l AQUATIC MICROBIAL ECOLOGY Vol. 19: 93-103,1999 Published September 6 Aquat Microb Ecol l

Lake KauhakG, Moloka'i, Hawai'i: biological and chemical aspects of a morpho-ectogenic meromictic lake

Stuart P. ~onachie'~*,Robert A. Kinzie 1112,Robert R. ~idigare~, Daniel W. Sadlerl, David M. ~arl'l~

'Laboratory for Microbiological Oceanography, 'Department of Zoology and 3Department of Oceanography, SOEST, University of Hawai'i, Honolulu, Hawai'i 96822, USA

ABSTRACT: We undertook the first combined microbiological and hydrochemical study of the 248 m deep meromichc Lake KauhakG. Situated at sea level 1.6 km from the sea in the crater of an extinct vol- cano on the island of Moloka'i, Hawai'i, USA, Lake KauhakB has the highest relative depth (ratio of depth to surface area, z, = 374 %) of any lake in the world. The upper 4.5 m were stratified (T=23 to 26'C, salinity = 6 to 24.5), but below a pycnocline at -4.5 m temperature and salinity were uniform (T = -26.25"C; salinity = 32). Seawater hkely intrudes by horizontal hydraulic conductivity through rock sep- arating the lake and the Pacific Ocean. Anoxia commenced below 2 m. Hydrogen sulfide was unde- tectable at 4 m. but averaged -130 PM between 5 and 28 m. Dissolved inorganic carbon concentrations ranged from -1.50 mM at the surface to -3.3 mM below 5 m. Total organic carbon peaked at 0.94 mM above the pycnocline hut rernaiced about 0.30 mM be!ow 5 m. Soluble reactive phosphorus and ammu- nium concentrations, nanomolar above the pycnocline, increased to -28 and l75 PM, respectively, at greater depth. Nitrate attained 3.7 PM in shallow water, but was -0.2 pM from the pycnocline to 100 m. Leucine aminopeptidase (LAPase) activity at the surface exceeded 1100 nmol of substrate hydrolyzed I-' h-' Activities of a- and P-glucosidase were lower, but showed depth distributions similar to that of LA- Pase. Surface waters hosted large and diverse picoplankton populations; chlorophyll a (chl a) exceeded 150 1-19 I-'. and heterotrophic bacteria and autofluorescent bacteria attained 2 X log and 9 X 109 1-l, re- spectively. Filamentous cyanobacteria and 'Proch1orococcus'-like autotrophs occurred only in the upper 2 m. Chl a was the dominant pigment above 2 m, but pigment diversity increased markedly in anoxic waters between 3 and 5 m. Lake Kauhako is a unique habitat for further studies, particularly of inter- actions among flora and fauna restricted to a shallow water column within a single basin.

KEY WORDS: Lake Kauhako - Meromixis - Moloka'i . Carbon . Nutrients . Pigments . Ectoenzyme Bacteria

'...the great intellectual fascination of limnology Lies in the comparative study of a great number of systems, each having some resemblance to the others and also many differences.' Hutchinson (1964)

INTRODUCTION & Macdonald 1946) (Fig. 1). At 248 m deep, it is be- lieved to be the 4th deepest lake in the United States. Lake KauhakG is situated in the crater of an extinct, The lake is meromictic, a condition here considered to late Pleistocene volcano on the Kalaupapa Peninsula, be morphogenically derived (Maciolek 1982), i.e. the on the north shore of Moloka'i, Hawai'i, USA (Stearns small surface area (3500 m') and relatively great depth hlit vertical mixing to the upper 2% of the water col- umn. The lake is bounded by high (-130 m), steep, and 'Present address: Environmental Microbiology Laboratory, Department of Microbiology, Snyder Hall, University of heavily wooded slopes, a topography that restricts Hawai'i, The Mall, Honolulu, Hawai'i 96822, USA. wind-driven mixing to a shallow surface layer and pro- E-mail: [email protected] motes the persistence of meromixis. As the lake is in

0 Inter-Research 1999 94 Aquat Microb Ecol 19: 93-103, 1999

In light of the conditions which prevail in such lakes and the fact that interest has heightened recently in environments considered physically and/ or chemically extreme, a more detailed study of Lake KauhakG appeared warranted. The lake is not easily accessible, and few data exist concerning its hydro- logy, particularly below a few meters. We applled a range of modern methods and measurement tech- niques to define aspects of the lake's chemistry and biology that have to date been undescribed or poorly documented.

