해양생태계 내 유기오염물질의 생지화학적 거동 평가 및 제일원리기법에 기반한 생태독성 반응 규명

학위논문(박사) -- 서울대학교대학원 : 자연과학대학 지구환경과학부, 2022. 8. 김종성.해양 환경은 육상 및 해양에서의 인간 활동으로 인해 광범위한 영향을 받는다. 해양 환경 중 조간대는 영양염류 (총질소 및 총인), 탄화수소 (유류 및 다환방향족탄화수소), 알킬페놀류, 스타이렌 올리고머를 포함한 유기 오염물질의 유입으로부터 육지와 바다 사이의 균형을 유지한다. 유기 오염물질의 거동은 물질의 물리화학적 특성에 크게 좌우된다. 더 큰 소수성 및 입자와의 반응성은 유기 오염물질이 퇴적물에 축적되게 만들고 그 결과, 퇴적물에서는 해수보다 몇 배나 더 큰 유기 오염물질의 농도를 유지한다. 해양 저서 퇴적물은 육상기인오염 물질의 최종 종착지이기 때문에 지속적인 해양 환경 생태서비스를 제공받기 위해서는 해양 환경의 유기 오염물 정화 능력과 생태독성 영향을 명확히 평가하는 것이 중요하다. 현재까지 조간대에서의 유기오염물질 정화 능력은 정량적으로 알려지지 않았고 정화 과정에서 발생하는 화학적, 독성학적, 생태학적 반응은 예측하기 어려웠다. 본 연구에서는 조간대 내 퇴적물이 정화되는 과정에서 발생하는 현상들을 실험적 규모 연구, 즉 메조코즘 연구를 통해 퇴적물의 생태위해성 평가법을 사용하여 정량적으로 확인하였다. 이를 위해 화학 분석, 생물 검정 및 저서 군집 구조 분석도 진행하였다. 그리고 인 실리고 연구를 통해 유기 오염물질의 물리화학적 특성을 분석하여 퇴적물의 정화 과정 속에서 발생하는 화학적, 독성학적, 그리고 생태학적 반응 원인을 제시하였다. 첫째로, 갯벌 퇴적물의 자정능을 평가하기 위해 0.2m3 부피의 유리 수조에 퇴적물을 이식하여 총인을 비롯한 고농도의 유기물질이 포함된 해수를 넣어 시간에 따른 변화 특성을 확인하였다. 갯벌 퇴적물은 총인 및 화학적 산소 요구량의 농도를 각각 2일 및 7일 만에 배경 농도 수준으로 제거하였다. 그리고 갯벌 퇴적물에 갈대를 심은 실험 구에서는 특히 용존 무기인을 빠르고 효율적으로 제거하였으며 이는 식생의 존재가 용존 유기 오염물질의 정화를 촉진하는 것을 의미한다. 또한 유기 오염물질의 침전은 생물학적 교란 효과가 작은 환경에서 우세한 것을 확인하였다. 둘째로, 자갈 조간대 내 유기 오염물질이 유입되었을 때를 가정하고 이를 회복하기 위한 물리적, 생물학적 기술들의 현황을 파악하고 효과를 분석하였다. 이를 위해 60일 동안의 메조코즘 연구로 잔류 유류 제거를 위한 물리적, 생물학적 기술, 그리고 자연정화능의 효과 및 영향을 비교하였다. 우선 고온고압 세척 처리는 잔류 유류를 최대 93% 제거하였다. 하지만 이와 같은 물리적 정화는 초기 정화 처리 기간 저서동물 군집에 악영향을 끼쳤다. 영양염, 유화제, 효소 활성제, 그리고 미생물 제제와 같은 생물학적 처리는 최대 66%의 잔류 유류를 제거하였다. ‘자연정화’는 다른 물리적 및 생물학적 기술들의 잔류 유류 제거 효과와 유사한 효율을 보여주었다. 특히 실험 기간은 미생물들의 천이를 확인하였으며 이는 시간에 따른 잔류 유류 성분의 변화 결과로 확인하였다. 자연정화는 다른 기술들만큼 유기 오염물질 제거에 효율적이었으며 특히 저서 군집에 대한 악영향을 최소화하는 특징이 있음을 확인하였다. 셋째로, 갯벌 퇴적물에서 잔류성 독성물질 특이적 정화와 생태독성 영향을 확인하기 위해 오염 퇴적물을 현장 갯벌에 이식하여 60일간 화학적, 독성학적, 그리고 생태학적 반응의 변화를 확인하였다. 그리고 머신 러닝을 통해 정화 과정 동안 오염 퇴적물의 특징을 4가지로 추출하였다. 대형저서동물과 식생이 함께 이식된 오염 퇴적물은 생물 관개 및 식물 정화 효과로 빠르게 회복되었다. 실험 기간은 잔류성 독성물질 내 다환방향족탄화수소, 알킬페놀류, 그리고 스타이렌 올리고머의 모물질들이 빠르게 감소하였는데 생물 관계 및 식물 정화로 인한 미생물의 활동으로 인한 결과임을 확인하였다. 넷째로, 매질 (해수, 퇴적물 및 해양 생물)에 대한 유기 오염물질의 반응성을 예측하기 위해 유기 오염물질의 물리화학적 성질을 분석하였습니다. 제1 원리를 활용한 밀도범함수이론을 통해 유기 오염물질과 매질 사이 반응을 정량적으로 확인할 수 있는 방향성 반응 인자를 고안하였다. 방향성 반응 모델은 유기 오염물질의 구조, 예로 벤젠 고리의 수, 메틸화 및 하이드록실 화와 같은 구조적 변형이 있는 크라이센의 동족체를 대상으로 계산하였고 특히 아릴 탄화수소수용체와의 반응 관계 중심으로 적용하였다. 본 반응 모델의 결과는 크라이센 동족체의 생물검정 결과와 일치하였다. 제1 원리에 기반한 인 실리코 연구는 유기 오염물질의 물리화학적 성질을 계산하였고 기존의 경험적 접근 방식을 보완하여 향후 해양 환경 내 매질 간의 반응성 예측에 활용할 수 있다. 끝으로, 독성 작용에 대한 반응 중심 접근 방법과 함께 16개의 다환방향족 탄화수소들의 생태독성 효과를 예측하기 위해 방향성 반응 모델을 응용하였다. 분자 역학 모델링 분석을 통해 아릴 탄화수소수용체와 다환방향족탄화수소의 결합 가능성을 확률적으로 계산하였고 이를 바탕으로 방향성 반응 모델과 연관시킨 방향성 반응-결합 인자를 고안하였다. 이 인자를 통해 유기 오염물질-수용체 결합을 예측할 수 있었고 나아가 생물검정 결과와 일치하는 것을 확인하였다. 잠재 독성 예측에 있어 방향성 반응-결합 인자는 유기 오염물질의 물리화학적 특성이 생물과의 독성 반응에 주요 요인이 될 수 있음을 요인이 될 수 있음 보여주었다. 향후 인 실리코 방법을 통한 유기 오염물질의 특성 분석은 생물뿐만 아니라 해수 및 퇴적물과 같은 해양 환경과의 반응 예측에 유의미하게 활용될 가능성을 확인하였다. 이상의 연구 결과를 종합해보면, 첫째, 먼저 조간대에 유입된 유기 오염물질의 농도는 초기 단계에서 전반적으로 급격한 감소를 했고, 모물질의 분해가 활발히 진행되었다. 둘째, 유기 오염물질 농도의 감소와 함께 퇴적물의 잠재 독성도 급격히 감소하였고, 유기 오염물질 농도의 감소 속도보다 더 빨랐다. 셋째, 저서 군집은 기하급수적으로 회복되었으며, 영양수준에 따라 각각의 회복 속도에 차이가 있었다. 넷째, 화학적, 독성학적, 생태학적 회복의 차이와 각 영향 관계는 유기 오염물질의 물리화학적 특성 영향에 기인한 것으로 확인되었다. 마지막으로, 본 연구의 방향성 반응 인자는 생물 내 잠재적인 독성을 예측하는 반응-결합 인자로 개발될 수 있으며 생물검정 결과와 상당히 일치한 것을 확인했다. 본 연구에서 활용한 통합적 접근은 해양 환경 내 유기 오염물질의 정화와 생태 독성학적 효과를 이해하는 데 있어 유용하게 사용될 수 있다. 