spectral fingerprinting of algal communities: a ... - Wiley Online Library

J. Phycol. 41, 439–446 (2005) r 2005 Phycological Society of America DOI: 10.1111/j.1529-8817.2005.04162.x

SPECTRAL FINGERPRINTING OF ALGAL COMMUNITIES: A NOVEL APPROACH TO BIOFILM ANALYSIS AND BIOMONITORING1 Chad Larson and Sophia I. Passy2 Department of Biology, University of Texas at Arlington, Box 19498, Arlington, Texas 76019-0498, USA

A new technique for spectral fingerprinting of major algal groups in the freshwater periphyton (i.e. cyanobacteria, green algae, and diatoms) was developed using confocal laser scanning microscopy. This technique used the differential spectral emission signatures of photosynthetic algae and allowed their spatially explicit quantification and community three-dimensional reconstruction. Algal biovolume measurements, carried out with this technique, are superior to existing protocols involving chl and ash-free dry mass assessments because they are nondestructive, localized, and specific at a group level. This technique can be used to generate depth profiles of the periphytic mat with various applications in aquatic ecology and biofilm analysis.

biofilm thickness: algal, bacterial, and exopolymer biofilm components (Lawrence et al. 1998a, Zhang and Fang 2001); live and dead biovolume distribution (Neu and Lawrence 1997); and the relationship between biofilm fractal structure and direction of water flow (Hermanowicz et al. 1995). Periphytic biofilms represent a unique system, where photosynthetic algae coexist within a multilayered structure composed of multiple species of various profiles, growth habits, and successional appearance. Periphyton is a major source of organic carbon in lotic ecosystems and a powerful indicator for ecosystem health. Yet, largely due to the lack of relevant technology, there is disproportionate scarcity of information on the three-dimensional (3-D) structure of live periphytic biofilms and how the environment affects it. Streamwater quality deterioration as a consequence of anthropogenic influence is a major concern of our society, and biomonitoring this process has been of paramount importance to a number of state and federal agencies. Biomonitoring protocols using periphyton have been developed and constantly improved but so far have been restricted to measuring algal biodiversity (species composition) and biovolume (cell density, chl a, and ash-free dry mass) (Barbour et al. 1999). These techniques frequently involve destruction of the biofilm and multiple steps preceding the final water quality assessment, including substrate scraping, laboratory processing, microscopy analyses (identification and counting), entering data into spreadsheets, and various metric calculations. Hence, there may be progressive error accumulation associated with these protocols, stemming from a number of reasons. First, not all cells are removed from the substrate because the number of missed cells depends on the amount of human effort and the roughness of the substrate (Becker et al. 1997), which can vary tremendously across sites even within the same survey. Second, not all cells are recovered after chemical processing, which sometimes requires multiple washing of the material. And finally, simple miscalculations of the metrics can result in erroneous conclusions. On the other hand, total biovolume measurements, which are less prone to some of the aforementioned errors, are destructive, nonspecific, depend on the efficiency of chl extraction, and are sensitive to the ratio of chl a to the other types of chl, often resulting in chl under- or overestimations. Our objective was to develop a technique based on confocal laser scanning microscopy (CLSM) that would

Key index words: benthos; biovolume; confocal laser scanning microscopy; phytoplankton; periphyton; three-dimensional reconstruction Abbreviations: 3-D, three-dimensional; ANOVA, analysis of variance; CLSM, confocal laser scanning microscopy; LM, light microscopy Recently there has been a growing interest in microbial biofilm structure and function because of their practical importance and implications for biotechnology and bioengineering. The structure of microbial biofilm communities has been shown to be related to their functional responses to the environment, to the nutrient uptake and efflux of wastes, to intrabiofilm mass transport dynamics, and to the interactions that occur in the communities (Tolker-Nielsen and Molin 2000). For that reason, much research has been devoted to revealing microbial biofilm structure, using LM, image analysis, and molecular approaches (Davies et al. 1998, Yang et al. 2000, Zhang and Bishop 2001). Confocal microscopy has become an indispensable tool in microbial biofilm studies because it permits the nondestructive examination of live biofilms and the detection of structural and compositional patterns at precisely defined distances from the biofilm surface. Thus, with the aid of a confocal microscope, the following attributes have been sequentially quantified within the

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Received 8 September 2004. Accepted 21 December 2004. Author for correspondence: e-mail [email protected].

