The Mono Lake Microbial Observatory

Mono Lake, California, is an alkaline, hypersaline lake that lies in the Eastern Sierra Nevada region of California. The lake was meromictic between 1995 and early 2004 (meromictic means that the lake is stratified and does not mix top to bottom in either fall or spring). As such, Mono Lake presents an ideal habitat for studing the proliferation and activity of microbes in a seasonal series of varying geochemical regimes. The primary driver of stratification in Mono Lake is the large density difference between surface and bottom waters. This density barrier is referred to as the chemocline. Monimolimion (=water beneath the chemocline) waters are permanently anoxic and reduced metabolites accumulate there. High concentrations of methane, ammonium and sulfide fuel diffusive fluxes of these materials from the monimolimnion through the chemocline into the mixolimnion (=water above the chemocline).

In addition to density-driven stratification, seasonal variations in temperature create additional stratificiation in the mixolimnion. Though the mixolimnion and monimolimion waters do not mix, seasonal temperature variations generate enough stratification to limit mixing within the mixolimnion during summer. Therefore, during winter, the oxycline (=dissolved oxygen gradient) and chemocline over lap. In contrast, during summer the oxycline tracks the theromocline (=temperature gradient) and may be 5-8m above the chemocline. The chemocline depth is currently 23-24m. The seasonal thermocline lies between 12-16m. Thermal and density driven stratification generates geochemical gradients; geochemical gradients generate variations in microbial community structure and activity.

As part of the National Science Foundation's Microbial Observatory Program, we are conducting a detailed study of the microbial biogeochemistry and molecular ecology of Mono Lake (the Mono Lake MObs Project). The lead PI on the project is Tim Hollibaugh and project co-PIs are Mandy Joye, Bob Jellison (UC Santa Barbara) and Jon Zehr (UC Santa Cruz). Joye lab members determine rates of microbially-mediated processes, such as, methane oxidation, sulfate reduction and nitrification, and correlate rate data with geochemical variables and with the distribution of specific microorganisms (e.g., methanonotrophs) and/or microbial groups (e.g., Archaea). Hollibaugh's group is studying the molecular ecology of bacteria and picophytoplankton; Jellison's group is studying primary production; and Zehr's group is studying the molecular ecology of nitrogen fixing microorganisms. Additional collaborators include Ron Oremland and Larry Miller at the USGS in Menlo Park and Sally MacIntyre at UC Santa Barbara. Oremland and Miller are studying arsenic and selenium cycling and MacIntyre is studying the physical limnology of Mono Lake, with an emphasis on quantifying micro-scale turbulence (internal waves).

In Joye's lab, PhD student Steve Carini and Research Scientist Vladimir Samarkin work on the Mono Lake project. Undergraduate students also participate in this work.

The Mono Basin lies at 1,946 m elevation and is approximately 760,000 yrs old. Mono lake was much larger in the past that it is presently. The current area of the lake is 150 km² and the average depth is 30m. In this aerial photo, ancient lake levels are visible as circles radiating out from the current lake edge. Mono Lake lies within the Long Valley Caldera and active volcanism occurs within and around the lake. Faults are present on the photo as (semi) straight lines and are particularly obvious on the upper right hand side of the image (N side of the lake).

As a terminal, closed basin soda lake, Mono Lake water is characterized by hypersalinity (salinity ~ 80 g L^-1), elevated pH (pH ~ 9.8) and high alkalinity (0.4 eq L^-1). The major cations are sodium (650 mM) and potassium (50 mM) and the major anions are carbonate (400 mM) and sulfate (120 mM). Mono Lake receives few inputs of nutrients and bioelements from creek inflow; most material is cycled internally numerous times and the lake functions as as a self-sustaining ecosystem. Mono Lake waters are rich in dissolved inorganic phosphorus (400 µM) but have very limited supplies of dissolved inorganic nitrogen in the surface waters (nM to low µM). Bottom waters contain equimolar concentrations of DIP and DIN (mainly as ammonium). Because of the hydrothermally-influenced nature of the Mono Basin, Mono Lake water also contains elevated concentrations of arsenic and selenium.

