The Biology ofZymomonas · BIOLOGY OFZYMOMONAS 3 and dry farm-made cider. The manufacturers encountered great difficulties, as sweet ciders easily develop a secondary fermentation,

BAcTURIowGIcAL Rxviuws, Mar. 1977, p. 1-46 Vol. 41, No. 1Copyright 0 1977 American Society for Microbiology Printed in U.S.A.

The Biology of ZymomonasJ. SWINGS AND J. DE LEY*

Laboratory of Microbiology and Microbial Genetics, Faculty of Sciences, State University,B-9000 Gent, Belgium

INTRODUCTION...................................................... 2OCCURRENCE OF ZYMOMONAS AND HISTORY OF ISOLATIONS ..... ....... 2

Ciders and Perries...................................................... 2Fermenting Agave Sap ...................., 5Beer.................... 5Fermenting Palm Sap.................... 6Fermenting Sugarcane Sap ......................... 8Ripening Honey ............................ 9Some Technological Applications ......................... 9Therapeutic Use ............................ 10History of Individual Strains ......................... 10

DETECTION, ISOLATION, AND IDENTIFICATION OF THE GENUS ZYMO-MONAS ........................ 10

Detection ........................ 10Isolation.................................................................... 10Identification................................................................ 11Some Commonly Used Media and Growth Conditions ....... ................... 11

TAXONOMY OF ZYMOMONAS................................................. 13Numerical Analysis of the Phenotype .......... ............................... 13DNA Base Composition and DNA Genome Size ........ ........................ 15Genome-DNA Relatedness ................ .................................... 15Similarity of Protein Electropherograms ......... ............................. 16Infrared Spectra of Intact Cells............................................... 16Serology ................................................................ 16Classification and Nomenclature ............ ................................. 18Relationship Between Zgmomonas and Other Genera ....... ................... 20

PHENOTYPICAL DESCRIPTION: MORPHOLOGY, GROWTH, PHYSIOLOGY,AND BIOCHEMISTRY ............... ................................. 22

The Cell ................................................................ 22Cell morphology ........................................................... 22Macromorphology.......................................................... 22Cellular composition........................................................ 22Resting cells and starvation ............... ................................. 23

Growth Response to Different Conditions ......... ............................ 23Growth in some ordinary media ........... ................................. 23Growth in the liquid synthttic medium of Kluyver and Hoppenbrouwers ...... 23Growth at different pH values ............. ................................. 23Growth at different temperatures ........... ................................ 24Thermal death point........................................................ 24Growth in the presence of ethanol ........... ............................... 24Growth in high glucose concentrations ...................................... 24Growth in the presence of KCN ............................................. 24Growth in the presence of NaCl ........... ................................. 24Growth in the presence of 0.01% Acti-dione ................................. 24Growth in the presence of oxgall ............ .................... 24Growth in the presence of 0.1% 2,3,5-triphenyltetrazolium chloride ..... ...... 24Growth in the presence of 0.01% thallium acetate ........ .................... 25Growth in the presence of 0.001% cadmium sulfate ....... ................... 25Growth in the presence of the vibriostatic agent 0/129 ........................ 25Tolerance to SO .................... ........................................ 25Growth in the presence of dyes ................... .......................... 25Reduction of dyes and HgC12............................................... 25Growth in the presence of antibiotics ............. .......................... 25

Carbohydrate Metabolism .................................................... 25Metabolism of glucose and fructose .............. ........................... 25Metabolism of sucrose: fermentation and levan formation ...... .............. 29Other carbon sources ...................................................... 29Formation of acetaldehyde................................................. 30

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Formation of acetylmethylcarbinol ............. .................. 30Molar growth yield ............................... 30Uncoupled growth ............................... 32Aerobic growth and metabolism .......... ..................... 32Cytochromes ............................... 34The pigment P-503 ............................... 34