앞으로 우리나라 해양 생태계 서비스의 가치를 재고하고 해안을 체계적으로 관리하기 위해서는 위와 같은 다학제적 접근이 지속해서 필요하다.The marine environment is subject to a broad range of adverse impacts from anthropogenic activities. Among the marine environment, an intertidal zone maintains a balance as a buffer between the land and the sea from the introduction of organic pollutants, including nutrients (total nitrogen, TN; total phosphorus, TP), hydrocarbons (oil and polycyclic aromatic hydrocarbons, PAHs), alkylphenols (APs+APEOs), and styrene oligomers (SOs). The fate of organic pollutants in the marine environment is highly dependent on their physicochemical properties. Due to their hydrophobicity and particle reactivity, many organic pollutants have concentrations in sediments that are several orders of magnitude greater than those in the surrounding water. As a result, sediments are frequently regarded as the final destination for pollutants in the environment. Thus, it is crucial to estimate the restoration capacity and ecotoxicological effect of organic pollutants in the marine environment to maintain environmental services. Until this study, the restoration ability of organic pollutants in the intertidal zone was not quantitatively known, and the chemical, toxicological and ecological reactions occurring during the restoration process were not predicted. In this study, the ecological processes occurring in sediments being purified were identified quantitatively using an enhanced integrated sediment quality triad (SQT) approach with an enclosed experimental scale study (mesocosm). Additionally, using an in silico study, by examining the physico-chemical properties of organic pollutants, the causes of the restoration and ecotoxicity were analyzed. For evaluating the specific recovery of the benthic community health from the organic pollutants, instrumental analysis, bioassays, and investigation of the benthic community structure were also implemented. The mudflat sediments significantly removed waterborne organic pollutants to background levels in ~2 and 6–7 days for TP and COD (chemical oxygen demand), respectively. This rapid removal of organic matters by natural sediments could be attributed to the microbe community degrading the corresponding pollutants. The temporal trend and removal efficacy for COD and TP were found to be similar between the bare tidal flat and salt marsh. Meantime, it was noteworthy that the salt marsh removed waterborne DIP much more quickly and efficiently, implying a high affinity of the halophyte on dissolved forms of organic matters. Of note, sedimentary organic sink prevailed in a defaunated condition under the lesser bioturbation effect. Physical and biological remediation techniques were compared to natural attenuation for the removal of residual oil using a 60-day mesocosm experiment with SQT. First, physical treatment of hot water + high pressure flushing maximally removed residual oils (max=93%), showing the greatest recovery among the SQT variables (mean=72%). Physical cleanup generally involved adverse effects such as depression of the microphytobenthic community during the initial period. Next, biological treatments, such as fertilizer, emulsifier, enzyme, and augmentation of the microbes, all facilitated the removal of oil (max=66%) enhancing the ecological recovery. Natural attenuation with “no treatment” showed a comparable recovery to the other remediations (max=54%). During the experimental periods, the dynamics of the benthic community