Because of the harsh chemical environment, Mono Lake is a microbially-dominated ecosystem. The only non-microbes living in Mono Lake are the brine shrimp, Artemia monica, and the brine fly (and fly larvae), Ge. sp. Our on-going studies of Mono Lake provide a nice complement to studies underway in deep ocean brine pools in the Gulf of Mexico (see Cold Seeps).

Study Sites
We have collected geochemical data and determined rates of microbial activity at six stations in Mono Lake (Stations 1, 3, 5, 6, 8 and 12, with a focus on Stations 3 & 6). These stations are a sub-set of those being surveyed monthly by Jellison as part of on on-going LA-DWP-funded project. Three of the MObs stations are deep and encompass the chemocline (>23m: 3, 6, and 12) and three are shallow, having bottom depths above the lake-wide chemocline (<23m: 1, 5, and 8). Two of the deep MObs stations, Stations 3 and 6, are the focus of process-oriented studies. Station 3 has been studied intensively in the past by MObs PIs as well as other investigators.

MObs field work commenced in May 2000 and continues (pending a renewal proposal). The Joye group is responsible for geochemical analsyes (nutrients, redox species, dissolved gases, stable isotopes) and for quantifying in situ rates of microbially-mediated processes. In the first 2.5 years of the project, we are working mainly in the water column to quantify rates of methane oxidation, nitrification, sulfate reduction and methanogenesis. We have also quantified rates of methane production and oxidation and sulfate reduction in lake bed sediments.

Mono Lake "clines"
One of the features that makes Mono Lake intersting is the variety of "clines" found in the water column. The term "cline" is used to describe regions of change. The chemocline refers to the region where the concentration of chemical constituents, like salt and ammonium, change significantly over depth (concentrations increase with depth across and below the chemocline). The chemocline is currently located at 23-24 m depth throughout the lake. The thermocline refers to the region where temperature changes significantly over depth. In summer, temperature decreases as depth increases and the summertime thermocline lies between 12-15m. During winter, inverse thermal stratification occurs with waters below the chemocline holding fast at 4ºC while surface waters may cool to 2ºC or less. This abnormal thermal structure does not lead to overturn because of the strong density gradient that persists between the monimnolimnion and the hypolimnion. However, winter mixing does gradually erode the chemocline, eroding it and pushing it deeper each year. The oxycline refers to the region where oxygen concenctration changes with depth. During winter, the oxycline tracks the chemocline, while during summer, the oxycline tracks the thermocline.

The figure below shows the summer "clines" of Mono Lake at Station 3 (W basin) and Station 6 (E basin) [see station figure above]. Station 6 is about 10m deeper than Station 3. Interesting features of the summer profile include a deep fluorescence peak (17m), illustrating a layer of phytoplankton positioned at the base of the thermocline and a deep layer that exhibits extremely low transmittance (24m). This layer corresponds to the chemocline.

Station 3, July 2000	Station 6, July 2000

Joye's research in Mono Lake strives to:

> Document rates of important microbially-mediated transformations, including methane production and oxidation, sulfate reduction and nitrification.

> Identify the physiological mechanism (pathway) of these processes.

> Determine the mediator (whether an individual microorganism or a consortium) of these processes.

Specific questions regarding methane cycling:

1. What affect has meromixis had on methane inventories and on methane oxidation rates?

2. How much of the methane flux from the bottom sediments is consumed by water column oxidation?

3. What is the relative importance of aerobic versus anaerobic methane oxidation?

4. What are the dominant microorganisms responsible for methane oxidation in Mono Lake oxic vs. anoxic waters and in the sediments?

5. What is the mechanism of anaerobic oxidation of methane in Mono lake bottom waters and sediments?

Methanogenesisis an obligately anaerobic process by which microorganisms generate methane, a potent greenhouse gas. Most of the methane in Mono Lake is produced biologically in lake bottom sediments. Previous studies of methanogensis in Mono Lake sediments documented production rates of up to 4 mmol CH₄ m^-2 d^-1 and bottom fluxes of up to 1 mmol CH₄ m^-2 d^-1 (Miller and Oremland 1988). The majority of the methane that fluxes from the sediments into the water column is oxidized and, thus, methane fluxes to the atmosphere are low.

