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Ron Caspi Microbial DiversityMarine Biological Laboratory

Woods Bole 1991

Abstract: the isolation of Mn and Fe reducing bacteria

Primary Project: Isolation of iron and manganese reducing bacteria,which use acetate, phenol or benzoate as carbon source, and iron and/ormanganese oxides as electron acceptors.

Procedure

Two different approaches were used to detect reduction:a. Growing the cultures in soft agar containing a suspension of metaloxide particles, and observing the clearance of the suspension.b. Growing the enrichments in liquid media, and measuring theconcentration of the different species of the metals byspectrophotometric methods.

Soft Agars

50 ml serum bottles were gassed with N2/C02, and inoculated with l5 mlof mud. The mud was mixed with 1% agar media and let cool untilsolidified. The rest of the space was filled with 0.75% agar mediacontaining 2mM Mn or Fe oxides. Acetate was added as a carbon source.Clear zones were detected in the agar after 2 to 5 days. Small aliquotsfrom the clear zones were transferred to agar tubes containing the samemedium with 0.75% agar. After several days different morphologies ofclear zones appeared in the tubes.

Liquid Media

50 ml serum bottles were gassed with N2/C02, filled with anaerobic mediaunder N2, and inoculated with l5 ml of mud. Carbon sources wereacetate, phenol, or benzoate at the respective concentrations of 15mM,1mM and 2mM. Mn and Fe oxides were added as the sole electron acceptor.Subsaxnples were taken every few days, and analyzed for concentrations ofMn2+,Fe2+, and total iron. Fe3+ was calculated as total iron — Fe2tWhen growth was observed, samples were transferred to agar tubes.As a control, mud samples were autoclaved, and used to inoculate bottlesunder the same conditions.

Plates

Colonies were transferred onto agar plates,which contained a top layerof soft agar with 2 mM Mn oxides.The plates were incubated in the anaerobic chamber, and after 4 dayscolonies began to appear, with clear zones around few of them. Thebacteria that formed these colonies were very small non—motile shortrods.An interesting phenomena was the presence of large (5 urn) bone shapedhighly refractile objects in many of the plates. It was not clear ifthese objects were living organizms or crystals of some source. Acridineorange staining caused the objects to fluoresce. Probing the objectswith fluorescent probes for archea, eukarya and eubacteria gave negativeresults. I assume that these are rodochrosite (MnCO3) crystals.

Microbial DiversityRon Caspi Marine Biological Laboratory

Woods Hole 1991

Isolation of iron and manganese reducing bacteria

Primary Project: Isolation of iron and manganese reducing bacteria,which use acetate, phenol or benzoate as carbon source, and iron and/ormanganese oxides as electron acceptors. This project was done togetherwith Sylvia Schnell.

Purpose

To isolate iron and manganese reducing bacteria from differentenviromnents.

Sources of inocula

1. The upper 5 cm of the black anaerobic zone from Sippewissett Marsh(SPl).

2. Anaerobic mud from the rizosphere of Spartina sp at SippewissettMarsh (SP2).

3. Anaerobic sediment from the Eel Pond (EP).4. Anaerobic sediment from the swamp near WHOI parking lot.S. Anaerobic sediment from acid mine drainage supplied by Joseph

Calabrese.

Materials and Methods

Preparations of oxides

Fe oxides A 0.4M Fed3 solution was neutralized with NaOH to pH 7.0. Theprecipitate was centrifuged and washed several times until no morechloride was present. Then the oxides were dried overnight at 60°c, andground to fine particles with a mortar.

Mn oxides A 30mM MnC12 solution was slowly mixed with a basic solutionof 30mM KMnO4. The precipitate was treated in the same manner as the Feoxides.

Media

The media used contained 75% filtered sea water, 25% deionized water, towhich 15mM NaIico3, 1 ml/l vitamin mix, 2mM Mn/Fe oxides, and theappropriate carbon source were added after autoclaving. In someexperiments apse be-as added to inhibit sulfate reducers. The mediumwas autoclaved in aWiSdel flask, cooled under N2 atmosphere, anddispensed under a constant flow of N2/C02.

Enrichment

Two different approaches were used to detect reduction:a. Growing the cultures in soft agar containing a suspension of metal

oxide particles, and observing the clearance of the suspension.b. Growing the enrichments in liquid media, and measuring the

concentration of the different species of the metals byspectrophotometric methods.