Amino Acid Metabolism ............................... 34Amino acid requirements................................................... 34Formation of higher alcohols............................................... 35Amino acid decarboxylases ....... ........................ 36

Growth Factor Requirements ....... ........................ 36H2S Formation and Sulfur Metabolism .............. ................. 38Various Features ............................... 38Presence of catalase ............................... 38Presence of gelatinase ............................... 38Presence of urease ............................... 38Oxidase reaction ............................... 38Hydrolysis of Tweens ............................... 39Nitrate reduction ............................... 39Formation of indole ............................... 39

CONCLUSIONS AND SUMMARY ........ ....................... 39LITERATURE CITED............................... 41

INTRODUCTIONThe alcoholic beverages in the Western

world, such as beer, wine, champagne, etc., areusually made by fermentation with yeasts. Themost commonly used organisms are strains ofSaccharomyces cerevisiae. In many tropicalareas of America, Africa, and Asia, other typesof alcoholic beverages are very popular andwidely used; these consist of plant saps un-

dergoing a mixed fermentation, containing bac-teria from the genus Zymomonas. In Europe,these bacteria occasionally grow in and spoilbeer, fermented apple juice (cider), and pearjuice (perry).

In the early 1950s, the genus Zymomonasacquired a certain fame among biochemists bythe discovery of Gibbs and DeMoss (see below)that the anaerobic catabolism of glucose followsthe Entner-Doudoroff mechanism. This was

very surprising, since Zymomonas was the firstexample (and still is one of the very few) of ananaerobic organism using a pathway occurringmainly in strictly aerobic bacteria (85). Thisbiochemical pathway in a gram-negative, non-

sporeforming, polarly flagellated bacterial rodsuggested the aerobic Pseudomonadaceae as

possible remote relatives of Zynomonas, not-withstanding its largely anaerobic way of life.

In spite of its extensive use in many parts ofthe world, its great social implications as anethanol producer, and its unique biochemicalposition, Zymomonas has not been studied ex-

tensively. Part of the available information hasbeen very briefly reviewed by Dadds and Mar-tin (50).We collected most of the available strains

from this genus and isolated several new ones.

For the last 4 years, we have examined some

40-odd strains from Zaire, Mexico, Indonesia,and Britain by modern taxonomical methods:deoxyribonucleic acid (DNA) base composition,DNA genome size, and genome similarity (163),protein electropherograms (164), and numericalanalysis of numerous phenotypic features (50).In the present contribution we shall integrateall available literature data with our own expe-rience and attempt to present a critical andcoherent monograph of the biology of the genusZymomonas. We shall not attempt to reviewhere the technological aspects of these bacteria,except where they are helpful in understandingthe biology.The results ofmodern relatedness analysis in

this genus, together with a critical examinationof the literature data, show that all strains ofZymomonas constitute but one species, Z. mob-ilis, with two subspecies, Z. mobilis subsp.mobilis and Z. mobilis subsp. pomaceae. Allstrains in our collection, except three, and moststrains from the literature belong in the formersubspecies. Three strains from our collectionand a few strains described in the literaturebelong to the subspecies pomaceae. The latterstrains induce typical cider sickness symptoms.We consider this nomenclature as the only real-istic one (50). On several occasions, we stillhave to use previous nomenclatures for a vari-ety of reasons. A synonymy Table 7 is providedfor the reader's convenience.