MATERIALS AND METHODS

All water samples were collected from the center of Lake KauhakG between 23 and 25 March 1997. Water at various depths in the upper 100 m was collected in a S l Go-Flo or 1 1 plastic bathymeter lowered by hand on a Kevlar line from a small dinghy. Vertical sampling resolution of the bathymeter was 12 cm. Samples were immediately returned to the shore and subsamples taken for determination of conductivity, dissolved oxy- gen, sulfide, nitrous oxide, dissolved inorganic carbon (DIC) and alkalinity, dissolved organic carbon (DOC), nutrients (soluble reactive and total dissolved phos- phorus [SRP, TDP], NO3-, NH,', total dissolved nitro- Fig. 1. Lake Kauhako is situated on the Kalaupapa Peninsula gen [TDN] and soluble reactive silica [SRSi]),eukary- on the north shore of Moloka'i, Hawai'i. The upper panel otic and prokaryotic pigments, microbial ectoenzyme shows a vertical profile along AB on the map. Scale interval in activities (leucine aminopeptidase [LAPase], and cc- hundreds of meters. Contour interval in meters. Redrawn and P-glucosidase), and cell numbers (heterotrophic from Maciolek (1982) and autotrophic Bacteria, other autotrophs, picophyto- plankton). Data for a temperature profile (0 to 100 m) close proximity to the Pacific Ocean, meromixis may were collected by a datalogger, which recorded tem- also be ectogenic (derived and sustained through perature as a function of pressure. inflow of both fresh- and seawater). Freshwater input Salinity. A hand-held conductivity meter (Oakton amounts to -1 m yr-' of rainfall (Giambelluca et al. WD-35607-10) with an integral temperature probe was 1986) over the lake surface and catchment area, the used to determine conductivity in samples to 100 m. latter comprising only the 0.26 km2 crater opening, and Salinities were derived from the conductivity and tem- groundwater intrusion from the freshwater table. The perature measured at 1 atmosphere, according to stan- lake surface is at sea level; in view of the local geology, dard procedures (Perkin & Lewis 1980). the lake's proximity to the Pacific Ocean (1.6 km) and Dissolved oxygen. Subsamples for dissolved oxygen the fact that the lake basin is fully cryptodepressed were collected to 4 m and were the first taken from the (below sea level), some exchange of water with the sea water sampler. They were collected in calibrated io- is likely. dine flasks and the dissolved oxygen chemically bound Meromixis in a water body is characterized by the in the field by addition of 1 ml each of MnSO,.H,O and formation of a layer of oxygenated surface water (the alkaline iodide. The floc was dispersed through vigor- mixolimnion) separated by a chemocline or pycnocline ous agitation (20 S) and the sample sealed and stored in from anoxic and denser water known as the moni- darkness until analysis within 5 h using the Winkler molimnion. In response to steep light intensity and/or titration method (Carpenter 1965). chemical gradients across depth intervals that may Sulfide. The presence of sulfide at 3 m was obvious cover only centimeters (Fry 1986), diverse and highly from the characteristic odor. At both 3 and 4 m, sulfide productive microbial populations develop above and subsamples were taken from the sampler after those in the vicinity of the chemocline (Pfennig 1967, Taka- for dissolved oxygen. From 4.5 to 20 m inclusive, sul- hashi & Ichimura 1970). fide subsamples were drawn first. Donachie et al.: Biological and chemical aspects of a meromictic lake 95