were presented. Artificial remediation techniques showed a better efficacy as indicated by the SQT parameters (mean=47%). Natural restoration was also often as efficient as most active restoration alternatives and was cost-effective while minimizing the impacts on benthic communities. Contaminated sediments were transplanted into the site tidal flats to confirm the PTS specific restoration in the tidal flat and their ecotoxicological effects. A 60-day in situ mesocosm study was implemented to quantify the restoration capacity using the SQT. Contaminated sediments recovered rapidly through bio-irrigation and phytoremediation (max. recovery: 71.2%). Machine learning classified the sedimentary qualities of the natural restoration process into four groups. During the 60-day sediment recovery period the benthic community changed through four stages. The reduction of parent compounds of PTSs (high molecular weight PAHs, STs, and APEOs) progressed primarily through bio-irrigation and phytoremediation. The results show that the presence of macrofauna and macrophytes in the tidal flat can promote the degradation of parent compounds with a rapid reduction of toxicity. The influence of the dipole-driven orientation and the resulting directional configuration of the organic pollutants on the predicted reactivity to the media (seawater, sediment, and marine organisms) were investigated. Using physico-chemical properties calculated by ab initio density functional theory, directional reactivity factors (DRF) were devised as the main indicators of reactivity, linking the interaction between the organic pollutants and the media. The directional reactive model was applied to predict the variation of the aryl hydrocarbon receptor (AhR)-mediated toxic potencies among homologues of chrysene with structural modifications such as the number of constituent benzene rings, methylation, and hydroxylation. The results of this study explain why the toxic responses of the parent and metabolites of the organic pollutants were different. Moreover, the results of the predictive models were consistent with the empirical potencies determined by the use of the H4IIE-luc transactivation bioassay. An experiment-free approach based on first principles would provide an analytical framework for estimating the molecular reactivity in silico and complements conventional empirical approaches for studying molecular initiating events in adverse outcome pathways. Because the advanced DRF model was calculated for the interaction between organic pollutants and media, quantitatively calculations for the dynamical mechanism were used for applying the potential toxicity prediction model. Using molecular dynamics (MD) analysis, given the possibility of AhR-organic pollutants binding (conjugated state), it was confirmed that the directional reactive binding factor (DRBF) could be a mechanistic predictive index linking molecular ligand-receptor binding to in vitro toxicity. The DR model accurately estimated the toxic potency of a set of 16 similar PAHs, as confirmed by the H4IIE-luc bioassay. The first application of DRF to the prediction of potential toxicity implies that the physico-chemical properties of organic pollutants can be a major driving factor in the reaction with a medium, and the in silico method will provide important basic data for predicting the restoration