OCCURRENCE OF ZYMOMONAS ANDHISTORY OF ISOLATIONS

Ciders and PerriesAt the turn of this century the taste of the

cider-consuming public changed; they began todemand a clear, sweet cider, instead of a rough

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and dry farm-made cider. The manufacturersencountered great difficulties, as sweet ciderseasily develop a secondary fermentation, i.e.,the "cider sickness" (see review [114]), perhapsdue to an infection from fruit (11, 29).Barker and Hillier (12) were the first to study

cider sickness extensively, and they gave a de-scription of the bacterium responsible for thetypical aroma and flavor. Outbreaks of the dis-order occurred from May until the end of sum-mer. The earliest symptoms are frothing andabundant gas formation. In a few days, the gaspressure causes the bottle to explode. The grow-ing bacteria change the aroma and flavor of thecider and reduce its sweetness. The cider at-tains a marked turbidity, which clears after-wards, with the formation of a heavy deposit.The susceptibility of cider to sickness dependson such factors as the low acidity of the ciderapples, the residual sugar in mature cider, anda high storage temperature.From the complex microflora of sick cider,

Barker and Hillier (12) isolated and purified abacterial strain (strain A [11]) in 1911. Theorganisms caused the typical strong aroma andflavor upon reinfection in sterile cider. Theywere motile rods, single or joined in pairs, 2 by1 Aum, with rounded ends; in old cultures, thecells were frequently longer; involution formswere frequently up to 200 Atm long, and theirends may be globular or dumb-bell shaped,with a diameter of 25 ,um; spores were absent;they were facultatively anaerobic, showing lim-ited and slow growth on solid media; growthwas creamy white and slimy; they vigorouslyfermented glucose and fructose with formationof ethanol and CO2; sucrose, maltose, and lac-tose were not fermented. The authors alsostressed that cider sickness is not identical withthe wine disorders known as "la pousse" and "latourne" in France. The discovery of representa-tives ofZymomonas is commonly attributed toLindner (see below) from pulque in Mexico in1924, but the real discovery of this genus wasmade in 1911 by Barker and Hillier. Unfortu-nately, they did not give a latin taxon name tothe new organisms.Barker (11) purified and studied two new

isolates from outbreaks of sickness in 1943 and1948; they were designated by him as strain Band strain C, respectively. Both strains inducedcider sickness. The cells were indistinguisha-ble, 2 by 1 ,m, occurring singly or in pairs, withinvolution forms in older cultures. The cells ofstrain B arranged themselves specifically inchains; strain C grew in complex, clumpy ag-gregates. Both strains were extremely motile.Strains B and C produced very little aroma;strain A produced a quite different, intense,

and characteristic aroma.In 1950 Millis isolated 33 strains from ciders

and from perries; 27 resembled Barker's strainB and six resembled Barker's strain C (114).The former strains were short rods, 2.5 by 1.5,um, or diplobacilli, 4 by 1.5 gm; they werepleomorphic and gram negative, without sporesor capsules. Actively motile when young, theirmotility ceased after 3 to 4 days. Motility de-pended on the medium; it was sometimes ab-sent in applejuice. The latter six strains formedrosettes. The isolates resembling Barker'sstrain B grew only very faintly aerobically onapple juice-yeast extract agar. Anaerobically,the colonies grew to a diameter of 0.3 mm in 2days and to 3 to 5 mm in 7 to 8 days. They werebutyrous, circular, regular, with an entireedge, convex to umbonate, opaque, and palegrey-buff. After 2 to 3 days on apple juice-yeastextract agar, a characteristic sickly sweetaroma was produced, which faded after a week.In the liquid apple juice medium, a compactcreamy deposit was formed. The colonies of theisolates resembling Barker's strain C weremore convex and less opaque; after 8 to 10 days,they had a curdled look. They spread and be-came irregularly circular. They, too, producedthe characteristic sickly aroma. In the liquidapple juice medium a granular flocculent de-posit was formed. In apple juice-gelatine stabsincubated without anaerobic precautions,growth with gas pockets occurred along thestab; there was no growth at the surface. Pep-tone could replace yeast extract as a source ofnitrogen in growth media, but the growth wasless vigorous: colonies on the former substratereached a diameter of 1 mm; on the latter, theywere 2 to 2.5 mm. Millis (114, 115) examinedthe biochemical characteristics of strain 1 fromcider and strain 2 from perry. Both strainsformed approximately 1.87 mol of ethanol permol of glucose, CO2 and some H2S, acetalde-hyde, and lactic acid. Lactose, raffinose, mal-tose, sucrose, galactose, rhamnose, arabinose,xylose, dulcitol, mannitol, or sorbitol was notfermented. Catalase was present. The tests forindole, methyl red, acetylmethylcarbinol, ni-trite from nitrate, ammonia, and gelatin lique-faction were negative. Strain 1 grew in the pHrange 3.75 to 7.9, its optimal pH range being 4.5to 6.5. The optimal temperature range wasfrom 25 to 310C. At 41°C, no growth occurred.To inhibit the growth of the cider sickness ba-cillus, 0.075 to 0.1% SO2 was necessary. Table 1compares strains 1 and 2 from Millis (114, 115)with Zymomonas anaerobia NCIB 8227 and Z.mobilis strains.

