenol A, BPA).
S140
Abstracts / New Biotechnology 33S (2016) S1–S213
Fungal batch reactor was colonized by P. ostreatus KRYOS obtained as a colonised straw waste material from commercial growing hall of Oyster mushroom. The fungus was refreshed by the addition of sterilized straw pellets. The volume of treated water was 6 L. Concentration of each EDC – estrone (E1), estradiol (E2), estriol (E3), 17a-ethynylestradiol (EE2), BPA, TRC in fortified wastewater was 60 ppb. An aliquot of treated water was extracted with ethylacetate. Analytes were derivatized with BSTFA and quantitatively analyzed by GC/MS. All EDCs were degraded in the range 42–86% during the first hour. After 24 h the concentrations of E1, E2 and TRC were below the limits of detection. The residual concentrations of EDCs were = 4.5 ppb. Moreover, a decrease in estrogenic activity of the degraded samples was observed under the conditions of the fungal batch reactor. Acknowledgement: This research was funded by Grant No. 1328283S from the Czech Science Foundation. http://dx.doi.org/10.1016/j.nbt.2016.06.1205
P14-24 Enzymatic C C bond formation by benzylsuccinate synthases – A key step in anaerobic microbial degradation of hydrocarbons Maciej Szaleniec 1,∗ , Johann Heider 2 1 2
Jerzy Haber Institute of Catalysis and Surface Chemistry, Poland Philipps – University of Marburg, Germany
Glycyl radical enzymes (GREs) catalyze some of the most remarkable and energetically challenging free radical reactions encountered in biology. One of these particularly important reactions is the addition of toluene to the double bond of fumarate, catalyzed by benzylsuccinate synthase (BSS) which has been mainly studied in the denitrifying species Thauera aromatica. Similar fumarate-addition reactions are also employed for other methyl-substituted aromatic hydrocarbons (e.g. p-cymene or 2-methylnapthalene) or even n-alkanes in denitrifying or sulfatereducing bacteria. BSS from T. aromatica is a heterohexameric enzyme (a2 b2 g2 ) with a characteristic catalytic dyad of Gly828 and Cys492 in the a subunit, which are part of the active site. The Gly radical is generated by hydrogen abstraction from Gly828 catalyzed by a GREactivating enzyme. After activation of the enzyme, the radical can be transferred from Gly828 to Cys492 , creating a catalytically relevant enzyme-bound thiyl radical. The reaction catalyzed by BSS is highly enantiospecific, producing only (R)-benzylsuccinate. Moreover, isotope-labeled studies revealed that the addition of toluene to the double bond proceeds with inversion of stereochemistry at the benzyl carbon atom and syn addition of a deuteron label upon quenching of the radical product. The reaction mechanism of BSS was studied by a combination of experimental (isotope labeling) and theoretical methods. The latter involved MD simulation of the enzyme–product complex, determination of the substrate binding mode, followed by QM studies of the reaction mechanism. These studies elucidated factors responsible for the enantiospecificity of the reaction and lead to a plausible mechanistic hypothesis consistent with the established model. http://dx.doi.org/10.1016/j.nbt.2016.06.1206
P14-25 Effluents from anaerobic digestion as potential media for microalgal culture: Evaluation of factors critical for photoautotrophic growth Przemysław Malec 2,∗ , Paweł Jedynak 1 , Jan Burczyk 2 , Sebastian Borowski 3 , Paweł Kaszycki 4 , Andrzej Waloszek 5 , Khongorzul 1 ˛ Mungunkhuyag 1 , Małgorzata Hałat-Ła´s 2 , Magdalena Kedra 1
Jagiellonian University, Faculty of Biochemistry, Poland Biophysics and Biotechnology, Poland 3 EKOSPOT Sp. z o.o., Laboratory of Biotechnology, Poland 4 Lodz University of Technology, Faculty of Biotechnology and Food Sciences, Poland 5 University of Agriculture in Krakow, Faculty of Biotechnology and Horticulture, Poland 2
Effluents derived from the anaerobic digestion of sewage sludge, obtained in the laboratory and industrial biogas plants, were analyzed for use as culture media for photoautotrophic microalgae. The liquids were generally alkaline (pH approx. 8.0) and contained all the minerals essential for the efficient growth of autotrophic microorganisms in concentrations exceeding the average physiological minima. In all liquids tested, ammonium ion was the dominant form of inorganic nitrogen. Liquids contained low levels of solids and an electrical conductivity in the range of approx. 