Subsamples for sulfide were collected in 125 m1 samples maintained at room temperature for several reagent bottles with ground glass stoppers. Sulfide weeks. TDP and TDN were measured after ultraviolet was immediately precipitated by addition of 1 m1 of a light photo-oxidation (Walsh 1989, Karl et al. 1993). Dis- zinc acetate solution in oxygen-free deionized water solved organic nitrogen (DON) was considered equal to containing 2 g 1-' gelatin (Fonselius 1983). The tops TDN - (NO3- + NH4+),and soluble nonreactive phos- were wrapped tightly with ~abfilm~,and the bottles phorus (SNP) considered equal to TDP - SRP. stored in darkness until analyzed. Sulfide was deter- Pigments. Total chlorophyll a (chl a) (i.e. monovinyl mined by titration with an acidic standard iodine solu- plus divinyl chl a) and accessory pigment concentra- tion, followed by back titration with standard sodium tions were determined in water samples collected from thiosulfate (Skoog et al. 1988). 0.5 to 100 m. Samples were filtered through 25 mm Nitrous oxide. Subsamples (0.1 to 5 m) for nitrous Whatman GF/F filters and stored at -20°C until ana- oxide (N20)were collected in 60 m1 glass bottles, fixed lyzed. Filters were placed in 3 or 6 m1 acetone, depend- with 100 p1 saturated mercuric chloride, and sealed ing on filter loading, and extracted for 24 h (O°C, dark). with rubber stoppers and aluminium crimp-seal caps. Prior to analysis, pigment extracts were vortexed and Prior to analysis the sample was equilibrated with an centrifuged to remove cellular and filter debris. Sam- equal volume of helium. Nitrous oxide was separated ples (200 p1) of a mixture of 0.3 m1 H20 and 1 m1 extract by gas chromatography and detected using an electron were injected into a Varian 9012 HPLC system capture detector (Elkins 1980). equipped with a Varian 9300 autosampler, a Timber- Dissolved inorganic carbon and alkalinity. DIC hecolumn heater (26"C), and a Spherisorb 5 mm ODS2 samples (0 to 100 m) were collected in combusted analytical (4.6 X 250 mm) column and corresponding 180 m1 glass bottles. After allowing the bottle to over- guard column. Pigments were detected with a Ther- flow twice, a small volume of sample was removed moseparation UV2000 detector (h = 436 and 450 nm) using a plastic pipette and 100 p1 of saturated HgC12 and analyzed after Wright et al. (1991) and Latasa et al. added. The bottles were sealed with rubber stoppers (1997). The HPLC method used cannot separate and aluminium crimp-seal caps. DIC was measured by monovinyl chl a from divinyl chl a. Concentrations of coulometric determination of extracted CO2 after these pigments were determined by monitoring the Johnson et al. (1987). Total alkalinity in each sample monovinyl chl a plus divinyl chl a peak at 2 wave- was determined by potentiometric titration with hydro- lengths (436 and 450 nm) and deriving their respective chloric acid (0.1 M HC1 in 0.6 M NaC1) after Winn et al. concentrations via dichromatic equations given by (1994) and calculated according to standard proce- Latasa et al. (1996). Pigment peaks were identified by dures described in DOE (1994). comparing retention times with those of extracts pre- Total organic carbon (TOC). Subsamples for TOC pared from algal cultures of known pigment composi- (0 to 100 m) were collected in sterile 50 m1 polypropylene tion (Latasa et al. 1996). Pigment concentrations were centrifuge tubes. The sampler spout was rinsed by al- calculated using pure external standards. lowing water to flow freely (-10 S), after which the tubes Cell numbers. Autofluorescent Bacteria, filamentous were filled without rinsing. These samples were imme- cyanobacteria and 'Synechococcus'-like autotrophic diately placed in a cooler, and frozen upright upon return picoplankton collected between 0.1 and 100 m were to the laboratory. They were stored frozen (Tupas et al. counted in 0.5 m1 samples fixed with paraformalde- 1994) until analyzed in an MQlOOl TOC analyzer (MQ hyde (final concentration 0.2 %) by epifluorescence Scientific Inc., Pullman, WA) after Qian & Mopper (1996). microscopy using DAPI (4'6-diamidino-2-phenylin- Dissolved nutrients. Subsamples for dissolved inor- dole) (Porter & Feig 1980). Samples for cell counts were ganic nutrient (NO,-, NO3-, NH,', SRP and SRSi; 0 to stored frozen in sterile 2 m1 polypropylene cryovials 100 m) determinations were passed from a sterile sy- (-20°C) until analyzed. Picoeukaryotes (e.g. crypto- ringe through combusted Whatman GF/F filters in a phytes) and other autofluorescent cells not considered Millipore disk filter holder. Filtrates were collected in Bactena, filamentous cyanobacteria, or 'Synechococ- acid-washed (3x with 1 M HC1) and rinsed (3x with cusl-like were counted as 'other autofluorescent cells'. double distilled H20) 125 m1 high density polyethylene Numbers of unpigmented Bactena plus Archaea (here- (HDPE) bottles, placed in a cooler, and frozen upon re- after referred to as heterotrophic bacteria) to 100 m, turn to the laboratory (-20°C) (Dore et al. 1996). They and of cells we tentatively consider as 'Prochlorococ- were analyzed according to Technicon Industrial sys- cusl-like to 2 m, were determined by flow cytometry tems (1973, 1977, 1979) after Armstrong et al. (1967) for (Monger & Landry 1993) in 1 m1 samples fixed in ster- N, Murphy & Riley (1962) for SRP, and Strickland & Par- ile 2 m1 polypropylene cryovials with paraformalde- sons (1972) for SRSi, with slight modifications to surfac- hyde (0.2 %). Prochlorococcus is undetectable by epi- tant concentrations, sampling rate, and pump tube size fluorescence microscopy due to its weak fluorescence, (T. Walsh pers. comrn.). SRSi was determined in thawed but may be enumerated by flow cytometry through a Aquat Microb Ecol 19: 93-103, 1999

0 10 20 30 400 2 4 6 8 100 5 10 15 salinity N20 (nM) Alkalinity (meq. L.')

