Biodegradation of pharmaceuticals using microalgae: Toxicity, metabolism and removal approaches

Abstract

수많은 의약품 오염 물질 (Pharmaceutical Contaminants, 이하 PCs)로 인한 전세계 수질 오염의 증가는 치명적인 생태 독성 및 관련 건강 문제로 인해 심각한 환경 문제가 되고 있다. 환경에서 인정되는 대부분의 PCs 의 농도는 ng/L ~ μg/L 수준이지만, 이것은 미생물 군집을 변화시키고, 미생물의 성장을 억제하며, 토양 내 미생물의 활성을 감소시키는 것과 같이 표적 생물 및 비표적 생물에 부정적인 생태학적 영향을 유발할 수 있다. 또한 오염 물질들의 발암성 때문에 인체에도 역시 심각한 건강 상 악영향을 초래한다. 현재 폐수 처리장의 평균 PCs 제거 효율은 일반적으로 10% 이하인데, 이는 폐수 처리장이 미량의 PCs 를 제거하기 위한 목적으로 설계되지 않았기 때문이다. 미세조류를 이용한 PCs 의 생물학적 정화 기술은 태양에너지를 기반으로 한다는 점과 조절 인자가 적어 운영이 수월하다는 점, 친환경적인 기술, 탄소 고정 및 전환의 역할을 한다는 장점들을 가지고 있으며, 또한 영양제와 화장품, 양식업을 위한 저가의 식품, 바이오 연료 및 동물 사료 생산을 위한 미세 조류 바이오매스와 같은 고부가가치의 제품을 동시에 생산할 수 있다는 점에서 최근 과학적 관심이 높아지고 있다. 본 연구에서는 미세조류를 이용하고 수중 매체에서 PCs 의 독성 평가 및 제거를 분석했다. 본 연구를 통해 미세조류를 이용한 PCs 의 제거효율을 향상시키기 위해 미생물 군집(microbial consortia), 환경 순응(acclimation) 및 공동 신진 대사(cometabolism) 를 포함하는 몇 가지 새로운 접근법을 요약하여 기술의 응용 가능성을 발전시킬 것이다.; The security and scarcity of clean water for the safe livelihood of human beings have drawn concern worldwide due to the contamination of water resources by various micropollutants, including pharmaceutical contaminants. These synthetic chemicals can be transported through the atmosphere and water and, in many cases, find their way into sediments and soils. Moreover, multidrug resistant efflux pumps are present in all organisms and can exist in large numbers within a single microorganism such as Staphylococcus aureus, Pseudomonas aeruginosa, and Acinetobacter baumannii. Low concentrations of PCs can be beneficial for bacteria by triggering specific transcriptional changes that are independent of the bacterial stress response pathways; this is especially pertinent for antibiotics, to which bacteria can develop resistance. PCs can accumulate in different trophic level organisms including human beings through biomagnification in food chains due to their hydrophobic and persistent nature. Conventional activated sludge processes in current wastewater treatment plants are not designed for the efficient removal of PCs, although several advanced treatment technologies are available for effectively removing PCs. The low removal efficiencies and/or limitations of these technologies motivated researchers to investigate better treatment strategies for the removal of these trace PCs. Considering its implications for human health and ecosystem processes, a comprehensive toxicity and environmental risk evaluation of these PCs and efficient and costeffective treatment technologies for their removal from the aquatic phase are essential. Microalgae-based biotechnologies is of growing scientific interest due to its advantages such as solar energy driven, relatively small amounts of operational inputs, ecofriendly, role in the fixation and turnover of carbon, and simultaneous production of high-value products such as nutraceuticals and cosmetics, low value food products for aquaculture, and microalgal biomass to produce biofuel and animal feed. Mixotrophic microalgae can switch their metabolism between autotrophic and heterotrophic depending on the availability of carbon sources and nutrients in the surrounding environment, which provides them with a great flexibility to survive and thrive in extreme environments. Such capability of microalgae can overcome some of the major limitations associated with bacteria and fungi that require carbon and other nutrients in stoichiometric balance for growth and degradation of pollutants. Such adaptability of mixotrophic microalgae makes them promising candidates for removal of contaminants in wastewater. Culturing of microalgae in wastewater substantially reduces the need for chemical fertilizers/nutrients and their related burden on the life cycle. A ‘zero-waste’ concept can be implemented through the utilization of wastewater as nutrient source for the cultivation of mixotrophic microalgae (wastewater treatment through microalgal remediation) followed by the subsequent utilization of produced biomass as a feasible feedstock for sustainable biofuel production to stimulate a more sustainable practice for the microalgae biomass based biofuel industry. Additionally, the growth inhibition test using different microganisms such as microalgae and cyanobacteria has been recommended as reliable method to predict/evaluate the ecotoxicity of pollutants by the Organization for Economic Co-operation and Development. In this research work, toxicity of several frequently found PCs (carbamazepine, sulfamethazine, sulfamethoxazole, levofloxacin, ciprofloxacin), and their biodgerdation, metabolic fate, and enhancement approaches for a better removal of PCs using microalgae were investigated. Toxicity of the PCs were studied according to their effects on microalgae growth, halfmaximum effective concentrations (EC50), microalgal biochemical characteristics (total chlorophyll, carotenoid, lipid eroxidation, antioxidant enzyme activities, fatty acid methyl esters, carbohydrate), elemental analysis, and Fourier-transform infrared spectroscope analysis. The results have demonstrated the growth of microalgae will negligibly influenced at relatively low concentrations of PCs and will be significantly decreased at higher concentrations of the investigated PCs. The calculated 96-h EC50 of carbamazepine (CBZ), sulfamethazine (SMZ), sulfamethoxazole (SMX), levofloxacin (LEV), and ciprofloxacin (CIP) using microalgae was 797 and 149 mg CBZ L-1 for Chlamydomonas