0.2–0.3 mS/cm. Redox values (rH) varied from weakly reducing (20) to neutral (25). The survival, proliferation and photosynthetic activity of Chlorella sorokiniana (strain 211-8k) and selected Chlorophyta strains isolated from eutrophic habitats in Poland were studied under standard conditions of photoautotrophic growth (fluence rate 50–70 mol m−2 s−1 , photoperiod 16L:8D) at ambient temperature (23 ◦ C), using post-fermentation effluents as a culture medium. It was found that the dilution of the digestates (25–50%) promoted an effective proliferation of both Chlorella sp. and other microalgal stains. This process has been accompanied by the accumulation of photosynthetic pigments and the increase in photosynthetic activity. The aeration of effluents resulted in the increase of rH and stimulated the survival of microalgae. The introduction of micro-algae caused a significant decrease of COD (approx. 50%) and the concentrations of both ammonium and phosphate in the period of 7 days. Our results show that the high initial ammonia concentration, low rH and low availability of phosphorus may limit the photoautotrophic growth of microalgae on digestates from anaerobic fermentation. http://dx.doi.org/10.1016/j.nbt.2016.06.1207
P14-26 Biodegradation of highly complex polyaromatic-hydrocarbon pyrene by Bacillus lenichiformis and Ralstonia sp from the coastal region of Saudi Arabia Alexis Nzila 1,∗ , Assad Thukair 1 , Saravanan Sankara 1 , Basheer Chanbasha 2 , Mohammed Musa Musa 1 1 2
King Fahd University of Petroleum & Minerals, Saudi Arabia Department of Life Science, Saudi Arabia
The contamination of the environment by polyaromatic hydrocarbons (PAHs) is common in The Kingdom of Saudi Arabia (KSA), as the result of exploitation of oil. PAHs, which are recalcitrant to
P14-20 Rhodococcus equi protects selected plant species from arsenic toxicity
S139
strate seems to be obligatory for decay of repeated doses of 4-chlorophenol. http://dx.doi.org/10.1016/j.nbt.2016.06.1203
Anna Kowalczyk 1,∗ , Dorota Tabor 2 , Dariusz Latowski 1 1 2
Jagiellonian University, Poland AGH University of Science and Technology, Poland
The new strain of arsenic resistant bacteria was isolated from soil contaminated with arsenic and identified as Rhodococcus equi. Isolated strain is capable to grow under laboratory conditions on LB medium supplemented with 2% (w/v) of pure As. Winter wheat as a starch-accumulating species; rapeseed (oil plant) and field pea (crude protein-rich species) were R. equi treated in order to test the bacteria influence on the plants tolerance to As. The final concentration of arsenic in culture medium was 0.25 mg/100 ml. The effect of R. equi on germination, average root and above ground parts length and biomass production were tested. The most tolerant to arsenic was winter wheat, whereas field pea was the least resistant. The supplementation with As in case of wheat and rapeseed slightly increased the number of germinated seeds, but inhibited pea germination. Average root and above ground parts length were reduced for all tested species. The inoculation of culture medium with R. equi increased the tolerance of plants to As, as the values of the controlled parameters increased. The treatment of wheat, rapeseed and pea with R. equi without arsenic demonstrated positive effect on measured parameters or the effect was not observed. The effect of R. equi on tolerance index and toxicity index of selected species to arsenic were tested. The presence of isolated strain in culture medium improved wheat and rapeseed tolerance to arsenic. The toxicity index demonstrated the increase of investigated plants tolerance, what indicates beneficial effect of R. equi on plants. http://dx.doi.org/10.1016/j.nbt.2016.06.1202
P14-21 Vanillic acid as the growth substrate in cometabolic biodegradation of 4-chlorophenol Izabela Gren´ 1,∗ , Justyna Michalska 2 , Klaudia Krzykała 3 , 1 ˛ Agnieszka Gaszczak
P14-22 Groundwater passive sampling and fungal biodegradation of psychopharmaceuticals Zdena Kresinova 1,∗ , Lucie Krejcova 2 , Klára Petru 3 , Tomas Cajthaml 1 1
Institute for Environmental Studies, Czech Republic Faculty of Science, Czech Republic 3 Charles University of Prague, Czech Republic 2