Fig. 2. (A) Salinity (0) and temperature (solid line) profiles of Lake KauhakE. (B)Concentrations of dissolved gases (0: oxygen, B: hydrogen sulfide, +: nitrous oxide) in Lake Kauhak6 . (C) DIC (*), TOC (0) and alkalinity (0)in Lake Kauhak6 . (Data below 1.0 m not shown) characteristic signature based on forward light scatter trophic nanoflagellates (Karner et al. 1994). Cell-spe- and chlorophyll fluorescence (Chisholm et al. 1988). cific activities described in this paper were calculated The flow cytometnc assignment of Prochlorococcus in on the basis of the combined numbers of hetero- Lake Kauhaka samples is based on previous experi- trophic and autofluorescent Bacteria, together with ence with Prochlorococcus in oceanic samples and those of the filamentous cyanobacteria, 'Prochlorococ- remains tentative, but the detection of marker pig- cus'- and 'Synechococcus'-like picoplankton; marine ments (divinyl chlorophylls) by HPLC analysis can heterotrophic bacteria and Synechococcus spp. may indicate the presence of these cells. express sirmlar levels of this enzyme (Martinez & Ectoenzyme activities. The potential activities (0.1 to Azam 1993) and there is uncertainty about the contri- 5 m) at -25°C of LAPase (EC 3.4.1.1) and a- and P-glu- bution, if any, to this activity from Prochlorococcus cosidase (EC 3.2.1.20 and EC 3.2.1.21 respectively) because this organism has not been available in pure were determined using fluorogenic substrate analogs culture until recently. Activities per cell of a- and (Hoppe 1983, Somville & Billen 1983, Christian & Karl P-glucosidase are based only on the numbers of het- 1995). Fluorescence was measured in a Perkin-Elmer erotrophic bacteria. LS-5 spectrofluorometer. Activities are potential since substrate analogs were applied at above trace concen- trations, 1 mM for LAPase, and 1.6 PM for the glucosi- RESULTS dases. LAPase activity in aquatic habitats is not solely of Hydrography heterotrophic bacterial origin and may be derived from photosynthetic Bacteria such as Synechococcus The upper 4 m of Lake Kauhaka were stratified with (Martinez & Azam 1993) and Eucarya such as phago- respect to temperature and salinity (Fig. 2A); euhaline Donachie et al.: Biological and chemical aspects of a meromictic lake 97

Fig. 3. Vertical distribution of (A) dissolved phosphorus (0.TDP, 0: SRP, X: SNP), (B)nitrogen species (H: TDN, 0: DON, 0: NH,+, 0: NO3-) and (C) soluble reactive silica in Lake Kauhak5 and isothermic water then extended to 100 m. Dicho- 5 and 20 m (Fig. 2C; data below 10 m not shown). Alka- thermy was pronounced, with a temperature minimum linity increased from 2.0 meq. 1-' at the surface to at 1 m. Salinity ranged from 6.4 at the surface to 34.4 3.5 meq. 1-' at 4 m. Below 5 m inclusive, alkalinity was between 5.5 and 80 m. A pronounced temperature and stable at -11.1 meq. 1-' (Fig. 2C). TOC peaked at salinity gradient around 4.5 m signaled the presence of 0.94 mM (11.2 mg C I-') at 1 m, the shallowest sam- a pycnocline. ple taken (Fig. 2C), falling sharply thereafter to 0.20-0.30 mM between 10 and 40 m (data below 10 m not shown). Dissolved oxygen, sulfide and nitrous oxide

Dissolved oxygen peaked at 365 pM (50% above air Nutrients saturation) at the surface (Fig. 2B). Water below 3 m was anoxic. Sulfide concentrations between 5 and SRP comprised the bulk of TDP at most depths 28 m were 126 to 133 pM (Fig. 2B). Surface water, with (Fig. 3A); it was depleted in the upper 4 m 7 nM N20, was -2% supersaturated with respect to (<0.08 PM) but attained >20 pM by 5 m and was atmospheric N20 (Fig. 2B). stable at -28 pM between 10 and 100 m (data below 10 m not shown). TDN measured 35 to 100 pM above 4 m, and -160 Carbonate system parameters to 200 PM below 5 m (Fig. 3B); DON con~prised up to 97 % of TEX iu 4 m, but only -7 to 14 % below DIC concentrations increased with depth in the the pycnocline (Fig. 3B). NH,' concentrations were mixolimnion but were stable at 3.1 to 3.5 mM between

