Independent Project Reportfor Microbial Diversity page 1

Independent Study Abstract(fl Microbial Diversity Summer Course

Tsute ChenDepartment of MicrobiologyUniversity ofMassachusetts

July30. 1993

I. Isolation of Purple Sulfur Bacteria from Purple Aggregates inGreat Sippewissett Salt Marsh, Massachusetts

Dense-packed purple bacterial aggregates were found in the sea water pools on a peninsula in atidal channel of Great Sippewissett Salt Marsh, Cape Cod, Massachusetts in 1992. Ecologicalsignificance of these so-called “purple berries” has been studied by Seitz, et al and purple sulfurbacteria (primary Thiocapsa roseopersicina) were found to be the major component in theaggregates. The purpose of this study was to isolate purple sulfur bacteria from these aggregatesusing agar-shalce tube method. Nine single colonies were isolated and cultured phototrophicallyin liquid medium containing Na2S and CO2. Two different cell morphological types (cocci androds) were found in these isolates and cells with the same morphological type possessed similarpigment analytical patterns. The tentative species name for these isolates are Thiocapsaroseopersicina and Chromatium violascens.. Aggregates were never formed by each singleculture, nor by the coculture of both types. Three other coccoid cultures (possibly also Thiocapsaroseopersicina) were isolated from purple sand layers on the sandbars of the tidal channeladjacent to the purple-berry pools. One of these three isolates devoloped from a small singlecolony (ca. 0.5 mm) to a larger purple aggregate (3 mm) similar to the natural aggregates,suggesting that purple aggregates might originate from the purple sand layer of the nearby tidalchannel.

II. Diversity of Purple Sulfur Bacteria in the Microbial Matat Great Sippewissett Salt Marsh, Massachusetts

Phototrophic purple sulfur bacteria occur in the light-penetrating and sulfide-containing layers ofnatural environments such as stratified lakes and microbial mats. Purple sulfur bacteria have longbeen found to be a morphological-diversed group and there are still a large number of them withunknown morphologies remained to be discovered. This study investigated the diversity of purplesulfur bacteria in the microbial mat from Great Sippewissett Salt Marsh. Six morphological typeswere isolated from the enrichment sample using general agar shake-tube method, although noneof them were considered to be new species. Four sulfur globule-containing bacteria with dipolarflagella, which were considered to be new species of photorphic purple sulfur bacteria, were also

Cobserved under phase-contrast microscope. However, after careful examination of coloniesformed in the shake tubes, none of these organisms was detectable. Whether they arephototrophic purple sulfur bacteria remains to be determined.

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I. Isolation of Purple Sulfur Bacteria from Purple Aggregatesin Great Sippewissett Salt Marsh, Massachusetts

Tsute ChenDepartment ofMicrobiologyUniversity ofMassachusetts

July 30, 1993

IntroductionDense-packed purple bacterial aggregates were found in the sea water pools on a peninsula in atidal channel of Great Sippewissett Salt Marsh, Cape Cod, Massachusetts in 1992. Ecologicalsignificance of these so-called “purple berries” has been studied by Seitz, et al and purple sulthrbacteria (primary Thiocapsa roseopersicina) were found to be the major component in theaggregates. The purpose of this study was to isolate purple sulfur bacteria from these aggregatesusing agar-shake tube method.

Materials and MethodsStudy MaterialsPurple aggregates (purple berries) were collected from a sea water pooi adjacent to the GreatSippewissett Creek channel. Purple sand was found and located on the sand bar in the samechannel.

(D Isolation of Purple Sulfur Bacteria and Incubation conditionsStandard agar shake-tube method described in the course syllabus and sea water version ofchromatium medium were used for isolation. Purple berries were smashed and suspended in sterilesea water and purple sand were shaken in the sterile sea water before introducing into a serial ofshake-tubes. Newly inoculated agar tubes were incubated in the dark overnight and incubatedunder tungsten light at room temperature. Capillary Pasteur glass pipets were used to isolatesingle purple colonies developed in the agar and single colony was resuspended again andtransferred through another serial of agar tube. The purified culture was then introduced into freshchromatium liquid in screw cap tubes and incubated under the same condition with periodicallychecking and feeding Na2S.