Millis (114) artificially induced cider sicknessin apple juice and cider. Apple juice infected

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TABLE 1. Comparison ofa few Zymomonas strains and their effect on cidera

Fermentation of: Celdpst HNames (as used by No. and origin of Motil -| r Cell delpusit pH Characteristics of ciderMillis) | strainb it) |sucrose |- Sore|l|medium growth after infection

Zymomonas anaero- Strain 1 (Millis) + _ _ + Compact 3.5-7.9 Typical aroma and flavorbia var. pomaceae = of sicknesscider sickness bacil- Strain 2 (Millis) + - _ - Flocculent 3.5-7.9 Typical aroma and flavorlus of sickness

Zymomonas anaero- NCIB 8227 + Flocculent 3.4-7.5 Putrid yeasty aroma; fla-bia vor similar to that of

sickness but not typicalZymomonas mobilis Strain 1 TH Delft - + (Gas) + + Flocculent Similar to sickness but

= ATCC 10988 not identicalStrain 2 TH Delft + + (Gas) - + Compact Foul aroma; flavor simi-

lar to sickness but nottypical

Strain 3 TH Delft + + (Gas) - + Flocculent Not characteristic of sick-ness; foul aroma; flavorunpleasant and harsh

a According to Millis (114, 115).b See also Table 5.

with cider sickness bacteria alone fermentedmore rapidly than when infected with the cidersickness plus the natural microflora. In thelatter competition conditions, the cider sicknessbacteria remained present in small numbersonly. There is some evidence that small num-bers of sickness bacteria are present in thefresh juice. This author also studied the devel-opment of cider sickness in the presence of cideryeasts. The yeast population declined as thesickness bacterium developed, but rose againafter day 45 (Fig. 1). When the cider sicknessbacteria alone were inoculated, only slight pop-ulation fluctuations occurred until day 130; inthe next 20 days, the cell count declined rap-idly. With a heavy yeast suspension (curve10:2), the sick aroma and flavor were lost after130 days. Millis commented: "The observationmade by the cider-makers that the aroma andflavour of sickness can be 'boiled' out of cider byencouraging a vigorous yeast fermentation hasbeen supported by these results." Probably adelicate ecological and physiological balanceexists between the cider yeast flora and thecider sickness bacteria, both competing for thesugar, the N source, and growth factors.To prevent the development of cider sickness

in ciders, a high acidity of the cider and lowstorage temperatures were recommended byBarker and Hillier (12). As a possible treatmentof the disorder Grove (77) proposed mixing withsharp ciders, addition of tartaric acid, or theaddition of brewer's yeast. Barker (11) proposedthe pasteurization of cider at 600C.Carr (28) stated that the cider disorder

known as "framboise" in France is caused by Z.anaerobia. It is questionable whether Zymo-monas is involved in every case of framboise.