and ecotoxicity of organic pollutants in the future. To summarize the above study results, first, the concentration of organic pollutants introduced into the intertidal zone showed a rapid decrease in the initial stage overall, and the decomposition of the parent material proceeded actively. Second, with the decrease of the concentration of the organic pollutants, the potential toxicity of the sediment also decreased rapidly, and the rate of decrease was faster than the decrease of the concentration of the organic pollutants. Third, the benthic cluster recovered exponentially, and there was a difference in each recovery rate according to the trophic level. Fourth, it was confirmed that the differences in chemical, toxicological, and ecological recovery were affected by the physicochemical properties of the organic pollutants. Finally, the DRF in this study could be developed into a DRBF predicting the potential toxicity and corresponded to the results of the in vitro bioassay. An integrated approach for understanding the restoration capacity and ecotoxicological effects of organic pollutants in this study can be useful for interpreting the chemical, toxicological, and ecological responses. In the future, to systematically manage coastal waters with severe contamination of benthic sediments, continuous development of ecological risk assessment techniques is required. In the future, to reconsider the value of ecosystem services in the intertidal zone of Korea and systematically manage coasts, the above interdisciplinary approach is continuously needed.CHAPTER. 1. Introduction 1 1.1. Backgrounds 2 1.2. Objectives 15 CHAPTER. 2. Natural Restoration Capacity of Tidal Flats for Organic Matters and Nutrients: A Mesocosm Study 18 2.1. Introduction 19 2.2. Materials and Methods 21 2.2.1. Study design and development of mesocosm system 21 2.2.2. Sampling and data analyses 22 2.3. Results and Discussion 28 2.3.1. Natural restoration under waterborne organic matters and nutrients in tidal flat 28 2.3.2. Comparison of restoration capacity between bare and vegetated sediment 31 2.3.3. Restoration capacity in Bongam tidal flat, Masan Bay 38 2.3.4. Comparison to other mesocosm studies for restoration capacity of tidal flats 40 2.4. Conclusions 44 CHAPTER. 3. Best Available Technique for the Recovery of Marine Benthic Communities in a Gravel Shore after the Oil Spill: a Mesocosm-based Sediment Triad Assessment 45 3.1. Introduction 46 3.2. Materials and Methods 48 3.2.1. Sample preparations 48 3.2.2. Mesocosm experimental setting 49 3.2.3. Instrumental analysis of residual TPH and UCM in gravel 57 3.2.4. Zebrafish (Danio rerio) embryo test 57 3.2.5. Vibrio fischeri (V. fischeri) biossay 58 3.2.6. H4llE-luciferase transactiviation bioassay 58 3.2.7. Identification of MPB individual 58 3.2.8. Bacterial metagenomic analysis 59 3.2.9. Measurements of benthic primary production 59 3.2.10. Multi-attribute utility theory (MAUT) analysis for selection of the best available technique for remediation of oil 60 3.2.11. Statistical analysis 60 3.3. Results and Discussion 62 3.3.1. Physical and biological remediations 62 3.3.2. Dynamics in bacterial communities 78 3.3.3. Effectiveness of remediation techniques 85 3.3.4. Best available remediation techniques 86 3.4. Conclusions 95 CHAPTER. 