建立真核微生物的富集培养从化学分层南极湖和固碳潜力评估
Summary
微生物的真核生物都在永久性冰雪覆盖的南极湖泊源的光合衍生的碳和顶级掠食性物种。本报告介绍了富集培养的方法分离代谢多功能微生物真核生物，从南极湖，湖波尼，使用核酮糖1,5 – 二磷酸羧化酶加氧酶（Rubisco活化酶）活性放射性同位素检测和评估无机碳固定潜力。
Abstract
波尼湖是众多永久位于麦克默多干谷，南极洲冰层覆盖的湖泊之一。常年冰盖保持化学分层的水柱，不同于其他内陆水体，主要是防止外部输入从河流的碳和营养。生物群暴露在众多的环境压力，包括全年的养分严重缺乏，气温低，极端的阴凉处，矿化，24小时黑暗，在冬季1。这些极端的环境条件限制在波尼湖生物群几乎完全微生物2。
单细胞微生物的真核生物（称为“原生生物”）是在全球生物地球化学循环的重要球员，在干热河谷湖泊的碳循环中发挥重要的生态作用，占小学和三级水生食物网中的角色。在干热河谷水产食品网站，修正我的原生生物organotrophic生物体的有机碳4，2 norganic碳（自养）的主要生产者。 phagotrophic或能异养细菌和较小的原生生物摄入原生生物作为食物网中的顶级掠食者。最后，未知的原生动物人口的比例是能够结合的混合营养代谢6，7。在原生生物mixotrophy涉及的能力结合猎物微生物phagotrophic摄入的光合能力。这种形式的mixotrophy不同于细菌混合营养代谢，一般涉及吸收溶解的碳分子。目前有极少数原生动物分离永久冰覆盖的极地湖泊，在这种极端环境中的原生生物多样性和生态的研究已经有限，8，4，9，10，5。的原生生物代谢的多功能性，将有助于更好地理解简单的干热河谷湖的食物网中的发展模式，为rOLE在全球碳循环中的原生生物。
我们采用隔离波尼湖潜在的光合和混合营养的原生生物富集培养方法。水柱采样深度被选为基于初级生产最大值和原生动物进化多样性4，第11的位置，以及影响原生动物的营养模式的主要非生物因素的变化：浅层取样深度为主要营养成分的限制，而更深层次的采样深度是有限的可用光。此外，湖水样品，辅以多种类型的生长介质，促进多种光合生物体的生长。
Rubisco活化酶催化的速率限制在CBB）卡尔文森Bassham（周期，主要途径，其中自养有机体修复无机碳和有机碳含量较高的营养水平，在水生和陆生食物链12步。在这项研究中，Wé应用放射性同位素法修改过滤样品13监察固碳潜力，并在湖的波尼富集文化的代谢多功能的代理最大的羧化酶活性。
Protocol
1。样品采集选择和准备采样点有一天前采样水柱。这将使水柱分层层扰动后，由于钻井和冰孔熔化改革。识别GPS演练现场的位置。 要访问的水柱，首先通过一个瞬间冰到了4英寸的jiffy的飞行延伸和钻头的螺旋冰钻了一个洞。为了防止冻结孔钻，尽量避免停止钻探约4-6英寸以上水柱顶部钻成液态水。 钻孔将需要从15至60厘米，直径扩大采样前。这是通过使用一个洞熔炉（Hotsy?…
Discussion
最近的分子研究报告通过一系列环境3，19，20高的单细胞真核生物的多样性;然而，由于缺乏整个完整的原生生物栖息地范围的分离，这些个别物种食物链的职能作用在很大程度上是未知之数。在这项研究中，我们所描述的方法，以丰富的真核生物的参展环境，从一个相对欠永久冰雪覆盖的南极湖泊的代谢多功能的微生物物种。从不同采样深度湖波尼丰富的文化表现差的固碳率，生长?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者感谢J. Priscu，A. Chiuchiolo和麦克默多LTER网络湖沼队在南极样品的收集和保全的协助。我们感谢Ratheon极地后勤支援服务和PHI的直升机。光显微镜在迈阿密的先进的显微镜和影像中心为中心的产生。这项工作是由国家科学基金会极地项目办公室资助0631659和1056396的支持。
Materials
| Name of the reagent | Company | Catalogue number | Comments |
| BBM | Sigma | B5282 | |
| BG11 | Sigma | C3061 | |
| F/2 | Sigma | G9903 | |
| GF/F filter, 25 mm | Fisher Scientific | 09-874-64 | |
| GF/F filter, 47 mm | Fisher Scientific | 09-874-71 | |
| Polyethersulfone filter, 0.45 μm pore, 47 mm | Pall Life Sciences | 61854 | |
| Sterile cell culture flask, 25 cm2 | Corning | 430639 | |
| Diurnal growth chamber | VWR | 35960-076 | |
| Zirconia/silica beads, 0.1 mm diamter | BioSpec Products | 11079101z | |
| Mini-Bead beater | BioSpec Products | 3110BX | |
| Screw-cap microcentrifuge tube (1.5 μL) | USA Scientific | 1415-8700 | |
| NaH14CO3 | ViTrax | VC 194 | Keep in aliquots of 400 μL at -20°C |
| RuBP | Sigma | R0878-100mg | Dissolve in 10 mM Tris-propionic acid (pH 6.5) |
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Tags
Microbial EukaryotesAntarctic LakeChemically StratifiedCarbon Fixation PotentialLake BonneyIce-covered LakesMcMurdo Dry ValleysEnvironmental StressesNutrient DeficiencyLow TemperaturesExtreme ShadeHypersalinityDarknessMicroorganismsProtistsBiogeochemical CyclingCarbon CyclingAutotrophyPrimary ProducersTertiary RolesFood WebPhagotrophic ProtistsHeterotrophic ProtistsMixotrophic Metabolism
Cite This Article
Dolhi, J. M., Ketchum, N., Morgan-Kiss, R. M. Establishment of Microbial Eukaryotic Enrichment Cultures from a Chemically Stratified Antarctic Lake and Assessment of Carbon Fixation Potential. J. Vis. Exp. (62), e3992, doi:10.3791/3992 (2012).