APPLIED MICROBIOLOGY, Dec. 1969, p. 1044-1049Copyright @ 1969 American Society for Microbiology

Vol. 18, No. 6Printed in U.S.A.

Numerical Taxonomy of Heterotrophic BacteriaGrowing in Association with Continuous-

Culture Chlorella sorokinianaCAROL D. LITCHFIELD', RITA R. COLWELL, AND J. M. PRESCOTT

Department ofBiochemistry and Biophysics, Texas A&M University, College Station, Texas 77843 andDepartmentof Biology, Georgetown University, Washington, D.C. 20007

Received for publication 16 July 1969

Twenty-nine cultures isolated from nonaxenic growths of Chlorella sorokiniana(Shihira and Krauss) and eight reference cultures were tested for 150 morphologicaland biochemical characteristics. The taxonomic data were subjected to computeranalysis from which five major clusters were identified. The bacterial isolates havebeen placed in the genera: Pseudomonas, Acinetobacter, Flavobacterium, andBacillus. Amino acid requirements of four strains from the test set were determined.

The problems associated with the mass cultiva-tion of algae have become increasingly importantwith the recent interest in algae for life-supportsystems in spacecraft and as potential sourcesof protein for man and animals (16). One of theseproblems involves the interactions of algae andbacteria, inasmuch as the maintenance of axeniccultures of algae is highly impractical, andpragmatism dictates that mixed cultures-perhapshighly complex ones-will be used for thesepurposes. The effects that individual species exertupon one another in mixed culture are difficultto assess, but it appears that bacteria growingin a mixed culture with algae could greatly affectthe algal cultures in a number of ways. Althoughseveral previous reports have dealt with theoverall effects of bacteria on population densitiesof algal cultures, (14, 17, 27), a limited numberof studies have been reported concerning thechemical, metabolic, and nutritional relationshipsbetween algae and bacteria (3, 26, 27). It appearslogical to expect that these relationships wouldbe influenced to a great extent by both the identityand the number of bacteria present.Ward and Moyer (27) and Ward, Moyer, and

Vela (28) reported on the isolation of bacteriafrom algal cultures and on the growth of algaeand bacteria in mixed culture. Their isolation ofbacteria, however, was restricted to the pre-dominant species to be found in various culturesof Chlorella pyrenoidosa TX71105, from whichthey isolated organisms of human origin and

I Present Address: University College of North Wales, BangorMarine Science Laboratories, Menai Bridge, Anglesey, GreatBritain.

therefore presumably of potential hazard to man,namely, Mima polymorpha, Pseudomonas aeru-ginosa, and Bacterium anitratum. Although thequantitatively predominant bacterial species areof obvious interest, it appeared relevant also toinvestigate a broader array of the types of bacteriato be found in a mass culture of an algal species.We report herein the results of a study in which29 bacterial isolates from a culture of Chlorellasorokiniana (Shihira and Krauss) were tested for150 characteristics and grouped by Adansoniantaxonomy. Also reported are data concerningthe amino acid requirements for some of thesestrains.

MATERIALS AND METHODSIsolation of bacteria from the algal culture. The

bacterial cultures were isolated from a sample ofcontinuous algal culture supplied by the Environ-mental Systems Division, USAF School of AerospaceMedicine, San Antonio, Tex. The algal culture was C.sorokiniana (Shihira and Krauss), which was isolatedby Sorokin and Myers (23) and formerly designatedas C. pyrenoidosa Chick, strain 7-11-05; recently, itwas reclassified as a separate species (20). It has beenthe subject of recent interest for potential use in lifesupport systems because of its rapid growth rate (upto 9.2 doublings per day) at 39 C. The alga wasgrown in a continuous culture apparatus with avolume of 6.2 liters on Knop's medium with KNO3as the nitrogen source (26). The dilution rate was 12ml/min and the steady state population density was2.1 g/liter. Appropriate dilutions of the algal culturewere made in 0.9% NaCl solution, and 0.1 ml wasspread upon the surface of Brain Heart InfusionAgar plates (Difco) which were then incubated for24 and 48 hr at 37 C. Incubation of a duplicate set

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HETEROTROPHIC BACTERIA IN ALGAL CULTURES

of plates in a candle jar for 24 hr, 48 hr, and 1 weekfailed to reveal, by visual inspection, any significantdifferences in colony types or numbers of coloniesper plate. This was not surprising inasmuch as aircontaining 5% CO2 had been continually bubbledinto the continuous culture system from which thesebacteria were isolated. Replicate plates from fourdifferent algal culture medium samples gave 2.0 X106 to 2.4 X 106 bacterial cells/ml of algal culturemedium. There were no significant changes in num-bers of bacteria per milliliter of culture mediumbetween the plates incubated for 24 hr and thoseincubated for 48 hr.

