إنشاء الميكروبية إثراء الثقافات حقيقية النواة من بحيرة القطب الجنوبي الطبقية كيميائيا وتقييم القدرة على تثبيت الكربون
Summary
حقيقيات النوى الجراثيم وكلاهما مصدر للكربون photosynthetically المشتقة وأعلى الأنواع المفترسة في بحيرات القطب الجنوبي بشكل دائم المغطاة بالجليد. ويصف هذا التقرير نهجا ثقافة تخصيب اليورانيوم لعزل عملية الأيض حقيقيات النوى الجراثيم تنوعا من القطب الجنوبي للبحيرة، بحيرة بوني، وغير العضوية ويقيم إمكانية تثبيت الكربون باستخدام فحص النظائر المشعة للنشاط ريبولوز-1 كربوكسيلاز ،5-bisphophate (RubisCO) أوكسيجيناز.
Abstract
Lake Bonney is one of numerous permanently ice-covered lakes located in the McMurdo Dry Valleys, Antarctica. The perennial ice cover maintains a chemically stratified water column and unlike other inland bodies of water, largely prevents external input of carbon and nutrients from streams. Biota are exposed to numerous environmental stresses, including year-round severe nutrient deficiency, low temperatures, extreme shade, hypersalinity, and 24-hour darkness during the winter 1. These extreme environmental conditions limit the biota in Lake Bonney almost exclusively to microorganisms 2.
Single-celled microbial eukaryotes (called “protists”) are important players in global biogeochemical cycling 3 and play important ecological roles in the cycling of carbon in the dry valley lakes, occupying both primary and tertiary roles in the aquatic food web. In the dry valley aquatic food web, protists that fix inorganic carbon (autotrophy) are the major producers of organic carbon for organotrophic organisms 4, 2. Phagotrophic or heterotrophic protists capable of ingesting bacteria and smaller protists act as the top predators in the food web 5. Last, an unknown proportion of the protist population is capable of combined mixotrophic metabolism 6, 7. Mixotrophy in protists involves the ability to combine photosynthetic capability with phagotrophic ingestion of prey microorganisms. This form of mixotrophy differs from mixotrophic metabolism in bacterial species, which generally involves uptake dissolved carbon molecules. There are currently very few protist isolates from permanently ice-capped polar lakes, and studies of protist diversity and ecology in this extreme environment have been limited 8, 4, 9, 10, 5. A better understanding of protist metabolic versatility in the simple dry valley lake food web will aid in the development of models for the role of protists in the global carbon cycle.
We employed an enrichment culture approach to isolate potentially phototrophic and mixotrophic protists from Lake Bonney. Sampling depths in the water column were chosen based on the location of primary production maxima and protist phylogenetic diversity 4, 11, as well as variability in major abiotic factors affecting protist trophic modes: shallow sampling depths are limited for major nutrients, while deeper sampling depths are limited by light availability. In addition, lake water samples were supplemented with multiple types of growth media to promote the growth of a variety of phototrophic organisms.
RubisCO catalyzes the rate limiting step in the Calvin Benson Bassham (CBB) cycle, the major pathway by which autotrophic organisms fix inorganic carbon and provide organic carbon for higher trophic levels in aquatic and terrestrial food webs 12. In this study, we applied a radioisotope assay modified for filtered samples 13 to monitor maximum carboxylase activity as a proxy for carbon fixation potential and metabolic versatility in the Lake Bonney enrichment cultures.
Protocol
Discussion
وأفادت الدراسات الجزيئية الأخيرة من درجة عالية من التنوع وحيدة الخلية حقيقيات النوى عبر مجموعة من البيئات 3، 19، 20، ولكن نظرا لعدم وجود عزلة عبر طائفة كاملة من الموائل أولاني الأدوار الوظيفية لهذه الأنواع الفردية في الشبكات الغذائية هي إلى حد كبير غير معروف. في …
Disclosures
The authors have nothing to disclose.
Acknowledgements
المؤلفان بالشكر J. Priscu، Chiuchiolo ألف وفريق ماك موردو المياه العذبة LTER للحصول على المساعدة في جمع وحفظ العينات في القارة القطبية الجنوبية. نشكر Ratheon خدمات القطبية ومروحيات PHI لتقديم الدعم اللوجستي. وقد تم توليد الميكروسكوب ضوء في مركز ميامي للفحص المجهري المتقدم ومركز التصوير. وأيد هذا العمل من قبل مكتب جبهة الخلاص الوطني من برامج المنح القطبية 0631659 و 1056396.
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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