Chlorophyll a (pg L") 0 40 80 120

Fig. 4. Vertical distribution of chlorophyll a in Lake Kauhak6

-175 pM between 10 and 100 m (data below 10 m not shown). No3 concentrations fell sharply with depth, from -3.75 pM at 0.5 m to

Pigments Time(minutes) Total chl a concentration peaked at over 150 pg 1-' at 0.5 m, and fell to -2 pg I-' at 4 m (Fig. 4). At 10 and Fig. 5. Reverse-phase HPLC chromatograms (436 nm) ob- tained for pigment extracts prepared from samples collected 40 m, chl a concentrations were -0.1 pg 1-'. A number at (A) 2 m, (B)3 m, (C) 4 m and (D) 5 m depth from Lake of oxygenic photoau.totroph accessory pigments were Kauhak5. Peak ~dentlties: (1) chlorophyll c (chromophyte present in the upper 2 m, including zeaxanthin marker), (2) fucoxanthln (diatom marker), (3) diadinoxanthin (cyanobacteria marker), alloxanth~n (cryptophyte (chromophyte marker), (4) alloxanthin (cryptophyte marker), marker), chl cand diadinoxanthin (chromophyte mark- (5) zeaxanthin (cyanobacteria marker). (6) monovinyl plus divinyl chlorophyll a, and (7) p-carotene ers), fucoxanthin (diatom marker), and P-carotene (Fig. 5A). A large number of pigment peaks were de- tected in samples collected from 3 to 5 m (Fig. 5B-D), but only a subset was found below 10 m (data not Sinking cells or cell debris may account for the divinyl shown). Divinyl chl a, a pigment so far found only in chl a at 3 and 4 m; this pigment was undetectable Prochlorococcus in aquatic ecosystems, was detected below 5 m. Most pigments detected between 3 and 5 m at 0.5 and 1 m, and comprised 2.1 and 5.6% of the chl a do not correspond to those found in oxygenic photoau- at these depths, respectively. This percentage dropped totrophs; they likely comprise bacteriochlorophyll and to -1.5% by 2 m and decreased to a trace level at 4 m. bacteriocarotenoid pigments. A lack of authentic bac- Donachie et al.: Biological and chemical aspects of a meromictic lake 99

B cells (X 10' L") 10 0 2 4 6 8 10 12 14

Fig. 6. Vertical distribution of (A) het- erotrophic (0) and autofluorescent (W) Bacteria, and (B) 'Synechococcus'-like cells (U), filamentous cyanobacteria (0) and 'other autofluorescent cells' (m) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 in Lake Kauhako filamentous cyanobacteria(X 10' L.')

tenopigment standards in our laboratory precluded they peaked at 3 m (Fig. 6B). No 'other autofluorescent identification of these compounds, which were distrib- cells' were observed below 5 m inclusive. uted heterogeneously with respect to depth, indicating Cells considered flow cytometrically as 'Prochloro- that the photosynthetic Bacteria taxa were segregated coccus1-like numbered 1.3 X 108 1-I at 0.5 m and 1.1 X into distinct layers. 107 1-' at 2 m (data not shown); their order of magni- tude reduction in abundance with depth accompanied a decrease in the divinyl chlorophyll concentration. At Cell numbers -1.0 pm diameter, these cells were larger than Prochlorococcus in oceanic samples (0.6 to 0.8 pm in The distribution of heterotrophic and autofluores- diameter and 1.2 to 1.6 pm in length; Chisholm et al. cent Bacteria contrasted with depth (Fig. 6A). Numbers 1992). of heterotrophic bacteria fell from -5 X log 1-' at 0.5 m to -1.9 X 10' 1-' between 5 and 100 m. Autofluorescent Bacterja numbers peaked at >8.0 X log 1-' in the vicin- Ectoenzyme activities ity of the pycnocline but were only -lo7rnl-' between 5 and 100 m. The highest total activity of each ectoenzyme was Filamentous cyanobacteria attained -1.0 X 10' 1-' at recorded at 0.1 m and fell with increasing depth 0.5 m (Fig. 6B). Filaments comprised 4 to 12 cells of -10 (Fig. 7). Total LAPase decreased by nearly 200-fold to 15 X 5 pm each, each filament terminating in a non- from -1.05 pm01 substrate hydrolyzed 1-' h-' at 0.5 m to autofluorescent heterocyst. These filaments were not -7.49 nmol 1-' h-' at 5.0 m; activity per prokaryote seen below 2 m. Cells of -1 X 0.75 I.lm, morphologically ranged from -122 to 4.70 am01 cell-' h-' at these similar to Synechococcus spp. (uniform autofluores- depths, respectively (Fig. ?A). Total a-glucosidase ac- cence, circular to ovoid), numbered 2.0 to 3.0 X 108 1-' tivity attained 10.79 nmol substrate hydrolyzed 1-' h-' between 0.5 and 2 m (Fig. 6B). They were not detected at 0.5 m, and ranged from 1.90 to 0.17 am01 h-' per het- below 3 m. Cells considered 'other autofluorescent erotrophic bacterium (Fig. ?B). Total P-glucosidase ac- cells' were not uniformly distributed with depth; low- tivity attained 13.96 nmol substrate hydrolyzed 1-' h-' est numbers occurred at the oxycline (1.65 X 10' 1-') but and ranged from 2.29 to 0.17 am01 cell-' h-' (Fig. 7C). Aquat Microb Ecol 19: 93-103, 1999