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allow 1) the spectral separation of algal groups for the study of the 3-D structure of intact biofilms in real-time and 2) a quick, reliable, spatially explicit, and noninvasive method for algal quantification for the purposes of stream ecology and biomonitoring. MATERIALS AND METHODS

Algal communities. An experiment involving natural biofilm communities was initiated in which 20 L of water were placed into each of three aquaria. The water was collected from a small stream flowing through the university campus. On the bottom of each aquarium, 49 49-mm porcelain tiles were placed equidistant from one another. A 400-W metal halide lamp, placed above the aquaria, provided light on a 14:10-h light:dark daily ratio. We allowed an initial period of colonization of 5 days, after which one tile was randomly retrieved from each aquarium and placed into an accompanying Petri dish with enough distilled water to cover the tile. The tiles were collected on days 5, 7, 10, 12, 14, and 17 of colonization and examined with the confocal microscope. For the depth profile study of algal colonization, the tiles were kept in aquaria for 26 days. Natural communities composed of a mixture of different algae were sampled from various sources, including drains and puddles. Artificial communities assembled from cultured algae, including Anabaena flos-aquae ´b.) Hilse (Lyngb.) De Bre´bisson and Navicula pelliculosa (Bre (from UTEX algal cultures, Austin, Texas, USA), and Oocystis sp. were used as controls in spectral analyses. Confocal microscopy. We used an Axioplan 2 LSM 510 META (Zeiss, Jena, Germany) with two single-channel detectors and a polychromatic 32-channel detector (META) for fast acquisition of lambda stacks (see below) and multiple adjustable pinholes for high-resolution multifluorescence imaging. The microscope was equipped with Ar laser (458, 477, 488, 514 nm) 30 mW, He/Ne laser (543 nm) 1 mW, and He/Ne laser (633 nm) 5 mW. Viewing was carried out with a 40 0.80 NA water-immersion objective. In the biofilm experiment, six random fields were examined on each tile. In vivo fluorescence of the algal communities was excited with 458- and 488-nm lasers, whereas their emission peaks were captured in the range of 580–740 nm. Spectral separation was achieved by means of a unique combination of spectral detection and analysis, performed by an emission fingerprinting device, the META detector, and Linear Unmixing software (Zeiss Advanced Imaging Microscopy Group, Jena, Germany) with the capability to separate up to eight emission channels. For each xy focal plane, the LSM 510 META measures the incremental emission every 10 nm and produces a stack of images, called lambda stack. Focusing through a specimen at desired increments on the z axis generates a Z-stack. An image stack with emission information in x, y, and z dimensions allows the determination of spectral signatures at any location of a specimen. Reference emission spectra for the algal classes were generated and stored in the Zeiss LSM Spectra Database using representative species from each class. The Linear Unmixing function uses reference spectra to separate the mixed signals in the image stack. It uses the entire emission spectrum of each fluorescing substance, which permits the separation of widely overlapping fluorescence emission signals and reduces the crosstalk between the different emission spectra. It should be noted that species within the same class were spectrally indistinguishable. Once a confocal image had been captured and the mixed signals separated with Linear Unmixing into separate channels, the biovolume (mm3 field 1) of each algal class in the image, which occupies a separate channel, was quantified using the 3-D for LSM software. A mean of the six random fields was