Reference cultures. Reference cultures of Bacilluslicheniformis (ATCC 12579), Escherichia coli (ATCC11303), Arthrobacter atrocyaneus (ATCC 1375), andVibrio neocystis (ATCC 14636) were utilized in theinvestigation. Also included as reference cultureswere B. anitratum (ATCC 10606) and M. polymorpha(ATCC 14291), inasmuch as previous workers hadisolated these two species as predominant organismsfrom similar cultures of algae. Herellea species (ATCC14290) was included among the reference culturesbecause of recent reports grouping B. anitratum andHerellea in the same genus (2, 8). All of above refer-ence bacterial cultures were obtained from the Ameri-can Type Culture Collection; in addition there wasutilized a strain of P. aeruginosa supplied by BillyG. Foster, Department of Biology, Texas A&MUniversity, College Station, Tex. All cultures weremaintained by transfer at 3-month intervals ontoBrain Heart Infusion Agar (Difco) slants and CystineTryptic Agar (Difco) stabs. After the appearanceof growth, which was examined for purity by staining,the stock cultures were routinely stored at 2 to 4 C.

Taxonomic studies. Biochemical tests were runby the methods described by Skerman (21) and inthe Manual of Microbiological Methods (13). Ingeneral, incubation was under stationary conditionsat 37 C. Appropriate time intervals were used forscoring each test, i.e., 24 hr, 48 hr, and 1 week.

Cell and colony morphology were determined bygrowth in Nutrient Broth and on Nutrient Agarand Brain Heart Infusion Agar (Difco). The BrainHeart Infusion medium, and King's Medium A andMedium B (11) were examined for pigment productionafter incubation for 24 hr at 37 C. Cultures werestained by the Gram, Ziehl-Nielson acid-fast, capsule,and malachite green spore staining methods (13, 21).Motility was determined in SIM (Difco) mediumas well as by examination of hanging drop prepara-tions from 18- to 24-hr Nutrient Broth cultures andby flagella stains prepared by a modification of theprocedure of Leifson (1).The following carbohydrates, or related sources

of carbon and energy, at 1% (w/v) concentrationwere tested for acid and gas production using aPhenol Red Broth Base (Difco) and inverted Durhamtubes: glucose, lactose, sucrose, maltose, galactose,ribose, ribitol, salicin, dulcitol, erythritol, inositol,inulin, melibiose, and glycerol. The ability to utilizecitrate was also determined by inoculation of Koser'sCitrate (Difco) medium. The oxidative or fermenta-tive capacity of each culture was determined by using

the Hugh and Leifson O-F medium (10) containingglucose. Methyl Red/Voges-Proskauer broth (Difco)was used to determine the methyl red and Voges-Proskauer reactions, 1% Tryptone Broth (Difco)was used in the test for indole production, and anaero-bic growth was determined by incubating inoculatedagar media in a 10% CO2 atmosphere (candle jar)as well as by growth in Thioglycollate Broth (Difco).Nitrate reduction and gas production were testedby using Nitrate Broth (Difco) in tubes containinginverted Durham tubes; powdered zinc was addedto confirm reduction beyond nitrate.The ability of the culture to grow on inorganic

nitrogen sources was determined by inoculation ina modified Knop's medium (26) supplemented with1% glycerol and in (NH4)2SO4 medium supplementedwith 1% glucose. Salt tolerance was estimated fromgrowth in Nutrient Broth with 0.1, 1.0, 3.0, 7.0, or10.0% NaCl added.Growth temperature ranges were determined by