4. Determining characteristics of Sediment Quality with Machine Learning to Evaluate the Natural Restoration of Persistent Toxic Substances in Contaminated Tidal Flat Sediments 96 4.1. Introduction 97 4.2. Materials and Methods 100 4.2.1. Sample preparation and In situ mesocosm experimental setting 100 4.2.2. Persistent toxic substances analysis (PAHs, APs, and SOs) 104 4.2.3. H4llE-luciferase transactivation bioassay 105 4.2.4. Bacterial metagenomic analysis 105 4.2.5. Identification of diatom individual 106 4.2.6. Identification of meiofauna individual 106 4.2.7. Measurements of benthic primary production 106 4.2.8. Self-Organizing Map (SOM) 107 4.2.9. K-means clustering via principal component analysis (PCA) 109 4.2.10. Statistical analysis 109 4.3. Results and Discussion 110 4.3.1. Chemical, toxicological, and biological responses in natural restoration process of tidal flat 110 4.3.2. Time series clustering in evaluating the recovery of each treatment with the self-organizing map (SOM) using machine learning 118 4.3.3. Effect of macrofauna (C. sinensis and M. japonicus) and salt marsh (P. australis) on the change of benthic community using K-means clustering: PCA 121 4.3.4. Correlation between composition of PTSs and bacterial community in each treatment groups 125 4.3.5. Evaluation to natural restoration of contaminated sediments & effect of bio-irrigation and phytoremediation in tidal flat by sediment quality triad approach (SQT) 130 4.4. Conclusions 132 CHAPTER. 5. Influence of Ligand’s Directional Configuration, Chrysenes as Model Compounds, on the Binding Activity with Ahr Receptor 133 5.1. Introduction 134 5.2. Materials and Methods 138 5.2.1. Selection of Model Chemicals 138 5.2.2. Density Functional Theory Calculations 140 5.2.3. C 1s NEXAFS Spectroscopy 140 5.2.4. H4llE-luc transactivation bioassay for evaluating AhR-mediated potencies and calculation of EC50 of homologues of chrysene 141 5.2.5. In silico analysis: quantitative structure-activity relationship (QSAR) and molecular docking model 144 5.3. Results and Discussion 145 5.3.1. Directional Reactive Modeling 145 5.3.2. DRF as an indicator of ligand-binding reactivity 147 5.3.3. AhR-mediated toxic potency of chrysene homologues 158 5.3.4. Comparison of predicted potencies: DR model vs. current in silico models 162 5.4. Conclusions 167 CHAPTER. 6. Comprehension of Bio-Physical Communication for Predicting Potential Toxicity of 16 Polycyclic Aromatic Hydrocarbons 169 6.1. Introduction 170 6.2. Materials and Methods 175 6.2.1. Selection of Model Chemicals 175 6.2.2. Density Functional Theory Calculations 175 6.2.3. Molecular dynamics for calculating the probability of binding in ligand:AhR 176 6.2.4. Aryl hydrocarbon receptor homology modeling 177 6.2.5. H4llE bioassay and calculation in median effective concentration 177 6.2.6. In silico toxicity prediction analysis 178 6.3. Results and Discussion 179 6.3.1. A comprehensive driving force of ligand to receptor calculated from DR model based on first principles 179 6.3.2. Probability of 16 PAHs binding to AhR and determination of potential toxicity for 16 PAHs 188 6.3.3. Experimental and predicted potential toxicity of 16 PAHs using in vitro and in silico testing 195 6.4. Conclusions 201 CHAPTER. 7. Conclusions 202 7.1. Summary 203 7.2. Environmental implications and Limitations 208 7.3. Future Research Directions 211 BIBLIOGRAPHY 213 ABSTRACT (IN KOREAN) 236박