Soft Agars

SO ml serum bottles were gassed with N2/c02, and inoculated with 15 mlof mud. The mud was mixed with 1% agar media and let cool untilsolidified. The rest of the space was filled with 0.75% agar mediacontaining 2mM Mn or Fe oxides. Acetate was added as a carbon source.Clear zones were detected in the agar after 2 to 5 days. Small aliquotsfrom the clear zones were transferred to agar tubes containing the samemedium with 0.75% agar. After several days different morphologies ofclear zones appeared in the tubes. Samples from these zones weretransferred in the anaerobic chamber onto agar plates, which contained atop layer of soft agar with 2 mM Mn oxides.The plates were incubated in the anaerobic chamber for 4 days beforecolonies began to appear. Some of the colonies appeared to clear thearea around them from Mn—oxide particles. Unfortunately, at this stagewe ran out of time.

Liquid Media

50 ml serum bottles were gassed with N2/C02, filled with anaerobic mediaunder N2, and inoculated with 15 ml of mud. Carbon sources wereacetate, phenol,or benzoate at the respective concentrations of 15mM,1mM and 2mM, and Mn and Fe oxides were added as the sole electronacceptor.Some of the bottles were provided with YE and peptone and let grow for 3days before transfer to the appropriate medium, in order to enrich thesamples with different kinds of bacteria before the selection, hoping tofacilitate growth rate and shorten the time required for the isolation.Subsamples were taken every few days, and analyzed for concentrations ofMn2+,Fe2+, and total iron. Fe3+ was calculated as total iron — Fe2+.When growth was observed, samples were transferred to agar tubes.As a control, mud samples were autoclaved, and used to inoculate bottlesunder the same conditions.

Spectrophotometric Methods

Fe2+: The method used was the method of Lovley and Phillips (1986). 0.2ml of well mixed subsample were added to 1.8 ml of HC1, and let sit foran hour at room temperature. then 0.9 ml of the sample were added to 3ml of ferrozine (lg/l) in 50 mM HEPES. After mixing for 15 5, the samplewas transferred to a couvette and the A562 was measured.

Total Fe: The method of Lovley and Phillips (1986) was modified after itfailed to reduce the oxides. 0.2 ml of well mixed subsample were addedto 1.8 ml of l.SM hydroxylamine in 0.5M Md and incubated on a shaker at60° for 3 days. Then samples were treated like Fe2+ samples.

2+. The method used was a modification of the method of Brewer andSpencer (1971). Subsamples were filtered with 0.2um filter, and 0.5 mlwere transferred to a vial containing 1.9 ml H20 and 0.5 ml mixedreagent (formaldoxime and 1:10 dilute NH4OH). The mixture was incubatedat room temperature for 20 minutes, and then the A490 was measured.

Results

Growth was observed in almost all of the inoculations. The visible

Oreduction of oxides was used successfully for the transferring ofcolonies. The colorimetric methods were not very efficient due to twomain factors:

l.The sampling of mud under anaerobic conditions proved difficult, andthere were different ratios of sediment to liquid in subsequent samples2. Autoclaving the mud samples appeared to catalyze reduction of oxides,resulting in similar reduction in inoculated bottles and in the sterilecontrols.The results of the colorimetric measurements are presented in tables 1and 2.However, although the time was short, Mn reducing colonies did appearboth in the agar bottles and in the agar tubes. Iron reduction occurred(as detected by the colorimetric analysis) but was not visible to theeye. It would probably require a much longer incubation time to observethat process.Colonies were transferred onto agar plates, and clear zones appearedaround few of them. The bacteria that formed these colonies were verysmall short rods, apparently non—motile. Since these colonies appearedone day before the end of the course, there was no time to characterizethem farther.An interesting phenomena was the presence of large (5 um) bone shapedhighly refractile objects in many of the plates. It was not clear ifthese objects were living organizms or crystals of some source. Acridineorange staining caused the objects to fluoresce. An attempt to probe theobjects with fluorescent probes for archea, eukarya and eubacteria wasdone during the last day of the course, but there was no positiveresults. My assumption is that these were rodochrosite (l4nCO3) crystals.