inoculation of tubes of Nutrient Broth incubated at3 to 5, 16, 26, 37, and 45 C. If no growth was observedafter 1 week, the broth was then incubated at 37 Cto determine whether the previous incubation tem-peratures had been bacteriocidal or bacteriostatic.Also, 24- to 48-hr Nutrient Broth cultures werefrozen at -20 C and left for at least 2 months. Thecultures were then thawed and used as inocula forfresh broths to determine survival at this low tem-perature.The presence of various enzyme systems was deter-

mined by the tests for Kovac's oxidase, catalase, ironporphyrin, lipase, urease, amylase, gelatinase, andcaseinase (21). "Patho-tech" (Warner-Chilcott Co.)reagent strips for urease, oxidase, phenylalaninedeaminase, and lysine decarboxylase were also used.Ulrich's Milk (BBL) was employed to determineacid, alkaline, rennin, peptonization, and reductionreactions in milk. Hemolysin was detected on preparedblood agar plates (Scientific Products Co.).

Sensitivity to the following antibiotics was deter-mined by using antibiotic discs (BBL): bacitracin(2 units), chloramphenicol (5 mg), colimycin (2 mg),dihydrostreptomycin (10 mg), nalidixic acid (5 mg),penicillin (10 units), tetracycline (5 mg), and erythro-mycin (2 units). "Bacto-Unidiscs" (at medium con-centration; Difco) of the following sulfonamideswere used: elkasin, gantrisin, sulfadiazine, sulfamera-zine, sulfamethoxypyridazine, sulfathiazole, thiosulfil,and triple sulfa.The results of all tests were scored as 0 (negative),

1 (positive), or 3 (not appropriate or not tested).The coded data were transferred to cards and sub-jected to computer analysis. Georgetown UniversityTaxonomy Programs (GTP- 1, 2, 4, and 5) were usedin the taxonomic analysis. The programs follow themethods of Colwell and Liston (6), Colwell (5), andMoffett and Colwell (15). The programs have beendocumented for the IBM Computer Users Library.The computations were carried out at the GeorgetownUniversity Computer Center on the IBM Systems360/40 equipped with disks and magnetic tape drives.

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1046 LITCHFIELD, COLWELL, AND PRESCOTT

RESULTS AND DISCUSSIONFrom the Brain Heart Infusion Agar plates

used for estimating the bacterial population, weselected 40 to 50 visually different colonies.Selection was made from both sets of aerobicallyincubated plates and those placed in the candlejar. The selected isolates from the latter werefound to grow under aerobic conditions andwere thereafter incubated under the same condi-tions as the other isolates. Two types of coloniespredominated-yellow pigmented and grey towhite types. Both general types were found invarious sizes ranging from pinpoint to 0.5 cm.Initial testing and purification resulted in theselection of 29 representative cultures which wereexamined for reactions to the various biochemicaltests. For comparison purposes, eight referencecultures were examined at the same time. Theresults from all the tests were coded and thestrain data subjected to computer analysis. Figure1 presents the graph of the S-value output forthe entire set of cultures, AF-1 through AF-35and the reference cultures. A high degree ofheterogeneity, based on S-values, was found toexist among the types of bacteria which cancoexist in a nonaxenic algal culture.

APPL. MICRoBIoL.

A grouping at S > 59% (-10%) resulted inthe major phenons bracketed in Fig. 1 as Athrough K. Phenon A consists of subgroups Iand II. In subgroup I, AF-il and AF-12, becauseof their high degree of similarity ( 2 80%), shouldbe considered to be the same species (5). Thesestrains exhibit the characteristics of the Pseudo-monas group I of Shewan et al. (19). SubgroupII contains AF-6 and P. aeruginosa; thus AF-6is identified as a strain of P. aeruginosa.Phenon B includes the majority of the isolated

strains, with polarly flagellated and peritrichouslyflagellated bacteria included in the phenon whichwas therefore further subdivided. Subgroup HImembers (AF-28 and AF-29) with S = 80%show significantly high overall similarity to beconsidered the same species, while AF-33 andAF-21 are also included because of their highdegree of similarity to each other and to AF-28and AF-29. These cultures are characterized bysmall colony size, slow growth rate in liquidmedia, and by lack of reaction in sugars; theyare gram-negative, nonmotile, and negativefor catalase, Kovac's oxidase, and nitrate reduc-tion tests. The six isolates in subgroups III andIV of phenon B, (namely, AF-1 and AF-2 and