Passive sampling is a rapidly developing technology which offers significant advantages over traditional grab sampling and it is being widely used for the monitoring of pollutants in different environments. In the present study, an analytical methodology for passive sampling of psychopharmaceuticals was evaluated and applied on contaminated locality in Czech Republic. Four pharmaceutical drugs (amitriptyline, dosulepin, chlorprothixene, butamirate) and five compounds which are either used during drug manufacturing or are created as by-products were detected in groundwater in the range from nanograms up to hundreds of milligrams per litter. A biodegrading potential of ligninolytic fungus Pleurotus ostreatus was tested towards a mixture of all detected contaminants. The initial concentration of individual compounds in the mixture was 2 mg L−1 . Five compounds were totally or partially degraded (>52%) during the in vivo cultivation of P. ostreatus on complex liquid medium. Applied acute toxicity tests with Vibrio fischeri indicated the potential formation of metabolites that exhibited higher toxicity than the one or more original substances. EC50 values in the acute toxicity of V. fischeri for individual substances ranged from 5.45 mg L−1 to 131.98 mg L−1 . The metabolites are studied using MS techniques. Acknowledgements: The research leading to these results has received funding from the Norwegian Financial Mechanism 2009–2014 and the Ministry of Education, Youth and Sports under Project Contract no. MSMT-23681/2015-1. http://dx.doi.org/10.1016/j.nbt.2016.06.1204
1
Faculty of Biology and Environmental Protection, Poland University of Silesia in Katowice, Poland 3 Silesian University of Katowice, Poland 2
Vanillic acid belongs to the group of phenolic compounds of plant origin which can serve as the source of carbon and energy for the cells of Stenotrophomonas maltophilia KB2. But simultaneously its concentration of 4 mM and more causes 50% inhibition of KB2 cells growth. Biodegradation of vanillic acid engages enzymes of protocatechuate degradation pathway and contributes to increase of KB2 cell biomass. While vanillic acid was introduced together with 4-chlorophenol as the additional source of carbon and energy time needed for its biodegradation was prolonged, but complete removal of 4-chlorophenol was observed in the presence of vanillic acid in the concentration of 3 mM. While 4-chlorophenol concentration was halved, its removal took place even in the presence of eight times more concentration of vanillic acid. Because this phenolic compound does not induce enzymes of 4-chlorophenol biodegradation, presence of phenol as the second growth sub-
P14-23 Degradation of endocrine disruptor compounds in wastewater by Pleurotus ostreatus fungal batch bioreactor Lucie Linhartová 1,∗ , Zdena Kˇresinová 2 , Alena Filipová 1 , Martin Ezechiáˇs 2 , Tomáˇs Cajthaml 1 1 Institute of Microbiology, Academy of Sciences of the Czech Republic, Czech Republic 2 v.v.i, Czech Republic
It has been previously reported that the ligninolytic fungus Pleurotus ostreatus is able to degrade selected endocrine disruptor compounds (EDCs) under both in vivo and in vitro conditions. In this work we demonstrate a convenient fungal batch reactor arrangement for the degradation of EDCs in real contaminated, as well as EDCs fortified wastewater. Concentration of EDCs in real contaminated wastewater from locality A was found to be in the range from 0.117 g L−1 (triclosan, TCR) to 0.288 g L−1 (bisphenol A, BPA).
S140
Abstracts / New Biotechnology 33S (2016) S1–S213
Fungal batch reactor was colonized by P. ostreatus KRYOS obtained as a colonised straw waste material from commercial growing hall of Oyster mushroom. The fungus was refreshed by the addition of sterilized straw pellets. The volume of treated water was 6 L. Concentration of each EDC – estrone (E1), estradiol (E2), estriol (E3), 17a-ethynylestradiol (EE2), BPA, TRC in fortified wastewater was 60 ppb. An aliquot of treated water was extracted with ethylacetate. Analytes were derivatized with BSTFA and quantitatively analyzed by GC/MS. All EDCs were degraded in the range 42–86% during the first hour. After 24 h the concentrations of E1, E2 and TRC were below the limits of detection. The residual concentrations of EDCs were = 4.5 ppb. Moreover, a decrease in estrogenic activity of the degraded samples was observed under the conditions of the fungal batch reactor. Acknowledgement: This research was funded by Grant No. 1328283S from the Czech Science Foundation. http://dx.doi.org/10.1016/j.nbt.2016.06.1205
P14-24 Enzymatic C C bond formation by benzylsuccinate synthases – A key step in anaerobic microbial degradation of hydrocarbons Maciej Szaleniec 1,∗ , Johann Heider 2 1 2
Jerzy Haber Institute of Catalysis and Surface Chemistry, Poland Philipps – University of Marburg, Germany