Total microbial countlog N/ml

100 _ _ _ _ ___

S

0. 50 10 5

Time in days

FIG. 1. Cider sickness bacteria in competitionwith cider yeasts in cider. A fermented cider wascentrifuged and inoculated. All cider samples wereinoculated with 2% (by volume) of an actively fer-menting culture of cider sickness bacteria. The sam-ple 0:2 received no cider yeast; the samples 2:2 and10:2 were inoculated with 2 and 10%, respectively, ofan actively fermenting cider yeast suspension. Theinoculated ciders were stored in a cellar. The ciderswere tasted by a panel. Abbreviations: S. Sick ciders;N, normal ciders showing no symptoms of sickness(data from Table 17 of Millis [114]).

Guittoneau et al. (80) found the fruity odor andtaste of "framboise" ciders to be due to highacetaldehyde concentrations. They attributedthe accumulation of this compound to a sym-biotic growth of yeasts and acetic acid bacteria.Bidan (24) isolated lactic acid bacteria andacetic acid bacteria from "framboise" ciders. Hestated that these disorders with more than 100mg of acetaldehyde per liter may be due tovarious organisms. Auclair (8) pointed to thestrict anaerobiosis and low rH values of the

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ciders developing the framboise disorder. Pol-lard (127) supposed that the "framboisement" ofciders is caused by two different agents, i.e.,the anaerobic cider sickness bacillus Zymo-monas and the aerobic acetic acid bacteria. Themeans to fight both causative agents of thedisorder of "framboisement" are therefore notnecessarily the same.

Fermenting Agave SapDuring his stay in Mexico in 1923 to 1924,

Lindner studied the aguamiel fermentation.Aguamiel is the sugary sap of the Agave; it isfermented to obtain pulque, an alcoholic bever-age containing approximately 4 to 6% ethanol.Lindner (102) discovered the causal organism ofthe fermentation, a bacterium which he calledTermobacterium mobile (now Zymomonasmobilis subsp. mobilis). This very motile bacte-rium was able to ferment sucrose, fructose, andglucose to ethanol, CO2 and some lactic acid.Maltose and lactose were not fermented (104).Lindner (105) suggested that the occurrence ofthese bacteria is restricted to tropical regions,where they account for the alcoholic fermenta-tions in palm wines, chica beer, etc., and forbread manufacture. They might even havebeen the causal organisms in the preparation ofthe "solid beers" of the Arabs and the ancientBabylonians.

Lindner's isolate was studied by Kluyver andHoppenbrouwers (89) and characterized as fol-lows. The cells occur as diplobacilli, 4 to 5 jumby 1.4 to 2.0 pum; they are motile with polarflagella and gram negative; they have no endo-spores. After 2 days at 300C on beer wort agar,the colonies are white, circular, convex, and 1mm in diameter. Very poor growth occurs onpeptone agar or peptone gelatin. There is goodgrowth on beer wort agar with 2% sucrose andin yeast water with glucose, particularly when2% CaCO3 is added. The pH of the peptoneglucose medium should be adjusted to 6.5 withlactic acid or phosphate solutions. The optimumtemperature is 300C. Catalase is present. Theorganisms are facultative anaerobes, but sup-port a certain degree of aerobiosis in the pres-ence of fermentable sugars. In anaerobiosis 45%of the glucose is transformed to ethanol, and45% is converted to C02, some lactic acid, andtraces of acetylmethylcarbinol. Glucose, fruc-tose, and sucrose are fermented; mannose, ga-lactose, maltose, lactose, raffinose, arabinose,dextrine, and mannitol are not. After repeatedsubculturing in glucose media, a lag phase of48h occurs upon reinoculating a sucrose medium.Concentrated glucose solutions of up to 25%were still fermented.

Neither Lindner nor Kluyver and Hoppen-brouwers (89) related the pulque bacteria withthe cider sickness organisms described muchearlier by Barker and Hillier (see above).