mexicana and Scenedesmus obliquus, 1.23 mg SMZ L-1 and 0.12 mg SMX L-1 for Scenedesmus obliquus, 58.6 mg LEV L-1 for Chlorella vulgaris, and 65 mg CIP L-1 for C. mexicana, which indicated that SMX was the most toxic contaminant for microalgae in current study. Total chlorophyll and carotenoid contents significantly changed with exposure to higher concentrations of PCs. The total chlorophyll content of C. mexicana significantly increased (p < 0.05) by 6%, 12%, and 19% at 10, 25, and 50 mg CBZ L-1, respectively, as compared to the total chlorophyll content in the control (73.5 mg g-1) at day 10, whereas, the total chlorophyll content of S. obliquus was significantly decreased (p < 0.01) by 9% and 12% at 100 and 200 mg CBZ L-1, respectively, The increased chlorophyll content in cells can serve as a protective mechanism to scavenge the accumulated reactive oxygen species in chloroplast. A reduction in photosynthetic pigments is also a common stress response in plants and microalgae that can be caused by the peroxidation of thylakoids lipid and the degradation of PSII complexes. The malondialdehyde (MDA, lipid peroxidation) content of C. mexicana was significantly (p < 0.01) increased by 152 and 295% at 60 and 100 mg CIP L-1, respectively, at day 11 compared to the control. In aquatic environments, microalgae are easily exposed to emerging contaminants such as detergents, aromatics, pharmaceuticals, pesticides and herbicides, which can disrupt the cellular metabolisms to induce the overproduction of reactive oxygen species (ROS). ROS in cells have strong oxidation potentials that can cause lethal damages to cell organelles. Photosynthetic organisms can scanvage the accumulated ROS by antioxidant enzymes such as, superoxide dismutase (SOD) and catalase (CAT) to decrease the ROS-induced cellular toxicity. These enzymes are known to provide the first line of defense against ROS toxicity and are commonly used as biomarker indicators. The SOD activity in C. mexicana was increased by 1.3-fold at low CBZ concentrations (50 mg L-1) to neutralize the free radicals. At higher concentrations (200 mg L-1), the SOD activity was decreased compared to the control, indicating the failure of microalgal cells to protect themselves from ROS toxicity. The activity of SOD in S. obliquus was significantly decreased at higher CBZ concentration compared to the control. The CAT activity in S. obliquus was significantly increased (p < 0.01) at increased CBZ concentrations. The percent compositions of the saturated (C14:0, C16:0, C18:0 and C20:0), monounsaturated (C16:1 and C18:1), and polyunsaturated (C18:2 and C18:3) fatty acids in S. obliquus ranged from 48.8-42.0%, 11.5-17.9%, and 39.8-40.1%, respectively, with increasing concentrations of SMZ and SMX (0-0.5 mg L-1). These results indicated that the percentage of fatty acids made up of saturated FAMEs decreased from 48 to 42% with increasing SMZ and SMX mixture concentrations, while that made up by unsaturated FAMEs conversely increased from 50 to 58%. The FT-IR spectra of S. obliquus cells obtained showed absorption bands over the wavenumber range of 3600-1000 cm-1 resulted from the adsorption peaks of various functional groups (O-H, C-H, C=O, C-O, N-H). Elemental analyses showed an improved percentage of nitrogen and sulfur in the microalgal biomass of S. obliquus as SMZ and SMX concentrations increased. Microalgae can serve as a potential sink for the removal of PCs due to their endogenous catabolic systems, heterotrophic capability and role in the fixation and turnover of carbon. After 10 days, C. mexicana and S. obliquus showed 37 and 30% total removal of CBZ, respectively, when the initial CBZ concentration was 1 mg L-1. C. mexicana was able to remove 13% of CIP (2 mg L-1). C. vulgaris removed 12% of 1 mg LEV L-1. S. obliquus removed 31.4-62.3% of the 0.025-0.25 mg SMZ L-1 and 27.7-46.8% of the 0.025-0.25 mg SMX L-1 in the mixture. Biodegradation, bioadsorption and bioaccumulation are three processes which are responsible for the removal of a contaminant via biological systems. The mass balance analysis of the current study demonstrated that biodegradation is the most effective way by which microalgae eliminate the organic contaminant from the aqueous phase. Most of the removals of PCs by microalgae were relatively low, current study has developed several enhancement approaches including acclimation of microalgal species and cometabolism to increase the remediation efficiency. For example, the acclimated C. vulgaris, which was pre-exposed to 200 mg L-1 of LEV for 11 days, exhibited enhanced removal of 1 mg LEV L-1 by 16% after 11 days of cultivation. C. mexicana showed 32, 32, 38 and 56% removal of CIP (2 mg L-1) at 0.5, 1, 2 and 4 g L-1 sodium acetate, respectively, comparing to the control (without organic substrate) after 11 days of cultivation. Different types of enzymatic reactions of the PCs by microalgae, such as hydroxylation, carboxylation, oxidation, hydrogenation, glycosylation, demethylation, ring cleavage, decarboxylation, dehydroxylation, and bromination, have been proposed in current study. This study supplied a good understanding of environmental toxicity of various PCs on microalgae, their removal and biodegradation mechanism, metabolic fate and enhancement approaches. The overall evaluation of microalgal bioremediation suggests that microalgae can be a potential candidate for the removal of PCs which offers a trustworthy outlook.