Total activity (-01 L.' hr.') B Total activity (nmol L"hr.') c Total activity (nmol L.' E') 400 800 1200 0 4 8 12 0 5 10 15

0 50 100 150 0.0 0.5 1.0 1.5 2.0 0.0 0.5 1.0 1.5 2.0 Activity ce~'(amol hi') Activity cell-' (am01 hi') Activity cell-'(amol hi')

Fig. 7.Vertical distribution of ectoenzyme activities in Lake Kauhaks. Solid symbols indicate total activity in water; open symbols indicate activity per cell

DISCUSSION have an -11 d transit time to the lake, and derive a sur- face fluctuation in a standing body of water 1.6 km We have described a range of hydrochemical and from the sea of 5 to 10% of the adjacent ocean tide (Oki microbiological aspects of Lake KauhakG, a lake only 1997). In Lake Kauhak6 this would equate to the 'few briefly reported in the limnological literature. The lake centimeters' reported by Maciolek (1982). The basin's is characterized by oxygenated, brackish water overly- extreme declivity and the protection from winds ing saline anoxic water. Our data for water column offered by the high crater walls ensure the persistence temperature and chemistry profiles are similar to those of meromixis. The freshwater table on the peninsula is of R. A. Kinzie 111 et al. (unpubl. data), who sampled located at 1.5 to 2.0 m (Oki 1997), the depth of the oxic Lake Kauhak6 in 1995 and 1996. These combined layer during our study. Meromixis in Lake KauhakG observations suggest that the stratification of Lake can therefore be considered morphogenic (Maciolek KauhakG is persistent. 1982) and ectogenic. Salinity through much of the lake was similar to that Limited aeolian influence will preclude mixing and of seawater. The lake surface is at sea level but does ensure that the oxic layer remains shallow. Oxic layer not respond synchronously with nearby ocean tides depth and the minimum depth at which sulfide was despite its proximity to the sea (c2 km). This observa- detected during our visit were each less than reported tion suggests the absence of an 'open' connection to by Maciolek (1982) based on a 1974 survey. Our salin- the sea. Anoxia in the monimolimnion also implies that ity, temperature and nutrient data indicate that mixing any connection to the ocean has little effect in terms of occurs at least between 4.5 and 100 m. Nitrous oxide is introducing large volumes of oxygenated water to the a greenhouse gas and is involved in ozone depletion lake. Exchange by horizontal hydraulic conductivity (Dore & Karl 1996); on the basis of N20 solubility in through the rock or fractures between the lake and the water (Weiss & Price 1980) surface water of Lake sea is possible; seawater passing through -150 m d-' of KauhakG was 2% supersaturated with respect to the the rock comprising the Kalaupapa Peninsula would atmosphere. The error associated with the analysis is Donachie et al.. Biological and chemical aspects of a meromictic lake 101

