A BACTERIUM CAPABLE OF USING PHYTOL AS ITS SOLECARBON SOURCE, ISOLATED FROM ALGAL SEDIMENT

OF MUD LAKE, FLORIDA *

BY K. B. HOAGt W. H. BRADLEY,t A. J. TousIMis,t AND D. L. PRICEt

BIODYNAMICS RESEARCH CORPORATION AND U.S. GEOLOGICAL SURVEY

Communicated April 28, 1969

Abstract.-A species of Flavobacterium that consistently attacks pure phytol andcan use it as a sole source of carbon has been isolated from the blue-green algalsediment of Mud Lake, Florida. Biochemical tests demonstrate that thisbacterium also readily uses various other organic compounds. This bacteriummay account for the degradation products of chlorophyll and its side chainphytol, which have been found in the Mud Lake algal sediment. Phytol and itsdegradation products play a role in Refsum's disease, but phytol is also the mostpromising precursor of the isoprenoid hydrocarbons found in oil shale of theGreen River Formation (Eocene) of Colorado, Utah, and Wyoming.The discovery of this species of Flavobacterium is a significant product of a pro-

tracted study of the bacteriology, phycology, zoology, and geochemistry of thealgal sediment forming in MIud Lake, which is believed to be a modern analogueof the kind of algal sediment that, through geologic time, became oil shale.

As a part of a protracted study of the origin of the oil shale in the EoceneGreen River Formation,1 the organic-rich algal sediments of several subtropicaland tropical freshwater lakes have been investigated in the hope of finding pre-cursors of the hydrocarbons that occur in the Eocene oil shale. One of the mostpromising sources of hydrocarbons is the phytol chain of the chlorophyll mole-cule.2 Indeed, isoprenoid compounds, presumably derived from chlorophyll,have been found in the Green River oil shale.3-9Mud Lake, a small, very shallow lake in the Ocala National Forest of north-

central Florida, has been studied intensively. Evidence of phytol degradationhas been found in samples of algal sediment from this lake. Bacteria wereisolated from samples of algal ooze as a part of our over-all studies on the bio-chemistry of this lake and to determine their possible role in the degradation ofphytol. Samples of algal ooze were taken at 15-cm intervals beginning at thesediment-water interface and continuing to a depth of 90 cm, which is close tothe base of the algal sediments. Because of our interest in phytol, it was sub-jected to attack by each species of bacteria isolated. One species of Flavobac-terium, occurring at several depths in the algal ooze, was isolated initially onphytol medium. This bacterium consistently attacks pure phytol and can use itas a sole source of carbon.

Materials and Methods.-A base medium, to which phytol would be added as the onlycarbon component, was prepared to examine for the breakdown of phytol by bacteria inthe Mud Lake sediments. The ingredients are as follows: distilled water, 1000 ml;K2HPO4, 0.2 gm; MgSO4 7H20, 0.05 gm; FeCl3 6H20, 0.15 ml of a 0.01% solution.To each test tube containing 7 ml of the above base medium, 0.05 ml of phytol (Nutri-

tional Biochemicals, Cleveland, Ohio) was added. Phytol remains suspended on the sur-748

MICROBIOLOGY: HOAG ET AL.

face as a large single droplet. For enrichment studies on solid medium, 15 gm of Difcoagar was dissolved in the above preparation, and 1.0 ml of phytol was added. Aftersterilization, the medium was thoroughly shaken by hand to disperse the phytol dropletsthroughout the medium. It was then poured into Petri dishes or solidified in the slantposition in test tubes. All preparations were autoclaved at 115 lb for 15 min.With a sterile syringe and needle, 0.5-ml portions of sediment were removed from the

cartridges used to collect the algal sediment, aseptically and anaerobically, from MudLake. Collections were made at 15-cm intervals from the sediment-water interface to adepth of 90 cm. The experiment was conducted on each series of seven sediment samplescollected in April, June, September, and December of 1968 and on water samples collectedaseptically in April, June, and September of the same year. After collection, all sampleswere brought to our laboratories where, within 3 or 4 days, cultures were prepared. Acontrol tube (base medium without phytol) and an experimental tube (base medium plusphytol) were each inoculated. The cultures were incubated at 280C for approximately 3weeks and were examined daily.Enrichment culture experiments, using the liquid and solid medium containing phytol,

