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.
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.
1. Sample Acquisition
2. Development of Enrichment Cultures
3. Cell Lysate Extraction from Filtered Enrichments
4. RubisCO Carboxylase Activity Filter Assay
5. Representative Results
We utilized enrichment culturing to isolate cold-adapted phototrophic and mixotrophic protists residing in the Antarctic Lake Bonney. To capture a greater diversity of organisms, we tested three growth media types: Bold’s Basal Medium (BBM) 15, F/2-Si Marine Medium 16, 17 and BG11 Medium 18. Visual inspection of the enrichment cultures by light microscopy revealed that the cultures were dominated by phototrophic protists (indicated by the presence of chlorophyll pigment in most cells) and harbored a variety of cell morphologies depending on the sampling depth from which the inoculum was taken and the type of media used in the culture (Fig. 1).
We measured maximum rates of carboxylase activity catalyzed by the enzyme RubisCO as a proxy for carbon fixation potential. Chl a abundance was also monitored as an estimate of phototrophic protist biomass (Fig. 2). Despite cultivation of all cultures under the same temperature/light regime (ie. 4 °C/20 μmol m-2 s-1), carbon fixation potential and phototrophic biomass varied dramatically between the enrichment cultures. Maximum RubisCO activity was observed in enrichment cultures growing in either BBM (Enrichments 4 and 6) or BG11 (Enrichments 13 and 14) growth media, while cultures enriched on F/2 growth medium (Enrichments 19, 21, 23) exhibited a 4- to 34-fold lower maximum carboxylase activities. These differences were not due to lower biomass levels in the F/2 cultures, as carboxylase activity was expressed on a Chl a basis. Moreover, RubisCO activity did not correlate with chl a concentration (r = -0.023, p>0.1; Fig. 2, inset). The BBM cultures inoculated with lake water from 6 m and 15 m (Enrichments 4 and 5) had the highest chl a levels, while all other cultures exhibited relatively low chl a, regardless of RubisCO enzyme activity (Fig. 2).
Figure 1. Representative microbial eukaryote enrichment cultures which have selected for the growth of different organisms. Identity of the culture is given in the lower left of the panels. All images represent light micrographs generated using oil immersion at 1000X magnification.
Figure 2. Potential carbon fixation capacity and extractable chlorophyll a levels in Antarctic microbial eukaryote enrichment cultures isolated from Lake Bonney and grown on various culture media. See Table 1 for culture details. Inset: Pearson correlation between chlorophyll a abundance and carbon fixation potential in Antarctic microbial eukaryote enrichment cultures.
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 different sampling depths in Lake Bonney exhibited differential carbon fixation rates that were growth medium-dependent. For example, low carbon fixation potential in cultures enriched in F/2 versus BBM or BG 11 growth media likely reflects differences in protist diversity and metabolic capability. BBM growth medium selects for green algal species (or chlorophytes) and our cultures appear to be a monoculture of a green algal species (Fig. 1A, B). These organisms are known to largely depend on photoautotrophy. One isolate from Lake Bonney exhibits pure photoautotrophic metabolism, having a strict requirement for light as its sole energy source 1. In contrast, F/2 growth medium is designed to enrich for a broad range of marine protists, including potentially mixotrophic organisms 12. While we did not fully explore the diversity of the F/2 cultures, microscopic views of these enrichments clearly show a consortium of protists of various cell morphologies (Fig. 1C, D). Thus, reduced RubisCO activity in the F/2 cultures could be due to utilization of alternative carbon/energy acquisition modes. In support of this prediction, pure cultures of photoautotrophic Antarctic isolates exhibit significantly higher maximum RubisCO carboxylase rates compared with Antarctic isolates that exhibit mixotrophy (Dolhi & Morgan-Kiss, unpublished results). Further studies of heterotrophic enzyme activity in these samples would help to fully characterize the metabolic versatility of the protist enrichment cultures and are currently underway in our laboratory. In addition, the physiological data described in this paper will be complemented with phylogenetic and metagenomic sequencing information as part of a large sequencing effort to characterize microbial communities across the globe called the Earth Microbiome Project (http://www.earthmicrobiome.org/).
The modified filter assay described in this report could also be applied to cell lysate extracted from filtered lake water samples. However, for new applications of this assay, optimal lysate volume should be determined by assaying three different volumes and using that which yields cpm at least 3 to 4 times above the negative controls. Analyses of carbon fixation potential in conjunction with enzymatic analyses of heterotrophic activity in natural protist communities would provide a more detailed view of the role of protist trophic ability and carbon cycling in aquatic environments. We are currently applying this approach to understand the role of abiotic environmental factors in protist metabolic versatility in Lake Bonney and other Antarctic lakes. The RubisCO carboxylase filter assay method will be a valuable tool to study potential carbon fixation and metabolic versatility in small scale enrichment cultures as well as whole lake systems.
Prior to the development of cultivation independent molecular tools, protists were traditionally identified by cultivation and taxonomically classified based on their cell morphology. Thus, there is a long history of enrichment and isolation of protists residing in a wide variety of habitats. Early investigations in particular are historically valuable for detailed taxonomic identification (reviewed in: 21, 22). While isolation of protists using enrichment-based approaches is relatively rare in Antarctic freshwater environments, several marine protist isolates from enrichment of Antarctic seawater are available 23-26. In addition, several culture collections are dedicated to protist and microalgal isolates (for eg: the Provasoli-Guillard culture collection, https://ncma.bigelow.org/). Dependable and cheap sequencing technologies have produced a massive genetic database of uncultivated protistan sequences. While these databases have been very informative regarding the diversity and distribution of this important group of microorganisms, there is a widening gap in linking sequence data with our understanding of the protist ecology and physiology. Thus, traditional cultivation techniques, particularly in undersampled environments such as polar aquatic habitats will continue to play an important role towards understanding the importance and ecological roles of protists in global geochemical cycling.
The authors have nothing to disclose.
The authors thank J. Priscu, A. Chiuchiolo and the McMurdo LTER limnology team for assistance in collection and preservation of the samples in Antarctica. We thank Ratheon Polar Services and PHI helicopters for logistical support. Light micrographs were generated in Miami’s Center for Advanced Microscopy and Imaging Center. This work was supported by NSF Office of Polar Programs Grants 0631659 and 1056396.
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) |