then multiplied by the number of fields in a square centimeter to obtain the biovolume of algae per square centimeter. The physiological state of an algal cell (particularly senescence) can affect its fluorescence, and in this study this was most evident in diatoms. Because of this, a small amount of the fluorescent signal emitted by senescing diatoms was at times detected in the green algae channel. However, this problem was largely alleviated by subtracting the signal emitted by one class of algae from the signal emitted by the other using the software functions. For the observations of artificial communities of cultured algae, two mixtures (A and B) were prepared by placing several drops from each culture into a depression slide. The three species were allowed to settle for 5 min after which a field was randomly chosen and the contents captured in a confocal image. After separating the different algal classes by applying the Linear Unmixing function, the biovolume for each algal class was quantified in 3-D for LSM as described above. Effect of formaldehyde additions on algal fluorescence. Approximately 25 mL of the A. flos-aquae, Oocystis sp., and N. pelliculosa cultures were placed into each of three scintillation vials. In one vial there was no addition of formaldehyde, whereas formaldehyde to a final concentration of 5% was added to the other two vials. Immediately after the addition of formaldehyde, the fluorescence of 10 individual cells was measured with the confocal microscope. The vial containing no formalin and one of the vials containing formalin were placed under constant illumination. The third vial containing formalin was placed in a drawer receiving no light. The fluorescence of algae was measured again after 1, 2, 3, 4, 7, and 14 days. LM. After observation with the confocal microscope, the biofilm on the surface of each tile was scraped off with a razor blade and a toothbrush, diluted, and preserved in 5% formaldehyde to a final volume of 40 mL. Biofilm ‘‘clumps’’ were separated with a pulse sonification device for 5 s. This was long enough to separate large clumps but short enough to avoid cell damage. After each 40-mL sample was uniformly mixed, a subsample was placed into a Palmer-Maloney counting cell (Wildlife Supply Company, Buffalo, NY, USA) and observed under a light microscope at 40 0.65 NA. The abundance of each algal class (cyanobacteria, green algae, and diatoms) was estimated by counting 20–30 fields and converting the number of counted cells to density per square centimeter of tile. Average cell volume for each species was determined in every sample by measuring the dimensions of all individuals when less than 20 were encountered and 20 when there were many individuals. The biovolume was calculated by incorporating the cellular dimensions in formulae for solid geometric shapes most closely matching the shape of the cells (Wetzel and Likens 1991). In each sample, cellular biovolume was determined by multiplying the number of cells of each species by its mean cell volume. Finally, total biovolume per class was derived by summing the respective cellular biovolumes of all encountered class constituents. For the examinations involving artificial communities of cultured algae, a digital transmitted light image of six fields was taken and immediately followed by the capture of a confocal image in each depression slide (mixtures A and B). The LSM viewing software allows the dimensions of objects within the image to be measured by outlining the object. Biovolumes of each class of algae in a field were obtained by measuring the dimensions of all objects and applying the aforementioned formulae. Statistical analyses. A three-way analysis of variance (ANOVA) with biovolume measurement method (confocal vs. transmitted light), algal class (cyanobacteria, green algae, and diatoms), and mixture (A and B) as factors and log10-transformed biovolume as the dependent variable was carried out on artificial

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RESULTS

FIG. 1. Emission spectra of three different algal classes obtained by CLSM. Each spectrum is an average of five measurements.

communities using SYSTAT 8.0 (SPSS Inc., Chicago, IL, USA). A repeated-measures ANOVA for two grouping factors (i.e. biovolume measurement method and algal class) and one within factor (i.e. day of colonization) was conducted on natural communities with SYSTAT.

Emission signatures of algal groups. Algal fluorescence was best induced after excitation with the Ar laser at 458 and 488 nm. The emission spectra of cyanobacteria were considerably different from green algae and diatoms. Emission of cyanobacteria peaked at 650 nm, whereas the emission of diatoms and green algae peaked at 675 and 690 nm, respectively (Fig. 1). This was also evident in the lambda stack showing algal emission in an incremental fashion (Fig. 2). The capability of the META detector for incremental emission reading made possible the spectral separation and subsequent quantification of diatoms and green algae despite the large overlap between their emissions (Figs. 3 and 4, a and b). Three-dimensional reconstructions of each algal component as well as the entire community were created for natural communities (Fig. 3) and biofilms, which were too thick, opaque, and packed with detritus to allow any coherent information to be gathered by LM (Fig. 4, c and d). Biovolume assessments. Biovolume assessments were carried out on natural and artificial communities. Natural communities on porcelain tiles were examined, and biovolume of each algal class was estimated by confocal microscopy directly from the tile and by Palmer-Maloney cell counting after scraping of the

FIG. 2. Lambda stack of the emission spectra of mixed algal community obtained by CLSM.

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FIG. 3. CLSM image illustrating the spectral separation into different channels and 3-D reconstructions of three algal groups in a mixed community. Each panel is also available as a movie clip for 3-D viewing at http://www.blackwellpublishing.com/products/journals/ suppmat/JPY/JPY04162/jpy04162sm.htm.

biofilm. Repeated-measures ANOVA demonstrated for each algal class that the biovolume across the six colonization days was not dependent on enumeration method (P 5 0.166, n 5 108). Analyses of artificial communities assembled from cultured algae supported these results. The confocal estimates of the biovolume of individual algal groups were again not significantly different from the Palmer-Maloney cell estimates across the two algal mixtures (three-way ANOVA, P 5 0.680, n 5 72). Effect of formaldehyde additions on algal fluorescence. The addition of formaldehyde did not imme-

diately alter the algal fluorescence (Fig. 5). As time progressed, the fluorescence of the fixed algae kept in the dark persisted but their emission spectra changed, which was most evident in cyanobacteria and green algae. All fixed algae continued to fluoresce with diminished intensity even after 14 days of exposure (data not shown). Fixed algae exposed to light from all three groups exhibited more or less strongly diminished fluorescence 1 day after fixation and negligible fluorescence by the end of the experiment (Fig. 5). Despite the continuous fluorescence of algae even after 14 days of fixation, their successful


