Establishment of Microbial Eukaryotic Enrichment Cultures from a Chemically Stratified Antarctic Lake and Assessment of Carbon Fixation Potential

Summary

Microbial eukaryotes are both a source of photosynthetically-derived carbon and top predatory species in permanently ice-covered Antarctic lakes. This report describes an enrichment culture approach to isolate metabolically versatile microbial eukaryotes from the Antarctic lake, Lake Bonney, and assesses inorganic carbon fixation potential using a radioisotope assay for Ribulose-1,5-bisphophate carboxylase oxygenase (RubisCO) activity.

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

1. Sample Acquisition

1. Choose and prepare the sampling site one day prior to sampling the water column. This will allow the stratified layers of the water column to reform after disturbance due to drilling and ice-hole melting. Identify location of drill site by GPS.
2. To access the water column, begin by drilling a hole through the ice with a Jiffy ice auger attached to a 4-inch Jiffy flight extension and cutting bit. To prevent freezing of the drill in the hole, try to avoid drilling into the liquid water by halting drilling approximately 4-6 inches above the top of the water column.
3. The drill hole will need to be widened from a diameter....

Discussion

Recent molecular studies have reported high diversity of single-celled eukaryotes across a range of environments ^{3, 19, 20}; however, due to a lack of isolates across the full range of protist habitats the functional roles of these individual species in food webs are largely unknown. In this study, we have described methodologies to enrich for microbial eukaryote species exhibiting metabolic versatility from a relatively undersampled environment, a permanently ice-covered Antarctic lake. Cultures enriched from .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
BBM	Sigma-Aldrich	B5282
BG11	Sigma-Aldrich	C3061
F/2	Sigma-Aldrich	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 Corporation	61854
Sterile cell culture flask, 25 cm2	Corning	430639
Diurnal growth chamber	VWR international	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, Inc.	1415-8700
NaH14CO3	ViTrax	VC 194	Keep in aliquots of 400 μL at -20°C
RuBP	Sigma-Aldrich	R0878-100mg	Dissolve in 10 mM Tris-propionic acid (pH 6.5)