Pigment AnalysesBacteriochlorophyll a was determined after extraction of the samples (either pure culture ornatural sample) in 99.5% acetone (Al, A2, A3, and Bl: overnight at 4°C; C and E: 4 hat roomtemperature). Individual aggregates were extracted after grinding in a homogenizer. Absorbencywas measured in a Shimadzu UV-3 101 PC spectrophotometer.

ResultsNine single colonies were isolated and cultured phototrophically in liquid medium containingNa2S and CO2 (Table 1). Four of them were coccoid (Al, A2, A3, and BI) without motility andthe other five were all rods. Similar morphological type (Al, A2, A3, and Bl; or C and E;)possessed similar pigment composition according to the pigment analysis pattern (Table 2 and

C Figure 4-6). These isolated were considered to be Thiocapsa roseopersicina (Al, A2, A3, andB1; Figure Ia) and Chromatiwn violascens (B2, C-F; Figure Ib).. Aggregation did not occureither in the single culture or in the coculture ofBl and C. Three other coccoid cultures (possibly

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also Thiocapsa roseopersicina; Table 1) were isolated from purple sand layers on the sandbars of(Th the tidal channel adjacent to the purple-berry poois. Surprisingly one of these three isolates

developed from a small single colony (ca. 0.5 mm) to a larger purple aggregate (3 mm) similar tothe natural aggregates, suggesting that purple aggregates originate from the purple sand layer ofthe nearby tidal channel.

DiscussionAlthough two different species of purple sulfur bacteria were isolated from purple berries and onespecies from purple sand. Microscopic examination of the isolates showed differences from thenatural sample either in term of color or the cell size in the cell clumps. However, a small berrywas formed in the liquid medium from a purple sand sample. The color of the berry looked similarto the natural berries but the color of the cell clumps looked differently (purple pink in the formerand yellowish in the later). Besides, there was no slime layer surrounding the cell clumps of theartificial berry while natural berry clumps are always surrounded by a thick (5-10 jim)polysaccharide layer (picture not shown). However, these differences could be due to thedifference between the laboratory conditions (medium, pH, temperature, etc.) and naturalconditions.

Pigment analyses (Table 2; Figure 4-6) also revealed that there were only two type of bacteriaamong these isolates. Analysis of isolates from purple sand (W, X, and Y) were not done becauseof the slow growing rate and there was still not enough cell mass for pigment extraction.Extraction of isolates C and E was much easier than of A1-3 and B1. Dark purple-violet colorappear almost right after adding of acetone in the case of C and E. But for the coccoid cells,there was only slightly color development in the acetone extract. Either the pigment in the cocciwas very sensitive to the light (although most of the extraction was done in the dark) or it degradeat room temperature or in the acetone. Other organic reagent should be tried in the future work.More direct approaches such as molecular probe specific for purple sulfur bacteria could be used

to verif’ whether these isolates are the major component of the purple berries. For example, 16 SrRNA genes from these isolates can be cloned by PCR and used as the probe for the in situhybridization with natural samples. Moreover, regeneration of purple berries under laboratoryconditions should also be tried to see if it’s repeatable since this phenomena was found toward theend of the course, leaving no much for further study.

References1. Nicholson, J.A.M., 3.F. Stolz, and B.K Pierson (1987) Structure of a microbial mat at Great

Sippewissett Marsh, Cape Cod, Massachusetts. FEMS Microbiol. Ecol. 45: 343-364.2. Pierson, B., A. Oesterle, and (IL. Murphy (1987) Pigments, light penetration, and

photosynthetic activity in the multilayered mats of great Sippewissett Salt Marsh,Massachusetts. FEMS Microbiol. Ecol. 45: 365-376.

3. Gibson, 0., E.R. Leadbetter, and H.W. Jannasch (1984) Great Sippewissett Salt Marsh: Asummary of projects carried out by students in the microbial ecology course of the MarineBiological Laboratory, Woods Hole, during summers 1972-198 1. In: Microbial Mats:Stromatolites (Cohen Y., LW. Castenholz, H.O., and Halvorson, eds.) Alan Liss, New York.p. 95-100.