S-VALUE

* 100 %

* 90-99%

E 80-89%

0 70-79%

0 60-69%

E 55-59%

\1 0D <55%

zrn1X 1

It IIDAt1 1o1 IlL IA Li7me A _S1iiLiI*9 8 2Z Z7 3 17 15 5 4 18 3526t9>soa ° 10 -25=-

FIG. 1. Full S-value output from a modified highest linkage sorting analysis ofSokal and Sneath (21), showingoverall similarities of the bacterial isolates from algal cultures and reference strains of bacteria. The externalbrackets indicate clusters of related organisms labeled as phenons A through K, whereas the internal bracketsassemble strains of the same species.

i\nPHENONS-

A

eb

293321

2162420714

B 98

222731715541835

C 26D 9E VibrioF ArthroboctorG r Herelleo

9BocterIumK I0H Bacillus

I 25d E. coliK Mima

S-I

.... -

1

c 11 6o 9321,C K 2 16 L4'20 7 1412

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HETEROTROPHIC BACTERIA IN ALGAL CULTURES

AF-21, AF-28, AF-29, AF-33) represent at least AF-16 through AF-35 (Fig. 1). In addition,two species, respectively, in the genus Acineto- AF-26 and AF-19 are included in the bracketbacter (24, 25). because of their overall similarity to the membersA cluster of 18 organisms in phenon B includes of phenon B. The subgroup V strains (AF-16

TABLE 1. Mean characteristics for major phenetic groupings of bacterial isolates

Genus Clusters of isolates from Selected characteristics positive for 50% or morealgal culture of the clustered strainsa

Pseudomonas

Pseudomonas/Xanthomonas

Acinetobacter

Unidentified

Flavobacterium

Bacillus

AF-6 and (AF-il, AF-12)b

(AF-5, AF-18), AF-26c

(AF-28, AF-29, AF-33, AF-21)and (AF-l, AF-2)d

AF-25

(AF-16, AF-20, AF-24) and(AF-7, AF-14) AF-8, AF-9,AF-22, AF-27, AF4 (AF-3,AF-17) AF-19

AF-10

Polar flagella; diffusible, fluorescent pigment;pyocyanin produced; (NH4)2SO4 as sole Nsource; NOs reduced; alkaline and peptonizedmilk reactions; lysine decarboxylase; 0.1 to 7.0%NaCl tolerance; 15 to 45 C tolerance; acid withglucose; catalase; iron porphyrin; growth inmodified Knop's + glycerol; Kovac's oxidase.

Beige to yellow insoluble pigment; polar tuft offlagella; Kovac's oxidase; growth in modifiedKnop's + glycerol; 0.1 to 7.0%0 NaCl toler-ance; 15 to 45 C temperature range; NOs re-duced; acid with glucose and lactose; hemo-lytic; lysine decarboxylase; iron porphyrin;catalase; (NH4)2SO4 as sole N source.

Gram-negative to gram-variable rods; translu-cent to opaque, unpigmented small colonies;0.1% NaCl tolerance; 25 to 37 C growth range;inhibited by 45 C, penicillin, bacitracin, chloro-mycetin, and tetracycline.

Gram-variable, motile, rod; translucent graycolonies except on potato; acid from glucosein both aerobic and anaerobic tubes; hemo-lytic; lysine decarboxylase; starch; acid fromlactose and maltose; 15 to 37 C temperaturerange; 0.1 to 1.0%/ NaCl tolerance; inhibitedby 45 C, penicillin, chloromycetin, tetracy-cline, and erythromycin.

Gram-negative rods, peritrichous or nonflagel-lated; generally yellow, opaque, entire edgecolonies; 0.1 to 3% NaCl tolerance; (NH4)2-S04 as sole N source; 15 to 45 C temperaturerange; milk reduced and alkaline; growth inmodified Knop's + glycerol; inhibited bytetracycline, dihydrostreptomycin, chloromy-cetin, colimycin, penicillin, and erythromycin.