were conducted to obtain a pure culture of the bacterium capable of utilizing phytol. Todetermine whether the bacterium isolated (from the phytol-enriched base medium) was apure culture and, further, to determine whether that organism was present in the originalalgal sediment, samples from these two sources were streaked on trypticase soy agar(TSA). Once a pure culture was obtained, biochemical tests were made on the isolatedbacterium. Bergey's Manual of Determinative Bacteriology was used to identify the iso-lated bacterium.

Samples for electron microscopy were prepared from fresh cultures of the isolated bac-terium grown on either solid base medium plus phytol or on TSA. Bacteria from eachmedium were washed three times in distilled water, and single drops of the washed orga-nism were mounted on each of several parlodium-coated, 300-mesh grids. The grids wereallowed to air-dry and were then lightly coated with carbon in a vacuum evaporator.Half of the grids, representing bacteria grown on each medium, were shadowed withuranium at a nominal angle of 45°. Electron micrographs of this bacterium were takenwith an A.E.I. model EM6-B electron microscope, operating at 60 kv.

Results.-Growth occurred in four to six days in liquid phytol cultures inocu-lated with samples from the sediment-water interface and from 15 cm deep in thesediment. The growth formed filmy sheets that surrounded the phytol dropletsat the surface of the medium. As incubation proceeded, the filmy growth be-came more pronounced until the entire surface of the medium consisted of suchfilms. Subsequently, similar growth became evident in tubes containing sedi-ment from the 30-, 45-, and 60-cm depth levels but was much less pronouncedthan in the above cultures. The typical filmy growth was not present at eitherthe 75- or 90-cm sediment levels from the samples collected in April, June, andSeptember of 1968. However, filmy growth was present at all sediment levels ofthe December collection, although again the cultures of the samples from thesediment-water interface and the 15-cm sediment depth were first to show thecharacteristic growth.No type of surface film was noted in any of the control cultures. Nor was the

filmy growth around the phytol droplets present in any of the tube cultures inoc-ulated with samples of M\oud Lake water.

Pure cultures, obtained by subcultures on phytol media, showed the sametypical growth. Slide preparations stained with crystal violet, when viewed bylight microscopy (oil immersion), displayed very minute phytol droplets, almosttotally surrounded by granulated rods. Bacteria, as they appear on phytoldroplets, are shown in Figure la (inset).

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TABLE 1. Biochemical activities of Flavobacterium sp.Medium Reaction

Gelatin No liquefactionLitmus milk PeptonizationStarch HydrolyzedNitrite Not produced from nitratesIndole Not producedH2S Not producedBlood agar ,3 HemolysisGlucose AcidSucrose Slightly acidLactose No reactionCellobiose Slightly acid No gasArabinose Acidd-Sorbitol Slightly acidMotility test medium Not motile

A variety of colonies appeared on TSA inoculated from liquid phytol cultures ofeach sediment sample of the four collections. This finding indicates that manyspecies of bacteria existed in algal sediment samples at the time of collection andinoculation and that these bacteria were growing on organic substances present inthe algal sediment itself or, perhaps, even on products derived from the degrada-tion of phytol. Through subculturing, the organic substances in the sedimentwere constantly diluted until they were eliminated entirely from the culturemedium, and bacteria were dependent on enrichments added to the culture.Only the one species of bacterium grew when phytol was the sole enrichmentadded to the medium.One distinct colony type was present on TSA plates inoculated with samples of

filmy growth from enrichment cultures. The same bacterium was obtainedfrom enrichment cultures made from each sample of the collections (April, June,September, and December of 1968). Colonies on trypticase soy agar werebright orange, round, and convex. This characteristic bright-orange pigmenta-tion was also present on nutrient agar and on gelatin agar. The bacterium was aGram-negative rod; granulation, which appeared when the bacterium wasgrown on phytol medium, was absent. Biochemical activities of this organismare presented in Table 1. No motility was observed in motility test medium oron slide preparations. The absence of flagella was confirmed by electron-microscope studies (Figs. 1 and 2). Using all available information, we classifiedthe bacterium as a member of the genus Flavobacterium.Discussion.-The study of Mud Lake has included work by many investigators