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FIG. 4. Transmitted light image (a) and CLSM image (b) of an artificial community, consisting of Anabaena flos-aquae, Oocystis sp., and Navicula pelliculosa (pseudo-colored in blue, green, and orange in b, respectively). Transmitted light image (c) and CLSM image (d) of a natural biofilm, consisting of cyanobacteria and green algae (pseudo-colored in blue and green in d, respectively). Images c and d are also available as movie clips at http://www.blackwellpublishing.com/products/journals/suppmat/JPY/JPY04162/jpy04162sm.htm, showing a focusing through the depth of the biofilm and biofilm 3-D reconstruction, respectively.

spectral separation was achieved only within the first 2 days of exposure to formalin. After 1 day of exposure, the spectral separation of the three species was as clear as with the live controls, that is, each algal group emitted only in one channel (as in Fig. 3). After 2 days, the green algae started to fluoresce in the diatom channel, but this noise was eliminated by the software subtraction function. At longer exposure durations, fluorescence of all three groups

was detected in all channels, which prevented their distinction. Depth profile. After confirming the accuracy of confocal microscopy in spectral separation and biovolume assessment of different algal groups, the 3-D structure of natural biofilms was examined during the course of their succession. Tiles with growing biofilm were retrieved from the tanks on days 3, 21, and 26 of the experiment, analyzed with CLSM, and

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FIG. 5. Temporal autofluorescence patterns of Anabaena flos-aquae, Navicula pelliculosa, and Oocystis sp. after preservation in 5% formaldehyde as measured with CLSM.

redeployed. Distinct community shifts were observed during biofilm development. In the early stages, the biofilm was homogeneous with diatom dominants and chlorophyte subdominants evenly distributed throughout the biofilm thickness (Fig. 6). In the intermediate and final stages, the biofilm became progressively stratified into chlorophyte-dominated and diatom-dominated layers (Figs. 6 and 7). Potential shortcomings of the CLSM fingerprinting technique. Capturing lambda and Z-stacks of thick biofilms is time consuming and may take up to 20 min. This may result in imprecise biovolume estimates of motile cells or colonies, which quickly traverse the field of view. If the biofilm is rich in motile species but not designed for redeployment, it should be fixed before confocal analysis; however, the examination should be carried out within 48 h of fixation. There are possible limitations imposed by the biofilm thickness and compactness. In less compact biofilms, composed of a mixture of algal groups with different profiles, the maximal encountered depth

was 360 mm, which was seen as an opaque mass in LM but was successfully spectrally dissected by CLSM. In highly compacted biofilms produced by the tight packing of filamentous algae such as nostocalean cyanobacteria, the CLSM lasers were able to penetrate only the top 200 mm. Preliminary analyses suggest that the technique may be ineffective in distinguishing algal groups having very similar pigment composition such as the chl b-bearing chlorophytes and euglenophytes or the phycobiliprotein-bearing cyanobacteria and rhodophytes. DISCUSSION

Periphytic biofilms have a complex structure and many of their properties are best understood in three dimensions. Important ecological matters such as colonization, population dynamics, interspecific competition, and herbivory exist only in the context of the spatial organization of the biofilm and species patch dynamics. Still, very few spatial aspects of its structure


FIG. 6. Depth profiles of natural biofilms on tiles after 3, 21, and 26 days of colonization.

and development have been investigated in reference to underlying physicochemical factors (Hoagland et al. 1982, Steinman and Mcintire 1986, Hudon and Legendre 1987, Steinman et al. 1989). A major reason for this has been our inability to study intact and growing biofilms in their entirety. The approaches available so far are almost exclusively based on SEM (Greenwood et al. 1999), which destroys the living cells, discriminates well only hard-shelled algae such as diatoms, and provides only snapshots of single vertical or horizontal planes. Light microscopy is inadequate for structural biofilm analysis because as they grow, biofilms quickly