Gram-positive to gram-variable, rod with sub-terminal spores, motile; 0.1 to 10% NaCltolerance; growth in modified Knop's + glyc-erol; fluorescent pigment on potato but whitecolony on nutrient agar; MR-VP; (NH4)2S04as sole N source; NO3 reduced; lysine de-carboxylase; hemolytic; iron porphyrin;starch hydrolysis; acid from glucose, glycerol,fructose, mannose, xylose, galactose, inulin,salicin, lactose, arabinose, and maltose; 25 to45 C temperature range.

* Feature frequencies computed from Georgetown Computer Program GTP-4 (17) which is based onthe Hypothetical Median Organism calculation (11).

b Parentheses indicate strains clustered at S > 70% level.* AF-26 by the Hypothetical Median Organism calculation is a separate species but is not included

with the strains identified as PseudomonasiXanthomonas.d AF-1 and AF-2 are placed in a separate grouping by the Hypothetical Median Organism calculation

(11). Overall similarities of these strains to AF-28, AF-29, AF-33, and AF-21 indicate membership inthe same genus.

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1048 LITCHFIELD, COLM

through AF-27) are either nonmotile or peri-trichously flagellated, produce yellow pigmenton Nutrient Agar and Brain Heart Infusion Agar,and are generally nonreactive toward carbo-hydrates. On the basis of overall characteristics,they have tentatively been assigned to the genusFlavobacterium (4, 7). Because of the generalconfusion concerning the taxonomy of the generaFlavobacterium and Cytophaga, the classificationof these cultures below the generic level or theassignment of the number of species representedis not attempted at this time. However, it canbe noted that groups labelled Va and Vb showedintra-S values of > 70%, again an indicationof a potential species level relationship (6).

Cultures in subgroup VI (AF-3 and AF-17)are nonchromogenic and nonmotile. Their generalcharacteristics would place them in the genusAchromobacter/Acinetobacter; however, bothstrains show S values of 64 and 60%, respectively,to AF-15, which is motile by means of a singlepolar flagellum and is assigned to Pseudomonasgroup IV (19).The cluster labelled subgroup VII (Fig. 1)

constitutes a group of polarly flagellated strains.Cultures AF-5, AF-18, and AF-35 of subgroupVII, with an overall similarity of S > 60%,demonstrate a positive oxidase reaction. On thebasis of the correlated characters available fromthis study, these strains have been placed in thegenus Pseudomonas. It should be pointed outthat these cultures might also be identified asXanthomonas species (9). Although it is recog-nized that a lack of pathogenicity for plants maynot exclude membership in the genus Xan-thomonas, it is temporarily expedient to placethese microorganisms in the genus Pseudomonaseven though plant pathogenicity studies havenot been done. Culture AF-4, which possessesperitrichous flagella, cannot be assigned to adefinite genus at this time despite the similarityto AF-5 at S 2 70%. Culture AF-26 demon-strated a 54% overall similarity to subgroup VIIand is probably a separate species; AF-19, atS = 58%, is also considered a separate species.

Strain AF-10 was found to be a gram-variable,spore-forming rod. This strain was placed in thegenus Bacillus and in Phenon H, even thoughAF-10 has only a 59% similarity to the referencestrain of B. licheniformis.A culture isolate which remains unidentified

is AF-25, a nonpigmented, peritrichously flagel-lated, gram-negative rod. This culture is rathernonreactive in the tests described above anddoes not attack carbohydrates.With the exception of P. aeruginosa, none of

the reference cultures matched with the unknown

IEILL, AND PRESCOTT APPL. MICROBIOL.

isolates at the species level of S ' 75%. Thisconfirms the decision from the taxonomic datathat none of the isolates are members of thegenera Mima, Bacterium, or Herellea. The refer-ence cultures of Herellea species and B. anitratumclustered together at S = 60%, phenon G, whichsuggests membership in the same genus as wasconcluded by other workers (2, 8).

In summary, except for strain AF-10, the algalculture isolates were found to be: gram-negativerods with round ends, 0.2 to 0.6 ,um in length by0.2 to 0.6 jim in width, motile, with a growthtemperature range of 25 to 37 C, and a tolerancefor 3% NaCl. In addition to these features, thestrains were generally nonreactive toward carbo-hydrates other than glucose, did not produceH2S from peptone, were oxidase-, lipase-, andgelatinase-positive, and produced yellow pig-mented colonies 1 to 5 mm in diameter, withentire, nonspreading edges. The features of themajor groups of bacteria isolated from the algalcultures are listed in Table 1.

