Chapter 6
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Application of Chlorophyll and Carotenoid Pigments for the Chemotaxonomic Assessment of Seston, Periphyton, and Cyanobacterial Mats of Lake Okeechobee, Florida 1
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Nancy M. Winfree , J. William Louda , Earl W. Baker , Alan D. Steinman , and Karl E. Havens 2
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Organic Geochemistry Group, Department of Chemistry and Biochemistry, Florida Atlantic University, 777 Glades Road, Boca Raton, FL 33431-0991 Ecosystems Restoration Division, South Florida Water Management District, 3301 Gun Club Road, West Palm Beach, FL 33406
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The presence of chlorophyll-a and its derivatives in an environmental sample indicates the presence of oxygenic photoautotrophs. The accessory (-b, -c) and alternate(bacteriochlorophylls-a,-b,-c,-d,-e) chlorophylls and the carotenoids provide additional biomarker specificity. That is, the presence of these pigments gives information about taxonomic structure. In the present study we utilized high performance liquid chromatography ( HPLC ) / ghotodiode array (PDA) methodology to investigate the photoautotrophic communities in Lake Okeechobee, Florida. Pigment distributions were measured in unispecific cultures and compared to literature reports on natural populations in order to generate equations for the estimation of the relative abundances of photoautotrophic taxa in seston, epiphytes, and benthic samples from 14 locations in the lake. The taxa and their specific biomarkers considered herein include: anoxygenic Eubacteria ( purple and green sulfur bacteria: bacteriochlorophylls-a / -c ), Cyanobacteria (myxoxanthophyll), Chlorophyta (chlorophyll-b, lutein), Chrysophyta (fucoxanthin), and Pyrrhophyta (peridinin). The estimate of each taxon was based upon the relative abundance of the specific biomarker pigment for that group and its quantitative relationship to chlorophyll-a. As examples of these methods, the technique of pigment-based chemotaxonomy is applied spatially and temporally to the photoautotrophic communities in Lake Okeechobee. The estimation of phytoplankton biomass using pigment data, with certain inference as to types (taxa), was first performed by Kreps and Verbinskaya during a 1930 study of the seasonal changes in diatom populations in the Barents sea (7). The utilization of © 1997 American Chemical Society
In Molecular Markers in Environmental Geochemistry; Eganhouse, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1997.
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MOLECULAR MARKERS IN ENVIRONMENTAL GEOCHEMISTRY
pigment 'colorimetry' for phytoplankton studies was popularized in 1934 by Harvey and included the first report of 'patchiness' in natural aquatic ecosystems (2). Spectrophotometric analyses of photopigment quantity and quality gained increasing utility with the advent of sets of equations with which to estimate the chlorophylls (-a, -h, -c) and total carotenoids in lipid extracts (3-6). Errors in these spectrophotometric methods have been reported as being due to the overlapping spectral bands of coincident pigments (7-11). The sensitivity and, to a certain degree, the selectivity of spectral estimates were enhanced by the utilization of fluorometric determinations (6,12-15). Reports of relatively large errors in this technique, due to overlapping fluorescence bands, have also appeared (16). The potential of reverse phase (RP)-HPLC was shown in 1979 by Brauman and Grimme with the separation of 15 pigments, including the epimeric forms of chlorophyll-a (17). The application of RP-HPLC to the analysis of phytoplankton communities, stressing the use of alloxanthin as a chemotaxonomic indicator for the cryptophytes, was reported by Gieskes and Kraay in 1983 (18). In that same year, Mantoura and Llewellyn reported on the inclusion of ion-pairing reagents (e.g. tetrabutylammonium acetate, ammonium acetate) for the enhanced separation of highly polar pigments (8). SCOR-UNESCO established a study ("Working Group '78") to compare methods and develop a standard method for the analysis of marine phytoplankton (19). Applications of HPLC-PDA analyses of pigment distributions for the study of aquatic systems are becoming more common. We