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become opaque and their algal constituents indistinguishable. Confocal microscopy is becoming increasingly popular in microbial biofilm studies (Lawrence et al. 1998b, Lawrence and Neu 1999). However, existing confocal microscopy methods for periphytic bio´ et al. 2001, film analysis (Lawrence et al. 1998a, Sole Neu et al. 2004) fail to distinguish between major algal groups that are spectrally similar, such as green algae and diatoms, and to produce direct and differential volumetric measurements of algal accumulation. Here we described a new technique for spectral fingerprinting of periphytic biofilms using confocal microscopy, which permits in vivo examination of biofilms, discrimination of the major algal groups, and the differential reconstruction of their 3-D structure. Moreover, periphytic biofilms that naturally occur on a wide variety of opaque substrates, such as stones, sand, bark, and snags, can be viewed just as easily as biofilms on glass slides. Finally, precise volumetric measurements render this technique a useful tool for aquatic ecology and biomonitoring. We outline several applications with great potential for future aquatic research. First, emission fingerprinting using confocal microscopy can be used in analyses of biofilm colonization patterns under different environmental conditions. Detection and quantification of fluorescence shifts within biofilm depth would indicate major community shifts throughout bioaccumulation resulting from abiotic and biotic interactions. The thickness of the biofilm can be partitioned into slices (Z-stack), where emission spectra of individual algal groups are captured (lambda stack). From both stacks the characteristic depth profile of each algal group can be reconstructed in three dimensions, thus revealing information about algal spatial arrangement within the biofilm. Such analyses are impossible with conventional LM or EM. Second, this technique enables higher resolution biovolume measurements (at an algal group level) than standard procedures for chl assessment. The separate biovolume quantification of each higher algal taxon presents significantly more detail about the makeup of an algal community than total biovolume alone. This is achievable without having to destroy the sample by extracting the various photosynthetic pigments. By eliminating the need to remove the attached biofilm from the substrate, there is no loss of material and analysis of the 3-D structure of the algal community can be performed. Spectral fingerprinting can be applied equally successfully in phytoplankton biovolume assessments. It may aid in biomonitoring of nuisance cyanobacterial blooms, assessments of phytoplankton, or periphyton primary productivity in any aquatic environment because of its discriminating power at an algal class level. Another possible application is in the analysis and quantification of grazers’ gut contents in real time. Live protozoans, crustaceans, and midge larvae fed on different diets can be viewed and their food vacuole or gut content spectrally analyzed, which is an essential

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FIG. 7. Transmitted light image of biofilm accumulation after 26 days (a). Depth profile reconstruction of the image in a showing the differential spatial distribution of algal taxa (b). Both images are also available as video clips at http://www.blackwellpublishing.com/ products/journals/suppmat/JPY/ JPY04162/jpy04162sm.htm.

advantage over the existing invasive techniques (Glozier et al. 2000). Grazing has important implications for algal accrual and succession, and as an intermediary step in the organic matter flow through the food webs, it is pivotal in ecosystem functioning. Therefore, the ability to track the food preference and amount in grazers’ diets is of great importance. In conclusion, we present a spectral fingerprinting technique using confocal microscopy for the analysis of biofilms that is as precise at estimating algal biovolume as conventional counting but more specific than existing methods for total biovolume assessment, such as chl a and ash-free dry mass measurements. It allows for repeated real-time examinations of changing community composition and 3-D structure of attached biofilms in response to a changing environment. This work was supported by the University of Texas at Arlington and the National Science Foundation under grant no. 0215852 to S. P. We are grateful to Jim Grover for insightful comments and discussions and to the two anonymous reviewers, whose constructive suggestions helped improve the manuscript. Barbour, M. T., Gerritsen, J., Snyder, B. D. & Stribling, J. B. 1999. Rapid Bioassessment Protocols for Use in Streams and Wadeable Rivers: Periphyton, Benthic Macroinvertebrates and Fish. 2nd ed. EPA 841-B-99-002. U.S. Environmental Protection Agency, Office of Water, Washington, DC, 339 pp. Becker, G., Holfeld, H., Hasselrot, A. T., Fiebig, D. M. & Menzler, D. A. 1997. Use of microscope photometer to analyze in vivo fluorescence intensity of epilithic microalgae grown on artificial substrata. Appl. Environ. Microbiol. 63:1318–25. Davies, D. G., Parsek, M. R., Pearson, J. P., Iglewski, B. H., Costerton, J. W. & Greenberg, E. P. 1998. The involvement of cellto-cell signals in the development of a bacterial biofilm. Science 280:295–98. Glozier, N. E., Culp, J. M., Scrimgeour, G. J. & Halliwell, D. B. 2000. Comparison of gut fluorescence and gut dry mass techniques for determining feeding periodicity in lotic mayflies. J. N. Am. Benthol. Soc. 19:169–75. Greenwood, J. L., Clason, T. A., Lowe, R. L. & Belanger, S. E. 1999. Examination of endopelic and epilithic algal community structure employing scanning electron microscopy. Freshw. Biol. 41:821–8.

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