4. Seitz, A., T.H. Nielsen, 3. Overmann (1993) Ecological Significance of purple sulfi.ir bacterialaggregates in Great Sippewissett Salt Marsh, Massachusetts, USA. (in press).

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II. Diversity of Purple Sulfur Bacteria in the Microbial Matat Great Sippewissett Salt Marsh, Massachusetts

Tsute ChenDepartment ofMicrobiologyUniversity ofMassachusetts

July 30, 1993

IntroductionPhototrophic purple sulfur bacteria occur in the light-penetrating and sulfide-containing layers ofnatural environments such as stratified lakes and microbial mats. Purple sulfur bacteria have longbeen found to be a morphological-diverse group and there are still a large number of them withnew morphologies remained to be discovered. This study focused on isolating a new species andinvestigated the diversity of purple sulfur bacteria in the microbial mat from Great SippewissettSalt Marsh.

Materials and MethodsStudy materialsA square of microbial mat sample was taken back from the Great Sippewissett Salt Marsh and putin a plastic basin with illumination during the day and fresh water flowing at night. Purple sulfurbacteria sample was picked from the pink or orange layer of the surface mud and examined undermicroscope and serial diluted in the agar tubes. Part of the Sippewissett mat was kept in a glassjar filled with sea water and incubated under a constant tungsten light source at roomtemperature. Purple layer was developed all over the glass wall toward the light with a thin greenlayer in the middle.

Isolation of purple sulfur bacteria and incubation conditionsStandard agar shake-tube method described in the course syllabus and sea water version ofchromatium medium were used for isolation. Purple berries were smashed and suspended in sterilesea water and purple sand were shaken in the sterile sea water before introducing into a serial ofshake-tubes. Newly inoculated agar tubes were incubated in the dark overnight and incubatedunder tungsten light at room temperature. Capillary Pasteur glass pipets were used to isolatesingle purple colonies developed in the agar and single colony was resuspended again andtransferred through another serial of agar tube. The purified culture was then introduced into freshchromatium liquid in screw cap tubes and incubated under the same condition with periodicallychecking and feeding Na25.

Phase Microscopy and PhotographyZeiss phase contrast microscope was used to examine cell morphology and Kodak Ektachromecolor side films (ASA16OT with reciprocal value = 5 and ASA 100 or 200 with recp. value = 0)were used for photography.

Results and DiscussionSix types of purple surlfl.tr bacteria (Table 3) were isolated from the microbial mat andcharacterized as some common species which have been described already. Besides, greatnumber of sulfur globule-containing bacteria were enriched and observed in the microbial mat andin the glass jar maintained under laboratory conditions. And many of them in these two particularenrichment possessed interesting morphologies (Figure 2, 3). For example, a dipolar flagellated

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spirillum (Figure 2a; when it moves it moves like spirillum although it doesn’t look like that) wasthe first one found and described from orange brownish layer of the mat. Many efforts had thenbeen tried to isolate this particular organism using general agar shake-tube method. But most ofthe cases, non-purple sulffir bacteria out competed purple sulfur ones and it was quite often thateven no single purple colony could be found in the agar tube. The only successfi.ul try was toincubate the agar tubes behind a gelatin filter specific for the wavelength specific for bacterialchlorophyll a and b. Even in this case, only one type of purple colonies were found and isolated(Table 3, isolates Z1-3). In few cases, even cyanobacteria bloomed inside the tube, makingaerobic conditions inhibiting the growth of anoxygenic phototrophs. Through the whole studyperiod and after screening hundreds of colonies, none of the colonies were found to have theseinteresting rnorphologies. This indicates that the medium used in this study was probably not rightfor enriching these bacteria. One other likely explanation was that these organisms arenonphototrophs at all. But even if this is true, they will still be new species in certain other groupssince no similar bacteria have ever been described or isolated before. One possible way todetermine whether these strange bacteria are purple sulfur bacteria is to use molecular probespecific for this group by doing in situ hybridization. Of course, useful probes such as 168 rRNAsequences specific for this group are needed to be developed first.