Biodegradation of Petroleum Hydrocarbons

Biodegradation of hydrocarbons in the environment is the natural way of cleaning the nature. The potential biodegradation of hydrocarbon contaminants by microorganisms is dependent on the environmental factors and the nutrients available. In this study culture conditions like temperature, pH, and nitrogen source were optimized by conventional one-factor at a time experimentation and the combination of other nutrients (nitrogen, phosphorus, magnesium, and sulfur) was optimized by using design of experiments (DOE) combined with grey relational analysis (GRA). Total petroleum hydrocarbons of oil sludge, light crude oil and heavy crude oil, degradation was studied for a period of thirty days using microbial strains isolated from the hydrocarbon contaminated sites. They have shown predominant results in the degradation of TPH’s under optimized culture conditions and prior addition of biosurfactants in the culture flask has enhanced the degradation process and microbial biomass yield

Characterisation of European Marine Sites: Mersey Estuary Special Protection Area. Occasional Publication of the Marine Biological Association 18

This report provides an overview of water and sediment quality within the Mersey Estuary European Marine Site (EMS) and examines evidence for their influence on bioloigcal condition. It has not been possible to determine adequately whether prevailing conditions in the Mersey impact on the interest features of the site as studies which address this issue have not been carried out. It is only possible to review the current level of knowledge regarding the biological and chemical status for the estuary, and extrapolate risks to the bird population. Often information relates to sites outside the EMS; where this is the case the authors have tried to appraise the general status of the estuary, based on best available knowledge

Habitats and Biota of the Gulf of Mexico: Before the Deepwater Horizon Oil Spill: Volume 1: Water Quality, Sediments, Sediment Contaminants, Oil and Gas Seeps, Coastal Habitats, Offshore Plankton and Benthos, and Shellfish

environmental management; marine; freshwater science

Science-based restoration monitoring of coastal habitats, Volume Two: Tools for monitoring coastal habitats

Healthy coastal habitats are not only important ecologically; they also support healthy coastal communities and improve the quality of people’s lives. Despite their many benefits and values, coastal habitats have been systematically modified, degraded, and destroyed throughout the United States and its protectorates beginning with European colonization in the 1600’s (Dahl 1990). As a result, many coastal habitats around the United States are in desperate need of restoration. The monitoring of restoration projects, the focus of this document, is necessary to ensure that restoration efforts are successful, to further the science, and to increase the efficiency of future restoration efforts

Sedimentos subaquáticos como fontes de bactérias anaeróbicas facultativas hidrocarbonoclásticas e produtoras de biossurfactantes