2 to 4 % so we cannot say if the lake constitutes either lakes (2 to 5 FM), while SRSi concentrations below the an N20 source or a sink, although both would be pycnocline compared to those in anoxic waters of insignificant considering the lake's small surface area. meromictic Lake Nordbytjernet (to 1.4 mM; Hongve The DIC concentration at the surface of Lake 1997). In the subtropical gyre north of Hawai'i, SRSi KauhakG was about 3/4 that in Hawaiian coastal concentrations of 0.15 to 0.16 mM between 2000 and waters (Winn et al. 1994), reflecting high primary pro- 4000 m have been reported, while concentrations did ductivity and rapid uptake of inorganic carbon. That not exceed 0.02 mM above 500 m (Roemmich et al. CO2 produced during nitrate or sulfate respiration by 1991). SRSi concentrations in seawater immediately off heterotrophic bacteria in anoxic waters may account the Kalaupapa Peninsula are unlikely to differ signifi- for the high DIC concentrations below the oxycline. cantly from open ocean surface values, so SRSi accu- Since CO2 must have a finite diffusive lifetime, the mulation in the Lake Kauhako monimolimnion may high DIC concentration at the oxycline implies that it is arise through removal from the surrounding rejuve- produced very rapidly indeed. As we did not collect nated-stage alkalic basalt and basanite rocks (Langen- dissolved nutrient samples at 4.5 m, we cannot say if heirn & Clague 1987) and dissolution of sinking diatom there were similar distributions of NO3-, NH4+and SRP. frustules. We observed diatoms by epifluorescence The rapid increase in alkalinity across the pycnocline microscopy in Lake KauhakG samples. Although our of Lake KauhakG is consistent with the production of samples for SRSi were frozen, a practice which may alkalinity in anoxic waters by organic matter oxidation lead to underestimation by 10 to 100% through poly- through sulfate reduction, as reported for the Black merization in saline waters with high SRSi concentra- Sea (Goyet et al. 1991). tions (MacDonald et al. 1986), extended storage at Although TOC peaked in the shallowest sample room temperature results in partial solubilization taken, we had no parallel sample from the pycnocline. (Walsh 1989). As the combined heterotrophic and autofluorescent Pigment diversity in the upper 2 m of Lake Kauhak6 Bacteria cell numbers increased 2.75-fold from 3 to was low and dominated by chl a. By 3 m chl a had 4.5 m it is likely that the TOC concentration increased largely been replaced by a diverse suite of bacterial at the pycnocline, especially since bacterial cells in pigments. The detection of Prochlorococcus spp. in anoxic lake waters may be larger than in oxic waters Lake Kauhako by flow cytometry was supported by the (Cole et al. 1993). Heterotrophic and photosynthetic presumptive detection of divinyl chl a (Goericke & activities in surface waters of Lake KauhakG probably Repeta 1992), with sinking cells likely accounting for keep NH,' and SRP low and maintain rapid turnover the trace levels of these pigments at 3 and 4 m. rates. Assuming a maximum photosynthetic uptake of Prochlorococcus has not been previously reported in 20 g C g-' chl h-' (Falkowski 1981) and a Redfield brackish waters such as those at the surface of Lake C:N:P molar ratio of 106:16:1, N and P demand would KauhakG. be 38 and 2.4 pm01 h-', respectively. Ammonium oxi- In the upper 4.5 m of Lake KauhakCi, heterotrophic dation by nitrifiers in the lake's surface waters may Bacteria numbers were similar to those in non-oligo- deplete NH4+to the low levels we detected, and deni- trophic lakes (Zehr et al. 1987, Miinster 1991). The fact trification (Codispoti et al. 1991) may also deplete NO3- that heterotrophic bacterial numbers were high in in anoxic waters above the pycnocline. anoxic water above the pycnocline suggests a switch to Order of magnitude differences in SRP concentra- nitrate respiration (Overbeck 1993). Chroococcoid tions between 0.5 and 4.0 m reflect the activities of dif- cyanobacteria similar to those described here as 'Syne- ferent microbial communities and the absence of verti- chococcus'-like are found in lakes (Cole 1983) and cal advective processes. The latter may occasionally oligotrophic seawaters (Campbell & Vaulot 1993). In affect productivity in surface waters of Lake KauhakG Lake Kauhaki5, large 'Synechococcus'-like unicells, through advection of bacterial cells from P-rich water. together with an unidentified filamentous cyanobac- Some lake sediments are an important P source when terium, dominated the picophytoplankton in the upper the entire water column mixes (Brooks & Edgington 2 m. Water samples from 4.5 to 4.75 m appeared 1994) but P from Lake KauhakG benthic sediments is brown, a coloration that may have arisen from brown unlikely to reach surface waters. Phosphorus (as P205) pigmented Chlorobium spp. (Guerrero et al. 1985, comprises -0.3% by weight of the Kalaupapa basalt Garcia-Gil et al. 1993). These bacteria usually occur in (Clague et al. 1982) and could constitute a relatively meromictic lakes in well-defined, colored 'plates' large supply to the water column if leached from sur- (Takahashi & Ichimura 1970, van Gemerden et al. rounding rocks. Additional utilizable P is likely found 1985). This work is the first report of autofluorescent in vegetation debris entering the lake (Vogt et al. 1986). Bacteria in Lake KauhakG. These cells peaked numer- SRSi concentrations in the upper meter of Lake ically at the pycnocline (4.5 m). Since most of the Bac- KauhakG exceeded those usually found in rivers and teria from 4 m observed by epifluorescence microscopy 102 Aquat Microb Ecol