adopted these methods to the study of the algal pigments in Lake Okeechobee, the largest lake in the S.E. United States. Algal blooms in Lake Okeechobee have been defined as occurring when chlorophyll-a concentrations meet or exceed 40 pg/L (20-21). These events have been dominated by cyanobacteria, and the largest bloom was recorded in 1987, covering about 700 Km (« 42%) of the lake's pelagic surface (22). Generally, since 1992 chlorophyll-a concentrations in the lake have averaged below 25 pg/L (23), and bloom frequencies are declining (20-21). The existence of algal (viz. cyanobacterial) blooms in this, or any other, lake could result in undesirable conditions for both man and the ecosystem. The present study was undertaken in order to assess the feasibility of applying HPLC/PDA based pigment analyses to the chemotaxonomic survey of the various photoautotrophic communities within Lake Okeechobee. In order to arrive at taxonomic conclusions from pigment data, we first had to address taxon-specific pigment ratios in (a) unispecific algal cultures, and (b) the literature. Using such data, equations could be derived which would allow taxon-specific chlorophyll-a contributions to be estimated. These equations would then provide an estimation of the relative importance of each taxon in any given sample. The goal of these studies is to provide a rapid, timely and reasonably accurate HPLC-PDA methodology with which to monitor short-term spatial and temporal changes in the photoautotrophic communities of Lake Okeechobee, Florida, with subsequent application to other environments. 2
In Molecular Markers in Environmental Geochemistry; Eganhouse, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1997.
6.
Application of Chlorophyll and Carotenoid Pigments
WINFREE ET AL.
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Experimental
Downloaded by UNIV MASSACHUSETTS AMHERST on October 4, 2012 | http://pubs.acs.org Publication Date: July 1, 1997 | doi: 10.1021/bk-1997-0671.ch006
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Site. Lake Okeechobee is a large ( 1,730 km ) shallow ( « 2.7m, * 04.5m) sub-tropical lake located at about 27°00'N x 80°00'W in the southern Florida peninsula. Wind fetch is in excess of 50 km and, coupled with its shallow depth, leads to resuspension of solids (20-21, 23-25). The lake has been described as consisting of five distinct ecological zones: (1) northern, (2) central, (3) southern ( "edge" ), (4) western-transitional, and (5) littoral. Sediments in these areas are predominately mud in the north and central regions, sand in the west/littoral and a mosaic of rock, marl and organic peat in the south (23-26). The littoral zone(s), mainly in the west, southwest and northwest fringes, are about 1-2 m in depth and support a wide variety of emergent macrophytes plus epiphyton. The littoral regions of the Lake Okeechobee amount to about 22% of its areal extent(20). Samples. Samples were collected on a quarterly basis from the 14 sites designated in Figure 1. Samples included seston, epiphyton and benthic phototroph mats. Benthic samples were collected using a coring device constructed of PVC pipe ( 7.34 cm cross section) and a one-way valve (27). These samples were extruded into polyethylene bags immediately upon retrieval and placed on ice untilfrozenabout 2-6 hours after collection. Prior to analysis, benthic mat samples were thawed to ice-bath temperatures (0-2°C) and collected onto GF/F filters in order to remove excess water. Epiphyte-containing macrophytes were removed from the lake and placed on ice. Epiphytes were removed from the host plant ( Scirpus sp., Eleocharis sp.) collected onto GF/F filters, placed into polyethylene bags, andfrozen.The surface area and weight of the host macrophyte were recorded at this time. Water samples were collected using a depth integrating technique. That is, a small pump, with tubing, attached to a rod was alternately raised and lowered through the water column to within 0.5 m of the surface and the bottom. Water was pumped into dark brown carboys and transported to shore, where the seston was collected by filtration onto GF/F filters and frozen. 2
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Solvents. All solvents were of Fisher Scientific "Optima" grade, or better. Water used was deionized at a resistivity of