Doutoramento em Engenharia QuímicaActualmente são conhecidas poucas estirpes bacterianas capazes de produzir biossurfactantes (BSFs) em condições de microaerobiose ou anaerobiose. Estas bactérias têm um papel importante não só em processos naturais (ex. formação de biofilmes ou de hidratos de gás), como podem ter diversas aplicações biotecnológicas (ex. estratégias de biorremediação e aplicações industriais). As bactérias produtoras de BSFs em condições de limitação de oxigénio, com capacidade para degradar hidrocarbonetos são de particular interesse para estratégias de biorremediação de locais contaminados com hidrocarbonetos de petróleo (PHs) e na recuperação microbiana de petróleo (MEOR). Neste contexto, o objectivo deste trabalho foi o isolamento, identificação e a caracterização de bactérias anaeróbias ou anaeróbias facultativas produtoras de BSF e degradadoras de hidrocarbonetos (hidrocarbonoclásticas) na perspetiva da sua aplicação biotecnológica em condições de limitação de oxigénio. Foram escolhidos dois ambientes contaminados com PHs como potenciais fontes de bactérias hidrocarbonoclásticas produtoras de BSFs: vulcões de lama (MV) de mar profundo do Golfo de Cádis (Oceano Atlântico) e o sistema estuarino da Ria de Aveiro (Portugal). Foram preparadas culturas de enriquecimento com sedimentos subaquáticos recolhidos nestes dois habitats, como potenciais inóculos de bactérias anaeróbias facultativas. Um design experimental fatorial foi usado para testar o efeito do crude como fonte de carbono, e de nitrato e/ou sulfato, como aceitadores terminais de eletrões. De forma a melhor compreender a estrutura das comunidades bacterianas envolvidas na biodegradação de PHs nos MV do mar profundo procedeu-se à sequenciação do gene 16S rRNA das comunidades bacterianas de culturas de enriquecimento com sedimento de dois MVs, um activo e outro inactivo, e com ou sem adição de crude e/ou nitrato. Detetou-se uma diferenciação entre as comunidades dos dois MVs, independentemente dos suplementos a que as culturas foram expostas, sendo que Alphaproteobacteria e Bacilli predominaram nas culturas com sedimentos de MV activo e inactivo, respectivamente. De uma forma menos acentuada, tanto o nitrato como o crude afetaram a composição das comunidades bacterianas. Géneros de bactérias que só foram detectados nos ensaios com adição de crude (ex. Erythrobacteraceae no MV activo e Acidimicrobiale no MV inactivo) poderão ser usados como indicadores da presença de hidrocarbonetos de petróleo nestes habitats. A biodegradação de PHs nas culturas com crude foi avaliada por cromatografia gasosa acoplada a espectrometria de massa. De uma forma geral, as comunidades de culturas do MV activo foram capazes de degradar n-alcanos de tamanho inferior a C13 e compostos monoaromáticos, enquanto as comunidades do MV inactivo apresentaram a capacidade de metabolizar vários tipos de hidrocarbonetos aromáticos policíclicos. A presença de nitrato apenas afectou positivamente a biodegradação de alcanos, e não teve efeito ou foi mesmo inibitória da biodegradação de outros hidrocarbonetos. A partir de todas as culturas, com todos os tipos de sedimentos, dos MVs do Golfo de Cádis e do estuário da Ria de Aveiro, foi possível isolar-se um total de 13 isolados capazes de sobreviver exclusivamente com crude como fonte de carbono e produzir BSF em condições de aerobiose. Destas, apenas duas não foram capazes de produzir BSFs em anaerobiose. A sequenciação do gene 16S rRNA dos isolados permitiu identifica-los como pertencendo aos géneros Pseudomonas, Bacillus, Ochrobactrum, Brevundimonas, Psychrobacter, Staphylococcus, Marinobacter e Curtobacterium, a maioria dos quais não tinha ainda membros conhecidos como produtores de BSF em anaerobiose. Os resultados obtidos com este trabalho permitiram caracterizar melhor as comunidades envolvidas na degradação de PHs em MVs de mar profundo. Conseguiu-se ainda isolar e identificar estirpes, tanto de mar profundo como de ambiente estuarino, capazes de degradar PHs e produzir BSFs em condições de anaerobiose. Estas estirpes apresentam elevado potencial biotecnológico para aplicações como MEOR e biorremediação em ambientes com escassez de oxigénio.So far, only few bacterial strains are known to produce biosurfactants (BSFs) under microaerobic or anaerobic conditions. However, these bacteria are not only involved in important natural processes (e.g. biofilm and gas hydrates formations) but can also be used in several biotechnological applications (e.g. bioremediation strategies and industrial applications). Bacteria able to produce BSFs under oxygen-limiting conditions that are also able to degrade hydrocarbons, are of particular interest to bioremediation strategies of sites contaminated with petroleum hydrocarbons (PHs) and microbial enhanced oil recovery (MEOR) strategies. In this context, this work aims at isolating, identifying, and characterizing BSF-producing and hydrocarbon-degrading (hydrocarbonoclastic) bacteria grown under anaerobic conditions, which can be used in biotechnological applications under oxygen limitation. Two environments contaminated with PHs were chosen as potential sources of hydrocarbonoclastic BSF-producing bacteria: deep-sea mud volcanos from the Gulf of Cadiz (Atlantic Ocean), and the estuarine system of Ria de Aveiro (Portugal). Enrichment cultures were prepared using subaquatic sediments from both sites, as potential sources of facultative anaerobic bacteria. A factorial experimental design was used to test the effect of crude oil as carbon source, and nitrate and/or sulfate, as terminal electron acceptors. Aiming at better understanding the structure of bacterial communities involved in PHs biodegradation at deep-sea MVs, sequencing of the 16S rRNA gene was performed for bacterial communities from cultures containing sediments from two MVs, active and inactive, and with or without crude oil and/or nitrate. A distinction between the communities of MVs with different activity, independent of the supplements was observed. Alphaproteobacteria and Bacilli were the predominant classes found in enrichment cultures inoculated with active and inactive MVs sediments, respectively. In a minor scale, nitrate and crude oil additions also affected the composition of bacterial communities. Therefore, genera that only appeared in cultures with crude oil. (e.g. Erythrobacteraceae in active MV cultures and Acidimicrobiale in inactive MV cultures) can be used as biosensors of the presence of PHs in these habitats. Biodegradation of PHs in cultures containing crude oil was assessed by gas chromatography coupled with mass spectrometry. Overall, communities from active MV cultures were able to degrade n-alkanes below C13 and monoaromatic hydrocarbons, while communities from inactive MV cultures presented the ability to metabolize several types of polycyclic aromatic hydrocarbons. The presence of nitrate only had a positive effect on the biodegradation of alkanes, and had no effect or even an inhibitory effect on the biodegradation of other hydrocarbons. A total of 13 isolates able to survive on crude as carbon source and produce BSF under aerobic conditions were obtained from all cultures either from sediments of the Gulf of Cadiz MVs or the estuarine system of Ria de Aveiro. Only two isolates failed to produce BSF under anaerobiosis. Sequencing of 16S rRNA gene was used to establish the identification of isolates as Pseudomonas, Bacillus, Ochrobactrum, Brevundimonas, Psychrobacter, Staphylococcus, Marinobacter and Curtobacterium. Most of these genera had never been described as able to produce BSFs under anaerobic conditions. The results obtained in this work allowed to better characterize the deep-sea communities involved in PHs degradation, as well as, to identify strains from deep-sea and estuarine sediments able to degrade PHs and produce BSFs under anaerobic conditions. These bacteria present high biotechnological potential for applications in oxygen-limiting environments, such as, MEOR and bioremediation of environments contaminated with PHs

