The Fate of Microbial Life Encased in Sea Ice: Tracking Organisms and Survival Strategies Through the Arctic Winter

每年秋天，随着大气温度下降和海冰的形成，北极高地的沿海开放水域开始再次关闭。随着冬季的推移，被困在冰层流体包裹体中的生物会被囚禁在越来越严酷的温度和盐分浓度(通常为0.20摄氏度和~20%的盐分)中。少数几项对冬季海冰的微生物研究表明，可以很容易地从中培养出适应寒冷(嗜冷)的异养细菌，代谢活性细胞与盐水中的颗粒物有关，胞外多聚体物质(EPS)可以作为低温保护剂和嵌入细菌的稳定基质。然而，在北极高地漫长而黑暗的冬季，特定微生物、种群或群落的命运是未知的；它们在极端条件下获得足够营养的战略只是猜测的主题。该项目建议在一个既定的跨学科研究地点，在冬季期间，在冰盖内以固定的间隔和垂直剖面跟踪微生物演替。荧光探针将用于在等温等盐溶液中融化的冰片的定量、原位杂交分析。将通过基于16S rRNA的环境克隆进一步评估选定样本的多样性。古生界和Cytophaga-黄杆菌-杆菌科将被作为最终成员种群(预计分别是低种群和高种群)和嗜冷属Colwell lia的成员作为可培养居民的代表。后者的选择也是因为完成了冷红假单胞菌34H株的全基因组序列，这是第一个被测序的嗜冷有机体。现有的知识导致了这样的假设，即盐水包裹体中的胞外蛋白水解酶活性是由EPS稳定的，从而能够向异养细菌供应富含氮的有机化合物作为越冬的燃料。这一概念和相关想法将通过在冰盖中进行现场培养来检验，使用34H菌株及其胞外蛋白酶和胞外聚合物的制剂作为海冰卤水的改良剂。研究平台将是加拿大的约翰·富兰克林号破冰船，它刚刚为冬季的科学行动进行了翻新。富兰克林号将在2003.2004冬季期间被冻结在富兰克林湾(加拿大群岛西北航道的西部入口处)，这是国际加拿大北极大陆架交换研究(CASES)在该地区长达一年的非凡探险的一部分。鉴于CASES平台，该项目的成果将在一个强有力的跨学科框架内进行解释。更好地了解冬季微生物的演替和与冰中有机化合物的联系将有助于澄清春季从冰到下面海洋的碳通量条件，这可能对区域和全球都具有重要意义。科尔韦利亚的环境数据将为未来复杂的基于基因组的研究奠定基础。冬季存活假说的现场测试结果将为生物医学、水产养殖和食品工业在冰冻条件下使用生物、酶和其他敏感有机化合物提供适用于社会关注的更实际问题的基本知识。案例越冬探险为所有参与者提供了一个独特的教育和培训机会。来自许多国家、当地因纽特人村庄和K-12教室的参与者将登上富兰克林号，进行实地、实验室、跨学科和跨文化的联合研究和培训。结果将在同行评议的文献中发表，纳入教学工作，并在旨在覆盖更广泛受众的场所传播，包括从海上发布新闻和信息，以及实地和船上经验的视频记录，供公众观看。

Abstract of Deming ProposalEvery fall, open coastal waters of the high Arctic begin to close again as atmospheric temperature drops and sea ice forms. Organisms trapped in fluid inclusions of the ice are then held captive to increasingly severe temperature and salt concentration (typically to .20degrees C and ~20% salt) as winter progresses. The few microbial studies of winter sea ice have shown that cold-adapted (psychrophilic) heterotrophic bacteria are readily cultured from it, that metabolically active cells are associated with particulate matter in the brines, and that exopolymeric substances (EPS) may serve as cryoprotectants and a stable matrix for embedded bacteria. The fate of specific microorganisms, populations or communities during the long dark winter of the high Arctic, however, is unknown; their strategies for acquiring adequate nutrition under extreme conditions, the subject only of speculation. This project proposes to track microbial succession, at regular intervals and in vertical profile within the ice sheet, through the winter season at an established, interdisciplinary study site. Fluorescent probes will be used in quantitative, in situ hybridization analyses of ice sections, melted in isothermal-isohaline solutions. Diversity in selected samples will be further evaluated by 16S rRNA-based environmental cloning. The domain Archaea and division Cytophaga-Flavobacteria-Bacteriodes will be probed as endmember populations (expected to be low and high, respectively) and members of the psychrophilic genus, Colwellia, as representatives of culturable inhabitants. The latter are also selected due to completion of the whole-genome sequence of C. psychrerythraea strain 34H, the first psychrophilic organism to be sequenced. Available knowledge leads to the hypothesis that extracellular proteolytic enzyme activity in brine inclusions is stabilized by EPS, enabling a supply of nitrogen-rich organic compounds to heterotrophic bacteria as fuel for the winter. This concept and related ideas will be tested via in situ incubations in the ice sheet, using strain 34H and preparations of its extracellular proteases and exopolymers as amendments to sea-ice brines. The research platform will be the Canadian icebreaker, John Franklin, newly renovated for science operations in winter. The Franklin will be frozen into Franklin Bay (at the western entrance of the Northwest Passage, Canadian Archipelago) during winter 2003.2004 as part of a remarkable yearlong expedition in the region for the international Canadian Arctic Shelf Exchange Study (CASES). Results from this project will be interpreted within a strong interdisciplinary framework, given the CASES platform. Better understanding of microbial succession during winter and links to organic compounds in the ice will clarify spring conditions for carbon flux, from ice to ocean below, which can be significant regionally and globally. Environmental data on Colwellia will set the stage for sophisticated genome-based research in future. Results of in situ tests of winter survival hypotheses will provide basic knowledge applicable to more practical issues of societal interest concerning use of organisms, enzymes and other sensitive organic compounds under freezing conditions in the biomedical, aquaculture and food industries. The CASES overwintering expedition provides a unique educational and training opportunity for all involved. Participants from many countries, local Inuit villages and K-12 classrooms will be aboard the Franklin for joint field, laboratory, interdisciplinary and cross-cultural research and training. Results will be published in the peer-reviewed literature, incorporated into teaching efforts, and disseminated in venues designed to reach much broader audiences, including news and information releases from sea and video documentation of field and shipboard experiences for public viewing.