In addition to the taxonomic study, all theisolates were tested for the ability to grow in amodified Knop's medium (26) supplementedwith 1% (v/v) glycerol and in filter sterilized,spent, algal culture filtrate also supplemented

TABLE 2. Amino acid requirements of selectedunknown isolatesa

Amino acid AF-7b AF-14b AF-18c AF-19c

Serine................ -d - +Glutamic acid........ - - +Methionine........... - - + +Glycine ..........- + +Alanine ..........-i _ + +Threonine ............ - + _Aspartic acid.........Proline...............- + -Valine. 4 +-: in.Isoleucine ............ +Leucine ......... 2+ - + -

Phenylalanine ........ - + + -

Lysine ....... -.. + + -

Histidine ............. - + -

Tryptophan ......... -_+ -

Pool of first five acids. 3+ NT NT

a Basal medium was a modified Knop medium(26). The 2% agar plates were incubated at 37 Cfor 3 days.

b Glycerol added at a concentration of 1%(v/v).

¢ No additional carbon source placed in themedium.

d (-) No growth, (i) slight growth, (+)growth, (2+) heavy growth, (3+) confluentgrowth, (NT) not tested.

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HETEROTROPHIC BACTERIA IN ALGAL CULTURES

with 1% glycerol. Those cultures showing growthonly in the tubes of spent algal culture filtratewere tested further for amino acid requirements.The results shown in Table 2 indicate that individ-ual and pooled mixtures of amino acids couldserve as the nitrogen source for AF-7 and AF-14when an additional carbon source was provided.On the other hand, AF-18 and AF-19 werecapable of utilizing amino acids as the solenitrogen and carbon source. Inasmuch as theamino acids listed in Table 2 have been foundto be excreted into the culture medium by C.sorokiniana (27, and unpublished data), it maybe significant that 14 of the 15 amino acids testedwere utilized by one or more of the selectedbacterial strains.As a result of the characterization and identifi-

cation of the bacteria growing in a culture withC. sorokiniana, it is now possible to present amore meaningful interpretation of the probablerelationships existing between algae and bacteria.The organisms which have been isolated arerather uniform in metabolic activity and require-ments for growth. That is, there is not a greatdiversity of bacterial types, but, at most, fourgenera represented among the bacterial isolatestested. The choice of isolation medium andcultural conditions may have been the controllingfactors, but this limited range of generic typeshas also been observed by Berland et al. (3) intheir studies on bacteria associated with marinealgae. Anaerobes and strict autotrophs were notenriched or selected for use in this investigation,and further study may show these bacterial typesto be significant in the algal-bacterial relation-ships. Finally, the more or less refractory typesof bacteria with minimally expressed metabolicactivities have been found to succeed undergrowth conditions provided by the associatedalgal cultures.

ACKNOWLEDGMENTS

This investigation was supported by contract F41609-67-C-101from the United States Air Force School of Aerospace Medicineand by National Science Foundation grant no. GB-6096X.

The helpfulness of B. Richardson and R. L. Miller in supplyingsamples from the continuous culture of C. sorokiniana is grate-fully acknowledged.

LITERATURE CITED

1. Baltimore Biological Laboratory. 1963. Flagella stain bulletinno. 04-104. Baltimore Biological Laboratory, Baltimore.

2. Baumann, P., M. Doudoroff, and R. Y. Stainer. 1968. Astudy of the Moraxella group. II. Oxidative-negative species(genus Acinetobacter). J. Bacteriol. 95:1520-1541.

3. Berland, B. R., M. G. Bianchi, and S. Y. Maestrini. 1969.Etude des bactdries associces aux Algues marines en cul-ture. I. Determination prdliminaire des espbces. Mar.Biol. 2:350-355.

4. Breed, R. S., E. G. D. Murray, and N. R. Smith. 1957. Ber-gey's manual of determinative bacteriology, 7th ed. Williams& Wilkins Co., Baltimore.

5. Colwell, R. R. 1964. A study of features used in the diagnosisof Pseudomonas aeruginosa. J. Gen. Microbiol. 37:181-194.