Ecological risk assessment of the Miri coast, Sarawak, Borneo: A biogeochemical approach

Ecological risk assessment was made along the Miri coast based on trace element concentrations (Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb, Rb, Zn) in the seawater, sediments, and aquatic biota (fish, shrimp, crabs, and bivalves). Prevailing major geochemical processes were identified. Contamination and risk assessment indices were estimated. Sediments were contaminated by Cu and Zn, but in the marine life the remaining metals were within the permissible limits set by international and national guidelines

Habitats and Biota of the Gulf of Mexico: Before the Deepwater Horizon Oil Spill: Volume 1: Water Quality, Sediments, Sediment Contaminants, Oil and Gas Seeps, Coastal Habitats, Offshore Plankton and Benthos, and Shellfish

environmental management; marine; freshwater science

Microbial Colonisation and Degradation of Plastic Pollution in the Marine Environment

Plastic pollution is ubiquitous in the world’s oceans and is predicted to increase by an order of magnitude by 2025. The environmental impacts of plastic pollution are well documented in terms of its effects on marine animals however, the impact plastic has on the natural physicochemical characteristics of seawater and the microbial composition of the world’s oceans remains largely unknown. In particular, the role that microbial communities play in the early stages of colonisation and decomposition requires more attention. In response to this knowledge gap, both laboratory-based and field work experiments were undertaken in order to gain further insights. A microcosm experiment using natural seawater amended with range of plastics, both synthetic and biodegradable, was carried out to evaluate the impact of these plastics on the marine microbial communities. To complement this, a one-year field exposure trial was undertaken to measure biofilm formation and growth, as well as changes in polymer characteristics resulting from degradation. Finally, a biodegradation study was undertaken to identify bacteria within seawater that may degrade plastic. The characterisation of marine bacterial communities revealed major shifts in composition when plastics were introduced into seawater. These microbiological shifts were due to changes in the concentration of dissolved organic carbon, pH and total nitrogen due to the degradation of plastic materials. Furthermore, the long-term field work exposure experiment showed that different plastic substrates selected for distinct colonising communities on their surfaces. The analysis of the biofilm communities revealed the presence of bacteria that could possibly be involved in the degradation of plastics. However, no significant degradation was measured for the in situ tested plastics, suggesting that biofilm formation may have limited degradation. An exploration of plastic degradation using distinct bacterial consortia isolated from seawater resulted in biodegradation (mineralisation) figures of 16, 9 and 7 % for polyvinyl chloride, high-density polyethylene and polyethylene terephthalate respectively. In summary, results from this thesis suggest that the presence of plastic in seawater affects the microbial community by changing the inherent chemical and biological properties of the water. In addition, plastics were shown to select for distinct colonising communities, which did not contribute to the plastic degradation. However, enriched marine bacterial communities demonstrated biodegradation ability that could be explored further in the future to gain additional insights into underlying biodegradation mechanisms