representing varied fields. In 1966 and 1967, C. E. AMize, who was then engaged

FIG. 1.-Electron micrographs of Flavobacterium sp. grown 48 hr on base medium with phytol(whole-mount preparations air-dried and coated with carbon). Unshadowed preparation.Note variation in density and the opaque inclusions (granules). X22,000. Inset: Compara-tive light micrograph of heat-fixed bacteria showing similar granular structure. X 1,920.(b) Shadowed with uranium. Vacuolar inclusions are clearly visible in this preparation (reverseprint). X 23,040.

FIG. 2.-Electron micrograph of Flavobacterium sp. grown 36 hr on trypticase soy agar.Whole-mount preparation coated with carbon and shadowed with uranium. Bacteria haveparallel sides and rounded ends and vary greatly in length. X 11,520.

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TABLE 2. Results of the analysis of algal ooze for certain organic products of chlorophylldegradation.

Depth belowsediment-water Phytanic Pristanic Phytanicinterface (cm) acid acid acid Pristane Phytane

I- (jg/gm dry weight of sediment)0 12.63 7.18 None 7.91 None30 14.7 0.0 None Trace None60 1.7 3.05 Trace None None90 2.74 0.75 None None None

in a study of Refsum's disease, a disease resulting from the inability of the patientto detoxify phytol,10 suggested to us that one or more bacteria present in therichly organic sediment of Mud Lake might be involved in attacking the phytolchain in the process of chlorophyll degradation.Samples of algal ooze taken from the lake were analyzed by Mize for various

organic acids and hydrocarbons that could have been derived from the phytol sidechain of chlorophyll (Table 2). Since the greatest volume of breakdown productsof phytol was found at depths in the algal sediment between 0 and 30 cm, it seemsapparent that initial breakdown of phytol in situ at AMud Lake occurs at thesedepths. These results were supported by our laboratory findings, althoughbreakdown of phytol by the same bacterium (Flavobacterium sp.) was observed toa lesser degree at other levels.Summary.-The medium utilized for this experiment provided an effective and

efficient means for determining the presence of bacteria capable of attackingphytol and for isolating the bacterium responsible for this breakdown. Theability of this bacterium to attack other organic substances was confirmedthrough biochemical tests. Although this species of Flavobacterium readily usesvarious other organic compounds, we have demonstrated that it can use phytolas its sole source of carbon.

We wish to thank Dr. Charles E. Mize, formerly of the National Institutes of Health,Bethesda, Maryland, for his suggestions, for analyzing samples of the Mud Lake algalooze, and for providing us with the results shown in Table 2.

* This investigation was made by Biodynamics Research Corporation, Rockville, Md.,under U.S. Geological Survey contract 14-08-0001-11214. Its publication is authorized by theDirector, U.S. Geological Survey.

t Biodynamics Research Corporation, Rockville, Md.I U.S. Geological Survey, Washington, D. C.I Bradley, W. H., U.S. Geol. Survey Prof. Paper 168 (1931), pp. 1-58, and Geol. Soc. Am.,

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Nature, 218, 1019-1024 (1968).4 Cummins, J. J., and W. E. Robinson, J. Chem. Eng. Data, 9, 304-307 (1964).6 Eglinton, Geoffrey, A. G. Douglas, J. R. Maxwell, and J. N. Ramsay, Science, 153, 1133-

1135 (1966).6 Robinson, W. E., J. J. Cummins, and G. U. Dinneen, Geochim. Cosmochim. Acta, 29,

249-258 (1965).7 Burlingame, A. L., and B. R. Simoneit, Nature, 218, 252-256 (1968).8 Burlingame, A. L., and B. R. Simoneit, Science, 160, 531-533 (1968).9 Leo, R. F., and P. L. Parker, Science, 152, 649-650 (1966).10Steinberg, D., J. Avigan, C. Mize, L. Eldjarn, K. Try, and S. Refsum, Biochem. Biophys.

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