Second Project: 165 rRNA sequencing of a manganese oxidizing bacterium

Strain $185 9A1, which was isolated by Bradly Tebo from Saanich Inlet onMn—containing minimal medium plates, is a gram negative eubacteriumwhich grows either heterotrophically on complex medium, ormyxotrophically with thiosulfate or methanol. When grown on low nutrientmedium containing lOQuM manganese this bacterium grows very slowly,while oxidizing the manganese to dark brown Mn oxides, which areprecipitated around the cells.In order to sequence the 165 rRNA, few colonies were used as a templatefor Polimerase Chain Reaction (PCR), with a universal primer (RP 1492)and a specific primer for eubacteria (FPL 8) for the corresponding gene.The product was then ligated into the phage 1413. The phage particleswere transformed into competant S. coli JM1OJ. cells, and the transformedcells were grown over night and plated on LB plates with x—gal. Sincethe insert would impair the galactosidase activity of the phage,positive inserts were easily detected by forming white plaques, whilephages with no inserts would form blue plaques. White plaques weretransferred into fresh tubes containing E. coli cells, and grownovernight.The bacterial cells were then harvested and removed, the viralparticles lysed and the DNA extracted. This DNA was used for a singlestranded DNA sequencing gel.Since the gel was run only one day before the end of the course, therewas no time to analyze the sequence and build the phylogenetic tree ofthis organism. However, I intend to continue with this project after thecourse, and expect to have the full sequence within a month.

n‘—I

Table 1: concentrations of Fe2+, total iron, Fe3+ and Fe2+/F’s3+

Fe2+ Total Fe

sample 7/13 7/15 7/23 7/13 7/15 7/23

Lake,ph 29.19 23.35 168.45 260.78 350.73 376.17

Lake,be 25.62 34.22 27.099 470.88 449.55 129.05

S1,ph 11.13 6.899 91.396 272.53 229.03 559.67

S1,be 6.678 6.923 65.948 240.11 241.72 347.18

Lake,ac 0.78 1.08 7.1757 112.2 149.55 195.28

Jo,ac not measured 12.465 286.5 247.15 265.99

S1,ac not measured 14.816 121.51 669.23 565.38

S2,ac not measured 21.81 136.53 241.16 222.54

EP,ac not measured 45.436 96.96 330.78 536.28

Lake,control 42.06 63.73 336.42 380.38 504.75 716.67

Jo,control 38.77 2.81 402.77 1339.5 739.84 1107.6S1,control 6.113 3.18 40.793 123.73 331.28 271.4852,control 9.775 36.03 50.608 95.297 275.69 253.91EP,control 5.95 0.0 120.14 972.77 204.37 727.59

Fe2+/Fe3+

sample 7/13 7/15 7/23 7/13 7/15 7/23

Lake,ph 231.59 327.38 336.52 0.13 0.07 0.5Lake,be 445.26 415.33 380.441 0.06 0.08 0.07S1,ph 261.4 222.131 152.374 0.04 0.03 0.6S1,be 233.432 234.797 259.562 0.03 0.03 0.25Lake,ac 111.42 148.47 160.9443 0.01 0.01 0.04Jo,ac not measured 225.815 not measuredS1,ac not measured 250.954 not measuredS2,ac not measured 210.1 not measuredEP,ac not measured 409.704 not measuredLake,control 338.32 441.02 29.83 0.12 0.14 11.28Jo,control 1300.73 737.03 10.64 0.03 0.0 37.85S1,control 117.617 328.1 221.207 0.05 0.01 0.18S2,control 85.522 239.66 198.372 0.11 0.15 0.26EP,control 966.82 204.37 455.88 0.01 0.0 0.26

A

Table 2: Mn2+ concentrations

sample 1/13 1/15 7/18 7/23

Sibe 459.26 370.76 124. 195.7

the 101.97 148.82 46.454 522.27

S1,ph 233.75 281.62 127.07 535.61

L,ph 109.01 89.907 60.053 165.48

tac 20.346 146.02 22.682 543.4

Jo,ac 16.33 75.106 40.925 534.61

S1.ac 20.476 58.578 137.82 526.66

S2,ac 18.919 180.36 198.73 516.76

EP,ac 22.881 257.34 192.3 532.57

L,controj. 74.969 141.34 27.982 546.94

Jo,control 62.369 62.837 545.81

51,control 45.064 98.087 475.47 541.6

S2,controj. 46.263 200.41 192.78 522.27

EP,control 38.858 228.36 108.71 485.46

C

References

1. Brewer, P. G. and Spencer, D.W. (1971). Lirnn. Oceanogr. 16: 107—112.2. Lovley, D. K. and Phillips, E. J.(1986). App. Environ.

Microbiol. 51:683—689.3. Lovley, D. K. and Phillips, E. J.(1987). App. Environ. Microbiol.

53:1536—1540.4. Lovley, D. K. and Phillips, E. J.(1988). App. Environ. Microbiol.

54:1472—1480.5. Lovely, IL R.,Phillips, E. J. and Lonergan, IL J.(1989). App.

Environ. Microbiol.55:700—706.6. Myers, C. K. and Nealson, K. H. (1988). Geochim. Cosmochim. Acta

52:2727—2732.7. Myers, C. K. and Nealson, K. H. (1990). J. Bacteriol. 172:6232—6238.