Glycyl radical enzymes (GREs) catalyze some of the most remarkable and energetically challenging free radical reactions encountered in biology. One of these particularly important reactions is the addition of toluene to the double bond of fumarate, catalyzed by benzylsuccinate synthase (BSS) which has been mainly studied in the denitrifying species Thauera aromatica. Similar fumarate-addition reactions are also employed for other methyl-substituted aromatic hydrocarbons (e.g. p-cymene or 2-methylnapthalene) or even n-alkanes in denitrifying or sulfatereducing bacteria. BSS from T. aromatica is a heterohexameric enzyme (a2 b2 g2 ) with a characteristic catalytic dyad of Gly828 and Cys492 in the a subunit, which are part of the active site. The Gly radical is generated by hydrogen abstraction from Gly828 catalyzed by a GREactivating enzyme. After activation of the enzyme, the radical can be transferred from Gly828 to Cys492 , creating a catalytically relevant enzyme-bound thiyl radical. The reaction catalyzed by BSS is highly enantiospecific, producing only (R)-benzylsuccinate. Moreover, isotope-labeled studies revealed that the addition of toluene to the double bond proceeds with inversion of stereochemistry at the benzyl carbon atom and syn addition of a deuteron label upon quenching of the radical product. The reaction mechanism of BSS was studied by a combination of experimental (isotope labeling) and theoretical methods. The latter involved MD simulation of the enzyme–product complex, determination of the substrate binding mode, followed by QM studies of the reaction mechanism. These studies elucidated factors responsible for the enantiospecificity of the reaction and lead to a plausible mechanistic hypothesis consistent with the established model. http://dx.doi.org/10.1016/j.nbt.2016.06.1206
P14-25 Effluents from anaerobic digestion as potential media for microalgal culture: Evaluation of factors critical for photoautotrophic growth Przemysław Malec 2,∗ , Paweł Jedynak 1 , Jan Burczyk 2 , Sebastian Borowski 3 , Paweł Kaszycki 4 , Andrzej Waloszek 5 , Khongorzul 1 ˛ Mungunkhuyag 1 , Małgorzata Hałat-Ła´s 2 , Magdalena Kedra 1
Jagiellonian University, Faculty of Biochemistry, Poland Biophysics and Biotechnology, Poland 3 EKOSPOT Sp. z o.o., Laboratory of Biotechnology, Poland 4 Lodz University of Technology, Faculty of Biotechnology and Food Sciences, Poland 5 University of Agriculture in Krakow, Faculty of Biotechnology and Horticulture, Poland 2
Effluents derived from the anaerobic digestion of sewage sludge, obtained in the laboratory and industrial biogas plants, were analyzed for use as culture media for photoautotrophic microalgae. The liquids were generally alkaline (pH approx. 8.0) and contained all the minerals essential for the efficient growth of autotrophic microorganisms in concentrations exceeding the average physiological minima. In all liquids tested, ammonium ion was the dominant form of inorganic nitrogen. Liquids contained low levels of solids and an electrical conductivity in the range of approx. 0.2–0.3 mS/cm. Redox values (rH) varied from weakly reducing (20) to neutral (25). The survival, proliferation and photosynthetic activity of Chlorella sorokiniana (strain 211-8k) and selected Chlorophyta strains isolated from eutrophic habitats in Poland were studied under standard conditions of photoautotrophic growth (fluence rate 50–70 mol m−2 s−1 , photoperiod 16L:8D) at ambient temperature (23 ◦ C), using post-fermentation effluents as a culture medium. It was found that the dilution of the digestates (25–50%) promoted an effective proliferation of both Chlorella sp. and other microalgal stains. This process has been accompanied by the accumulation of photosynthetic pigments and the increase in photosynthetic activity. The aeration of effluents resulted in the increase of rH and stimulated the survival of microalgae. The introduction of micro-algae caused a significant decrease of COD (approx. 50%) and the concentrations of both ammonium and phosphate in the period of 7 days. Our results show that the high initial ammonia concentration, low rH and low availability of phosphorus may limit the photoautotrophic growth of microalgae on digestates from anaerobic fermentation. http://dx.doi.org/10.1016/j.nbt.2016.06.1207
P14-26 Biodegradation of highly complex polyaromatic-hydrocarbon pyrene by Bacillus lenichiformis and Ralstonia sp from the coastal region of Saudi Arabia Alexis Nzila 1,∗ , Assad Thukair 1 , Saravanan Sankara 1 , Basheer Chanbasha 2 , Mohammed Musa Musa 1 1 2
King Fahd University of Petroleum & Minerals, Saudi Arabia Department of Life Science, Saudi Arabia
The contamination of the environment by polyaromatic hydrocarbons (PAHs) is common in The Kingdom of Saudi Arabia (KSA), as the result of exploitation of oil. PAHs, which are recalcitrant to