Methane oxidation occurs under oxic and anoxic conditions. The aerobic methanotrophs are obligate aerobes that require molecular oxygen to oxidize methane. This group of microorganisms has been thoroughly studied and their biochemistry and molecular biology are well documented. The aerobic methanotrophs are chemolithotrophs and are metabolically similar to the nitrifying bacteria, in terms of the enyzmes involved in methane oxidation. There are two primary groups of aerobic methanotrophs, the type I organisms which cluster within the alpha proteobacteria and the type II microoraganisms which cluster in the gamma proteobacteria. In aerobic methane oxidation, methane is sequentially oxidized to methanol (via methane monooxygenase), formaldehyde (via methanol dehydrogenase), formate (via formaldehyde dehydroganse) and finally to carbon dioxide (via formate dehydrogenase):

CH₄ -> CH₃OH -> HCOH -> HCOOH -> CO₂

Formaldehyde (HCOH) is shunted into cellular biosynthetic pathways; type I methanotrophs use the serine pathway and type II methanotrophs use the RUMP pathway. The activity of methanotrophs is impacted by primary substrate availability (methane and oxygen), the presence of alternate/competitive or inhibitory compounds (e.g., ammonium, methanol, methylated amines, or hydrogen sulfide), and environmental variables (e.g., tempature, salinity, and pH).

Anaerobic oxidation of methane (AOM) has been documented in a variety of environmental settings; however, most examples of anaerobic methane oxidation come from marine sediments. AOM also occurs in anoxic water bodies, such as the Black Sea, the Cariaco Trench and even in Mono Lake. The microorganisms responsible for anaerobic methane oxidation are unknown and the mechanism of the process is hotly debated. In all likelihood, anaerobic methane oxidation involves a syntrophic association of two or more microorganisms that exchange one or more metabolic intermediates. The most frequently cited mechanism for AOM involves an association between a CO₂-reducing methanogen and a sulfate reducer (cf. Hoehler et al. 1994). This hypothesis has received a lot of attention because AOM has been documented frequently in the vicinity of the sulfate-methane transition zone. This zone is the interface between the portion of the sediment column dominated by sulfate reduction and that dominated by methanogenesis. Basically, the transition zone is where sulfate concentrations are low, but still high enough to permit sulfate reduction. Methane concentrations are low, but high enough to support oxidation. Within this zone that sulfate is depleted completely, which results in a lower depth horizon that is dominated by (net) methanogenesis. The contemporaneous activity of both groups of microorganisms is required to support AOM.

So, what is the methanism of AOM in Mono Lake?

We don't know...yet. The water column of Mono Lake, like other pelagic habitats where AOM has been documented, does not have a sulfate-methane transition zone. The water column sulfate concentration is approximately 100 mM throughout the water column though there is a surface to bottom gradient in methane concentration (higher in anoxic bottom waters, as shown on the figures above). There is probably a sulfate-methane interface in Mono Lake sediments, but most of the lakewide AOM occurs in the water column. In the Mono Lake water column, AOM occurs in a region where sulfate concentration does not change, but where methane concentrations are gradually increasing. This makes us think that the mechanism of water column AOM in Mono Lake might be very different from that observed in the sulfate-methane transition zone of marine sediments.

We are currently studying interactions between AOM, methane production, and sulfate reduction in Mono Lake and hope to pin down the mechanism by quantifying rates of processes, as well as the concentrations of possible intermediates (e.g., hydrogen, acetate, formate, and a few others).

At all stations in the lake, we collect samples from a depth profile to describe surface to bottom variations in physical (temperature, salinity, density, PAR, transmittance), biological (bacterial counts, chlorophyll, molecular samples), and chemical (organic and inorganic nutrients, bulk DOC, volatile fatty acids, major ions, hydrogen sulfide and dissolved gases (methane, hydrogen, oxygen, nitrogen, nitrous oxide and carbon monoxide). We determine rates of methane oxidation, methanogenesis and sulfate reduction using standard radioisotope tracer techniques (for methane oxidation either C³H₄ and ¹⁴CH₄ is used, depending on the concentration). We also quantify the stable carbon isotopic composition of methane and dissolved inorganic carbon using gas chromatography-isotope ratio mass spectrometry.

We thank the National Science Foundation's Microbial Observatory Program for supporting this work.

**Disclaimer** The content of this page is based in part on work supported by the National Science Foundation. Any opinions, findings, and conclusions or recommendations expressed here are those of the author (Mandy Joye) and do not necessarily reflect the views of the National Science Foundation.