- Email: [email protected]
Biodegradation of highly complex polyaromatic-hydrocarbon pyrene by Bacillus lenichiformis and Ralstonia sp from the coastal region of Saudi Arabia
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
Abstracts / New Biotechnology 33S (2016) S1–S213
degradation, can be removed by biodegradation with microorganisms. We report on the isolation and characterization of bacteria that biodegrade the 4-ring PAHs pyrene (PY), from soil contaminated with petroleum products. The enrichment in liquid culture in presence of PY as sole source of carbon has lead to the isolation of a consortium of PY-biodegrading bacteria (PY-Con). This consortium growth in presence of PY can reach up to 108 CFU/ml (colony forming units), within 30–40 days. To characterize individual bacterium that form this consortium, PY-Con was cultured in agar solid plate (after streaking), and a total of 2 different colonies, PY-2A and PY-4B, were identified. 16S RNA sequencing analysis and the BLAST program for homology analyses have shown that PY-2A was Bacillus licheniformis, (GI: 540360856) and PY-4B is Ralstonia sp (GI: 540360857). Further studies have shown that the maximum growth of PY-con is attained at salinity of 1%, pH7, 37 ◦ C, and each species can biodegrade efficiently the 3-ring aromatic phenanthrene, anthracene; the 2-ring naphthalene, and the 1-ring salicylate and catechol. Investigations are underway to identify PY metabolites produced by these strains using GC–MS, and their expressed enzymes during PY-degradation by proteomics analysis. The combination of metabolite identification and proteomic analysis will lead to propose biochemical pathways of PY degradation in these 2 species. http://dx.doi.org/10.1016/j.nbt.2016.06.1208
P14-27 Withdrawn
http://dx.doi.org/10.1016/j.nbt.2016.06.1209
P14-28 Evaluation of bioremediation of soil highly contaminated by petroleum hydrocarbons Pola Lomza ∗ , Krzysztof Poszytek, Aleksandra Sklodowska, Lukasz Drewniak University of Warsaw, Poland Soil contamination with petroleum released from leaking fuel storage tanks are a worldwide environmental problem and can pose significant ecological risks. Among various technologies that have been used to purify contaminated areas, bioremediation has proven to be an economic and environmentally friendly approach. Bioremediation of contaminated soil is usually done by biostimulation or bioaugmentation. Biostimulation involves the addition of nutrients, final electron acceptors, and auxiliary substrates (e.g. vitamins) to increase the activity and stimulate the growth of indigenous microbial populations. In turn, bioaugmentation refers to the introduction of autochthonous or exogenous contaminant-degrading microorganisms. This study evaluated the effectiveness of biostimulation (through the addition of oxygen, nitrogen and phosphorous) and bioaugmention (by exogenous consortium of bacteria with enhanced capabilities to degrade complex organic compounds) of soil exposed to long-term pollution from leaking underground fuel storage tanks. The biodegradability studies were performed on two samples of soils with different content of crude oil contamination (∼6000 and 68,000 mg/kg) obtained from refinery in Plock, Poland.
S141
To monitor biodegradation efficiency the manometric respirometric BOD OxiTop® method was used. The obtained results showed that higher biodegradation rate was received for bacteria community representing less polluted soil, in which BOD/ThOD after 28 days of incubation reached ∼25% in biostimulation variant. In turn, the maximum biodegradation rate (∼10%) of highly contaminated soil was achieved when bioaugmentation with exogenous microflora was performed. http://dx.doi.org/10.1016/j.nbt.2016.06.1210
P14-29 Comparative study of biodegradation of aged organochlorine pesticides in soil through native bacteria Maria Kopytko Universidad Pontificia Bolivariana, Colombia DDT and others organochlorine pesticides have been extensively used in agriculture at Agustín Codazzi, Colombia between 1970s and 1980s in cotton crop, then were buried in the soil, after being banned in 1999. The purpose of this work was assess biodegradation of DDT and its transformation products DDD and DDE which have been persisting during 16 years in soil at Agustin Codazzi. Biostimulation, bioaugmentation were carried out under anaerobic and aerobic sequential conditions, compared with the natural bioremediation of soil. The bioremediation process lasted 8 weeks under anaerobic conditions and subsequent 12 weeks under aerobic environments. Biostimulation consisted in adding of nutrients to optimize the proportion of C:N:P into the soil. For bioaugmentation, a bacterial consortium of four strains Burkholderia cepacia, Pseudomonas fluorescens, Aeromona caviae, and Bacillus sp, which were selected from the native bacteria bioaugmented in a laboratory and inoculated at soil with a concentration of 108 UFC/ml was established. Bacterial strains were identified by its morphological characteristics, and 16S rDNA sequencing. The concentration of DDT, DDD and DDE were determined by gas chromatography. The best results were obtained during the anaerobic phase for the reductive dechlorination of organochlorines. The bioaugmentation process was the most effective for the removal of DDT, DDD and DDE at 56.2%, 17.1% and 44.5% respectively. During 12 sequential weeks of aerobic process, they increased amount of DDT and its metabolized products (DDD and DDE) due to desorption development. After that, biodegradation process has been detected by reduction of concentration of these substances. http://dx.doi.org/10.1016/j.nbt.2016.06.1211
P14-30 Prospects for native arbuscular mycorrhizal fungi community to assist in phytoremediation of soil hydrocarbon contaminants Monika Rajtor 1,∗ , Zofia Piotrowska-Seget 2 1 2
Faculty of Biology and Environmental Protection, Poland Univeristy of Silesia in Katowice, Poland
Phytoremediation is considered as a prominent method for hydrocarbon removal. In this process plants interact with the rhizosphere microflora which encompass hydrocarbon degraders and plant growth-promoting microorganisms, including arbuscular mycorrhizal fungi (AMF). AMF mycelium acts as a biofilter of