6. Colwell, R. R., and J. Liston. 1961. Taxonomic relationshipsamong the pseudomonads. J. Bacteriol. 82:1-14.

7. Floodgate, G. D., and P. R. Hayes. 1963. The adansoniantaxonomy of some yellow pigmented marine bacteria.J. Gen. Microbiol. 30:237-244.

8. Gilardi, G. L. 1968. Morphological and biochemical differen-tiation of Achromobacter and Moraxella. Appl. Microbiol.16:33-38.

9. Hayward, A. C. 1966. Methods of identification in the genusXanthomonas. In B. M. Gibbs and F. A. Skinner (ed.).Identification methods for microbiologists, part A, p. 9-14.Academic Press Inc., New York.

10. Hugh, R., and E. Leifson. 1953. The taxonomic significance offermentative versus oxidative metabolism of carbohydratesby various gram negative bacteria. J. Bacteriol. 66:24-26.

11. King, E. O., M. K. Ward, and D. E. Raney. 1954. Two simplemedia for the demonstration of pyocyanin and fluorescin.J. Lab. Chin. Med. 44:301-304.

12. Liston, J., W. Wiebe, and R. R. Colwell. 1963. Quantitativeapproach to the study of bacterial species. J. Bacteriol. 85:1061-1070.

13. Manual of microbiological methods. 1957. Society of Ameri-can Bacteriologists. McGraw-Hill Book Co., New York.

14. Mayer, A. M., U. Zuri, Y. Shain, and H. Ginzbuig. 1964.Problems of design and ecological considerations in massculture of algae. Biotech. Bioeng. 6:173-190.

15. Moffett, M. L. and R. R. Colwell. 1968. Adansonian analysisof the Rhizobiaceae. J. Gen. Microbiol. 51:245-266.

16. Myers, J. 1963. Algal cultures. In Encyclopedia of chemicaltechnology, vol. 1, p. 649-688. Interscience Publishers, NewYork.

17. Myers, J., J. N. Phillips, Jr., and J. Graham. 1951. On themass culture ofalgae. Plant Physiol. 26:539-548.

18. Quigley, M. M., and R. R. Colwell. 1968. Properties of bac-teria isolated from deep-sea sediments. J. Bacteriol. 95:211-220.

19. Shewan, J. M., G. Hobbs, and W. Hodgkiss. 1960. A deter-minative scheme for the identification of certain genera ofgram-negative bacteria, with special reference to the Pseu-domonadaceae. J. Appl. Bacteriol. 23:379-390.

20. Shihira, I., and R. W. Krauss. 1963. Chlorella: physiology andtaxonomy of forty-one isolates, p. 30-31. University ofMaryland Press, College Park, Md.

21. Skerman, V. D. B. 1967. A guide to the identification of thegenera of bacteria, 2nd ed. Williams & Wilkins, Co., Balti-more.

22. Sokal, R. R., and P. H. A. Sneath. 1963. Principles of numeri-cal taxonomy. W. H. Freeman and Co., San Francisco.

23. Sorokin, C., and J. Myers. 1953. A high-temperature strain ofChlorella. Science 117:330-331.

24. Thornley, M. J. 1967. A taxonomic study of Acinetobacter andrelated genera. J. Gen. Microbiol. 49:211-258.

25. Thornley, M. J. 1968. Properties of Acinetobacter and relatedgenera. In B. M. Gibbs and D. A. Shapton (ed.), Identifica-tion methods for microbiologists, part B, p. 29-50. Aca-demic Press Inc., New York.

26. Vela, G. R., and C. N. Guerra. 1966. On the nature of mixedcultures of Chlorella pyrenoldosa TX71105 and various bac-teria. J. Gen. Microbiol. 42:123-131.

27. Ward, C. H., and J. E. Moyer. 1968. Ecologic relationshipsbetween bacteria and algae in mass culture, p. 117. In Bio-regenerative systems. NASA SP-165, Washington, D.C.

28. Ward, C. H., J. E. Moyer, and G. R. Vela. 1964. Studies onbacteria associated with Chlorella pyrenoidosa TX71105 inmass culture. Dev. Ind. Microbiol. 6:213-222.

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