Spectroscopic and Microscopic Characterization ofPhototrophic Isolates and Mat Communities

Sherry Dollhopf

Microbial Diversity1999

Abstract

Phototrophic bacterial mats are common in many environments and play animportant role in sulfur and carbon cycling in many aquatic environments, as well asbeing excellent models for the development of ecosystems on the early earth. However,our understanding of the community structure ofbacterial mats and the factors thatdetermine that structure is incomplete. The composition of bacterial mats maintained inthe lab under different environmental conditions was ascertained by single colonyspectrophotometry, pigment extraction, and microscopy. In addition, similar techniqueswere used to examine the diversity of phototrophic colonies in primary agar dilutiontubes of mat material. Dilution tubes and sea-table enrichments yielded differentorganisms in all cases, and water turbulence, sulfide source, inoculum source and lightconditions effected the phototrophic species that appeared. Absorption spectra andmicroscopy from mat material identified five thiferent green sulfur bacteria, one purplesulfur bacterium, and seven different non-sulfur bacteria.

Introduction

Phototrophic bacterial mats are common in many environments and play animportant role in sulfur and carbon cycling in many aquatic environments, as well asbeing excellent models for the development of ecosystems on the early earth. However,our understanding of the community structure ofbacterial mats and the factors thatdetermine that structure is incomplete. The dominant organisms in the uppermost layerof a phototrophic mat vary, but the most common organisms are Cyanobacteria. Othermat structures include those dominated by Chioroflexus species or diatoms. Only a fewphototrophic communities have been described that have high numbers of purple sulfurphototrophs or other phototrophs near the surface of the mat. The reason for thepredominance of Cyanobacteria in most mats is unclear, and can not be explained by onefactor, such as light intensity (Ward, D. M.,1989).

The overall phenotypic and genotypic diversity within structured bacterial matshas not been fully explored. The area around Woods Hole, MA provides an excellentopportunity to sample a number of marine microbial mats that can be used for suchdiversity studies. Although genotypic techniques are veiy useful, simple phenotypicapproaches to studying microbial diversity can also give new information and do notalways require the isolation of pure organisms. In vivo and in vitro absorption spectra ofphototrophic communities and organisms can provide information about the organismspresent and their possible phenotypes, as can microscopy. In this study, the compositionof bacterial mats maintained in the lab under different environmental conditions wasascertained by single colony spectrophotometry, pigment extraction, and microscopy. Inaddition, similar techniques were used to examine the diversity of phototrophic coloniesin primary agar dilution tubes of mat material.

Materials and Methods

Maintenance of microbial mats. Microbial mats from the Great Sippewissett SaltMarsh were removed and placed into 15 gallon aquaria or large plastic containers.

*4

Seawater directly from Martha’s Vineyard Sound was dripped onto the mats and drainedoff slowly, maintaining approximately 1 to 5 inches of water above the mat surface. Alarge microbial mat from Trunk River was maintained by flowing sulfide rich water intoa tupperware container. The sulfide was generated by allowing seawater to pass througha bed of rotting seagrass, naturally reducing the sulfate present and producing sulfidicwater. Smaller Trunk River mats were maintained in glass sampling dishes filled withsulfidic mud and fed a constant flow of seawater. These smaller mats were covered withlight filters that allowed the passage of light of wavelenths 715-780 rim or 450-520 nm.A no light control was also constructed, however small amounts of light allowed for thedevelopment of some phototrophic organisms.

Phototrophic agar dilution tubes. Media was prepared as described in the coursehandout (Hanselmann, 1999). Briefly, three types of media were prepared. A brakishmineral salts medium plus 0.9 % agar was prepared and amended with different electrondonors for phototrophic enrichments. Non-sulfur phototroph cultures contained 5 mMeach of succinic acid, glutaric acid, malic acid, and acetic acid as electron donors. Greenand purple sulfur phototroph enrichments contained 2.5 and 1 mM sulfide, respectively.Mat material was homogenized and diluted in sterile seawater before addition to moltenagar shakes. After inoculation, the enrichments were incubated at room temperature andilluminated with a 40 watt light bulb. Subsequent transfers were made in the samemedia.

Single colony spectrophotometrv. Single colonies of 1999 isolates were removedfrom agar dilution tubes under a stream of nitrogen using sterile pasteur pipettes. Isolatesfrom the 1998 microbial diversity course were first streaked on agar slants of the mediadescribed above to obtain pure colonies before analysis. The cell material was placed ona glass microscope slide and a glass coverslip was gently placed over the cell material tocreate a thin layer of cells. The area of the slide containing the cell material was thenplaced in the light path of the Cary 50 spectrophotometer and a scan from 1100 nm to300 rim was performed. Background absorption from the glass slide was calculated byscanning the slide and coverslip without cell material. This background was thensubtracted from the spectrum.

Pigment extraction. Mat material was placed in a 1.2 ml eppendorf tube and

extracted under a stream of nitrogen using a 7:2 ratio of acetone:methanol until theremaining cell material was gray in color. Extracts were then scanned immediately withthe Cary5O spectrophotometer.