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
Abstracts / New Biotechnology 33S (2016) S1–S213
degradation, can be removed by biodegradation with microorganisms. We report on the isolation and characterization of bacteria that biodegrade the 4-ring PAHs pyrene (PY), from soil contaminated with petroleum products. The enrichment in liquid culture in presence of PY as sole source of carbon has lead to the isolation of a consortium of PY-biodegrading bacteria (PY-Con). This consortium growth in presence of PY can reach up to 108 CFU/ml (colony forming units), within 30–40 days. To characterize individual bacterium that form this consortium, PY-Con was cultured in agar solid plate (after streaking), and a total of 2 different colonies, PY-2A and PY-4B, were identified. 16S RNA sequencing analysis and the BLAST program for homology analyses have shown that PY-2A was Bacillus licheniformis, (GI: 540360856) and PY-4B is Ralstonia sp (GI: 540360857). Further studies have shown that the maximum growth of PY-con is attained at salinity of 1%, pH7, 37 ◦ C, and each species can biodegrade efficiently the 3-ring aromatic phenanthrene, anthracene; the 2-ring naphthalene, and the 1-ring salicylate and catechol. Investigations are underway to identify PY metabolites produced by these strains using GC–MS, and their expressed enzymes during PY-degradation by proteomics analysis. The combination of metabolite identification and proteomic analysis will lead to propose biochemical pathways of PY degradation in these 2 species. http://dx.doi.org/10.1016/j.nbt.2016.06.1208
P14-27 Withdrawn
http://dx.doi.org/10.1016/j.nbt.2016.06.1209
P14-28 Evaluation of bioremediation of soil highly contaminated by petroleum hydrocarbons Pola Lomza ∗ , Krzysztof Poszytek, Aleksandra Sklodowska, Lukasz Drewniak University of Warsaw, Poland Soil contamination with petroleum released from leaking fuel storage tanks are a worldwide environmental problem and can pose significant ecological risks. Among various technologies that have been used to purify contaminated areas, bioremediation has proven to be an economic and environmentally friendly approach. Bioremediation of contaminated soil is usually done by biostimulation or bioaugmentation. Biostimulation involves the addition of nutrients, final electron acceptors, and auxiliary substrates (e.g. vitamins) to increase the activity and stimulate the growth of indigenous microbial populations. In turn, bioaugmentation refers to the introduction of autochthonous or exogenous contaminant-degrading microorganisms. This study evaluated the effectiveness of biostimulation (through the addition of oxygen, nitrogen and phosphorous) and bioaugmention (by exogenous consortium of bacteria with enhanced capabilities to degrade complex organic compounds) of soil exposed to long-term pollution from leaking underground fuel storage tanks. The biodegradability studies were performed on two samples of soils with different content of crude oil contamination (∼6000 and 68,000 mg/kg) obtained from refinery in Plock, Poland.
S141
To monitor biodegradation efficiency the manometric respirometric BOD OxiTop® method was used. The obtained results showed that higher biodegradation rate was received for bacteria community representing less polluted soil, in which BOD/ThOD after 28 days of incubation reached ∼25% in biostimulation variant. In turn, the maximum biodegradation rate (∼10%) of highly contaminated soil was achieved when bioaugmentation with exogenous microflora was performed. http://dx.doi.org/10.1016/j.nbt.2016.06.1210
P14-29 Comparative study of biodegradation of aged organochlorine pesticides in soil through native bacteria Maria Kopytko Universidad Pontificia Bolivariana, Colombia DDT and others organochlorine pesticides have been extensively used in agriculture at Agustín Codazzi, Colombia between 1970s and 1980s in cotton crop, then were buried in the soil, after being banned in 1999. The purpose of this work was assess biodegradation of DDT and its transformation products DDD and DDE which have been persisting during 16 years in soil at Agustin Codazzi. Biostimulation, bioaugmentation were carried out under anaerobic and aerobic sequential conditions, compared with the natural bioremediation of soil. The bioremediation process lasted 8 weeks under anaerobic conditions and subsequent 12 weeks under aerobic environments. Biostimulation consisted in adding of nutrients to optimize the proportion of C:N:P into the soil. For bioaugmentation, a bacterial consortium of four strains Burkholderia cepacia, Pseudomonas fluorescens, Aeromona caviae, and Bacillus sp, which were selected from the native bacteria bioaugmented in a laboratory and inoculated at soil with a concentration of 108 UFC/ml was established. Bacterial strains were identified by its morphological characteristics, and 16S rDNA sequencing. The concentration of DDT, DDD and DDE were determined by gas chromatography. The best results were obtained during the anaerobic phase for the reductive dechlorination of organochlorines. The bioaugmentation process was the most effective for the removal of DDT, DDD and DDE at 56.2%, 17.1% and 44.5% respectively. During 12 sequential weeks of aerobic process, they increased amount of DDT and its metabolized products (DDD and DDE) due to desorption development. After that, biodegradation process has been detected by reduction of concentration of these substances. http://dx.doi.org/10.1016/j.nbt.2016.06.1211
P14-30 Prospects for native arbuscular mycorrhizal fungi community to assist in phytoremediation of soil hydrocarbon contaminants Monika Rajtor 1,∗ , Zofia Piotrowska-Seget 2 1 2
Faculty of Biology and Environmental Protection, Poland Univeristy of Silesia in Katowice, Poland
Phytoremediation is considered as a prominent method for hydrocarbon removal. In this process plants interact with the rhizosphere microflora which encompass hydrocarbon degraders and plant growth-promoting microorganisms, including arbuscular mycorrhizal fungi (AMF). AMF mycelium acts as a biofilter of