were associated with particles, this part of the lake Chnstian JR, Uarl DM (1995) Bacterial ectoenzymes in marine may be a major site of organic matter recycling. waters: activity ratios and temperature responses in three Proteolytic enzyme activities in Lake KauhakG ex- oceanographic regions. Limnol Oceanogr 40:1042-1049 Clague DA, Dao-Gong C, Murnane R,Beeson MH, Lanphere ceeded those in meromictic Mahoney Lake (Overmann MA, Dalrymple GB, Friesen W, Holcomb RT (1982) Age et al. 1996) and Mekkojarvi (Munster 1991). Although and petrology of the Kalaupapa basalt, Molokai, Hawaii. we applied substrate analogs at concentrations con- Pac Sci 36:411-420 sidered saturating in oceanic samples, they may not Codispoti LA, Friederich GE, Murray JW, Sakamoto CM (1991) Chemical variability in the Black Sea: implications have been saturating in any or all our Lake KauhakB of continuous vertical profiles that penetrated the samples; enzyme activities may therefore have been oxic/anoxic interface. Deep-Sea Res 38:S691 -S710 underestimated. Total glucosidase activities were high Cole GA (1983) Textbook of lirnnology, 3rd edn. CV Mosby, St compared to those found in other aquatic habitats Louis (Munster 1991, Karner & Rassoulzadegan 1995) al- Cole JJ, Pace ML, Caraco NF, Steinhart GS (1993) Bacterial biomass and cell size distributions in lakes: more and larger though per cell activities of each enzyme were within cells in anoxic waters. Lirnnol Oceanogr 38:1627-1632 the range reported for pelagic marine bacteria (Do- DOE (Department of Energy) (1994) Handbook of methods for nachie et al. unpubl.). the analysis of the various parameters of the carbon diox- KauhakG Crater '...contains one of the finest exam- ide system in sea water; version 2. Dickson AG. Goyet C (eds) ORNLKDIAC-74. U S. Department of Energy ples of ... windward dryland forest [in] the state of Dore JE, Uarl DM (1996) Nitrification in the euphotic zone as Hawaii' (Medeiros et al. 1996) and was designated a a source for nitrite, nitrate, and nitrous oxide at station Special Ecological Area in 2994. Anthropogenic ALOiiA. Limnoi Oceanogr 4i:i619-i628 impact on the lake may be minimal; it represents a Dore JE, Houlihan T, Hebel DV, Tien G, Tupas L, Karl DM unique site in which to study interactions among rela- (1996)Freezing as a method of sample preservation for the analysis of dissolved inorganic nutrients in seawater. Mar tively isolated flora and fauna, much of which appears Chem 53:173-185 restricted to a shallow water column within a single Elkins JW (1980) Determination of dissolved nitrous oxide in basin. aquatic systems by gas chromatography using electron- capture detection and multiple phase equilibration. Anal Chem 52:263-267 Acknowledgements. We are indebted to Supt Dean Alexan- Falkowski PG (1981) Light-shade adaptation and assimilation der of Kalaupapa National Historical Park (KNHP) for permit- numbers. J Plankton Res 3:203-216 ting us to visit Lake KauhakG, and Mr hck Potts (KNHP) for Fonselius SH (1983) Determination of hydrogen sulfide. In: logistic assistance. We thank Chris Carrillo for conducting the Grasshoff K, Ehrhardt M, Krernling K (eds) methods of DIC and alkalinity analyses, and Stephanie Christensen for seawater analysis. Verlag Chemie, Weinheim, p 73-80 the pigment analyses. John Casey, Scott Larned, Ken Longe- Fry B (1986) Sources of carbon and sulfur nutrition for con- necker and Scott Murakarni provided assistance in the field. sumers in three meromictic lakes of New York State. Lim- We gratefully acknowledge the valuable suggestions of 2 no1 Oceanogr 31:79-88 anonymous reviewers. 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Editorial responsibility: Tom Fenchel, Submitted: February 6, 1998; Accepted: March 17, 1999 Helsing~r,Denmark Proofs received from author(s): August 24, 1999