My suggestion for further isolation of these bacteria is to use light filter specific for bacterialchlorophyll filters all the time and to try more different incubation conditions such as different pH,Na2S concentration, and maybe organic carbon source could also be tried. Probably some ofthem would grow heterotrophically in the dark and after single colony was pick out, one couldthen try to characterize whether or not they are phototrophs.

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Appendix

C Legends of Figures:

Figure 1. (a) Cells of purple berry isolate Al Thiocapsa roseopersicina; (b) Cells of purple berryisolate C: Chromalium violascens. Inner bar: 20 wn.

Figure 2. Sulfur globule-containing bacteria found in the enrichment from microbial mat. (a)Dipolar flagellated spirillum = Figure 3 (5). (b) Another longer dipolar flagellatedspirillum = Figure 3(1). Inner bar: 20 j.tm.

Figure 3. (1)-(5) Sulfur globule-containing bacteria found in the purple enrichment frommicrobial mat of Great Sippewissett Marsh.

Figure 4-5 Pigment absorbency of purple sulfrr isolates and natural samples: Fig 4. Purple berryextraction (left) and purple berry isolates Al-3, Bi (right). Fig 5. Purple berryextraction (left) and purple berry isolates C and E (right). Fig 5. Purple berryextraction (left) and purple sand extraction (right).

C

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Table 1. Properties of Purple Sulfur Bacteriafrom Purple Berries

Isolate Cell Color Motility Sediment Aggregate Swirming Tentativeshape at bottom formation toward H2S species

Purple berry isolatesAl sphere purple-pink 1.5—2 — + — +“ 7 roseopersicinaA2 sphere purple-pink 1.5-2 — + — +‘‘ T. roseopersicineA3 sphere purple-pink 1.5—2 — + — +“ T. roseopersicinaSI sphere purple-pink 1.5—2 — + — + T. roseopersicina82 rod purple-red 2.5—3 x 5-4 + + — + C. violascensC rod purple-red 2.5—3 x 5—S + + — + C. violascensD rod purple-red 2.5-4x5S + + + C. violascensE rod purple-red zs-xs-s + + — + C. violascensF rod purple-red as—a * s—s + ÷ — + C. violascens

Purple sand isolatesW sphere chalky pink 1.5—2 — + — — T. roseopersicinaX sphere chalky pink 1.5—2 — + — — T. roseopersicine

Y sphere chalkyplnk 1.5—2 — + + — T.roseopersicina

0

Table 2. Pigment Analysis of Purple Sulfurand Original Samp!cs

Isolates

Sample Color of SchI a Bchl b Carotenoid ChI aacetone extract

Purple berry purple-violet + — +++ -1-4-

Al, A2, A3, & 81 light-pink -H- — ‘44+ —

C & E purple-violet -f-H- — ++ —

Purple sand yellow green ++ — +++ +

isolatedSize(psn)

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* Particulate turbidity observed.

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Table 3. Properties of Phototrophic Purple Sulfur Isolates

Isolate Cell Color Size (jim) Motility Light Tentative Morphologyshape x L source species

U sphéi-e orange-pInk 2 — W’ T. roseopersicina ê&e

N pt.eve-ovold purple-pInk 1.5 x 2 — W Thiocapsa sp.

. 0080 sphere-ovoId purple-pInk 1.5 x 2 — W Thiocapsa sp.

S sphere-ovoid purple-pink 2 x 3— ? W Thiocapsa sp. 4 o o

t40

P curve rod orange-plnk 1 x 3 + W C. minutissimum

01Q curve rod orange-pink 1x3 + W C.minutissimum 0 4

R curve rod orange-pink 1 x 3 + W C. minutissimum

ZI rod chalky pink 2—3 x 5 ++ FBChW2 C. minus a

9iZ2 rod chalkypink 2—’3x5 ++ Ft1 C.minus ? 0

Z3 rod chalky pink 2-3 x 5 ++ FBChIL, C. minus

V rod purple-violet2.5—3 x 6—7 + W C. violascens gin.

V-I rod purple-violetas—3 x 6—7 + W C. violascens

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