Oxygen and sulfide determination. Both oxygen and sulfide concentrations weremeasured using CHEMetrics Vacu-Vials photometric analysis kits (Sulfide 2 and Oxygen2).

Results

Comparison of sea-table mats. Many of the mats maintained in the lab changeddramatically after a few days. Cyanobacterial mats taken from the Sippewissett SaltMarsh and maintained on the sea-table in 15 gallon aquaria developed into light greenmats. In vivo spectral analysis revealed that only bacteriochiorophyll c was present inthis light green mat, and organisms similar to Chioroherpeton species were identified bymicroscopy (Fig. 1). Almost no cyanobacteria could be found in the mat after 3 to 4weeks in the lab. Mat material transferred to the lab from a high iron site in theSippewissett also changed drastically and developed an olive green mat that wasintermixed with light green spots. The olive green material contained onlybacteriochlorophyll c, however the morphology of the organisms were very different thanthose from the other bacterial mat, and resembled Pelodictyon chlathratforme (Fig. 2).Both green sulfur bacterial mats had no detectable sulfide in the water directly above themat, and up to 96 j.tM oxygen was present, indicating that this mat was effectivelyremoving all the sulfide diffusing up from the sulfidic mud below and was tolerant to lowlevels of oxygen (Table 1).

Only one mat kept its natural appearance in the lab. Trunk River mat materialmaintained with flowing sulfidic water contained a variety of Cyanobacterial cells thatcould be identified by microscopy, including Plectonema-like cells, Microcoleus species,and Lyngbya species (Fig. 3 and 4). The in vivo absorption spectrum of the Trunk Rivermat material confirmed the presence of Cyanobacteria. Only chlorophyll a andphycocyanin were detected. In contrast to the Sippewissett mats, which were maintainedunder low flow conditions with only sulfidic mud as a sulfide source, the water above the

Trunk River mat contained 0.84 uM total sulfide and 5 uM oxygen.

Phototrophic isolates. Colonies obtained from non-sulfur phototroph dilutiontubes in the 1998 and 1999 microbial diversity had a variety of morphotypes, howevertheir in vivo absorption spectra, as determined from single colony spectral analysis, werequite similar (Fig. 5-8). The most unique spectra were seen in the 1998 colony obtainedfrom a dilution of pink sand material, which has a slightly shifted bacterioch.lorophyll aabsorption, and the 1999 brown non-sulfur colony 4, which has an additional absorbancepeak at 883 (Fig 5 and 8).

Sulfur phototroph dilution tubes from 1999 yielded three different colonies (Fig.9-11). Colonies one and two had spectra consistent with green sulfur bacteria. 16SrDNA sequence analysis done by Jackie Aislabie confirmed that these two organismswere related to green sulfur bacteria. The closest relative to colony 1 wasProsthecochioris aestuarii, and both the morphology and absorption spectra seen in thiscolony agree with this identification. Colony 2 was most closely related to Chiorobiumphaeobacteriodes, which again agrees with both the morphology and absorption spectra(Fig 12). No 16S rDNA sequence information is available for the third colony obtainedfrom the sulfur phototroph dilution tubes, however its spectrum and morphology indicatethat it is a member of the Chomatiaceae.

Effects of light ciualitv. Selective light filters enriched for different chlorophyllsand light harvesting accessoiy pigments from the Trunk River mat material. The matsthat received green light (450-520 nm) and low light were enriched in accessory lightharvesting pigments that absorb light below 500 nm, with a ratio of 410:665 ofapproximately 3 (Fig. 13). The 7 15-780 light selected for chlorophyll a containingorganisms (Fig. 14).

Discussion

The development of green sulfur bacterial mats from Sippewissett mat materialwas unexpected. The reason for the apparent disappearance of Cyanobacteria from themats under the lab conditions is not understood; however, it is possible that the steady

supply of sulfide from beneath, and the relatively undisturbed nature of the mats in thelab allowed green sulfur bacteria such as Chioroherpeton and Pe1odicyon to outcompeteCyanobacteria for light. The sulfide and oxygen measurement of the water overlying themats does not sufficiently explain the absence of Cyanobacteria, as there is actually moreoxygen present in the water above the green sulfur mats than there is above thecyanobacterial mat from Trunk River. The observation that the green sulfur mats wereeasily disturbed by turbulence in the water may provide a clue as to why this type of matis not commonly seen in the environment. The long, filamentous gliding Cyanobacteriaare much better adapted to form a solidified mat that is resistant to water turbulence andother physical disturbances that occur often in a natural setting, thus giving theCyanobacteria the advantage in the environment. The selection of different pigmentsunder different light conditions worked as expected and allowed organisms that areusually found below the surface of mats to predominate at the surface.

The sulfur and non-sulfur phototrophic organisms cultured in dilution tubes ofmat material were fairly diverse; however, the mat enrichments on the sea-table yieldedorganisms that were not detected in the dilution tubes. This indicates that both methodscan enrich for different organisms and that culture independent methods are needed toassess the importance of the organisms in the natural environment. This does notdiscredit the culture methods, which provided excellent information about the generaldiversity of phototrophs in the mat.

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