BeerShimwell (150, 151) isolated Zymomonas for

the first time from beer, from the surface ofbrewery yards, and from the brushes of cask-washing machines. The cells were gram-nega-tive plump rods, 2 to 3 by 1 to 1.5 ,um in youngcultures and longer in old cultures, withoutendospores. Motile or nonmotile strains oc-curred in beer from the same brewery. For thenonmotile strains the name Saccharomonasanaerobia var. immobilis was proposed. Motil-ity was lost within 3 days. The cells formedrosette-like clusters. The surface colonies, incu-bated in CO2 on glucose beer agar, were irregu-larly circular, 1 mm in diameter, cream col-ored, sometimes with translucent margin, con-vex, entire edged, butyrous, and granular. Thedeep colonies were lenticular. In glucose beeragar, a dense filiform-to-beaded growth oc-curred along the stab, but no surface growth. Innutrient broth, yeast extract, sugar-free beer,or in any medium without glucose or fructose,there was no growth. In glucose yeast extractbroth, the cells grew with a deposit on the wallof the tube and a dense deposit on the bottom.No growth occurred on aerobically incubatedagar slopes. The optimum temperature was30°C. The thermal death point was 600C for 5min. The cells grew between pH 3.4 and 7.5.Maltose, sucrose, and lactose were not fer-mented. In glucose-containing beer, ethanol,CO2, a little acetaldehyde, and H2S were pro-duced, but neither acetylmethylcarbinol nor di-acetyl. The indole reaction and the nitrate re-duction were negative. Hopping had no effecton growth. The culture died off quickly whenthe fermentation stopped.

Microbial beer spoilage is restricted to a fewgenera of bacteria and yeasts. According toAult (9), the most frequently occurring spoilagebacteria in breweries are Bacillus, Sarcina,Micrococcus, Clostridium, Lactobacillus, Pedi-ococcus, Acetobacter, Gluconobacter, coliformbacteria, Flavobacterium, and Zymomonas.Priest et al. (130) pointed to Citrobacter freun-dii, Enterobacter aerogenes, Enterobacter cloa-cae, Hafnia alvei, Klebsiella aerogenes, Hafniaprotea, Serratia, Achromobacter, Acinetobac-ter, and Pseudomonas as wort contaminants.

Recently, Zymomonas has been recognizedas a serious beer contaminant. In cask or kegbeers, Zymomonas infections can occur becauseof anaerobiosis and the presence of priming

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sugars. These bacteria produce a heavy turbid-ity and an unpleasant odor of rotten apples dueto traces of acetaldehyde and H2S (9). In warmweather, spoilage can occur within 2 to 3 days(9, 132). Harrison et al. (81) stated that thefeatures mentioned above make Zymomonas,"a particularly important organism to eradicatefrom high-speed keg beer processing plants."High acetaldehyde concentrations in beers aredue to Zymomonas (34) (see below).Zymomonas has not been reported in lager

beers. Its presence during the lager fermenta-tion should not be excluded since glucose, su-crose and fructose are present. However, thelow temperature of the process, 8 to 12'C, isunfavorable for the development of these bacte-ria.The ecology ofZymomonas in breweries has

been studied by Dadds (33).

Fermenting Palm SapPalm wines are alcoholic beverages obtained

throughout the tropics from the spontaneousfermentation of the sugary sap of palm trees.The different tree species that are tapped forthe preparation of palm wines are given inTable 2. Distillates from palm wines are knownin many areas around the world. The chroni-

clers of the early Euro-African contacts alreadymentioned palm wine and its preparation (Pi-gafetta and Lopes in 1591 [125], Capelle in 1641[27], Jean-Frangois de Rome in 1648 [52], andZenobio da Firenze in 1820 [37]).

Different modes of tapping the palm sap arepracticed: (i) the tree of the oil palm Elaeisguineensis Jacq. is felled, and the sap is col-lected in a shallow notch in the stem or bycutting the terminal bud. (ii) After a prelimi-nary clearing of the tree, the bracts of the maleinflorescense are cut off and a hole is made atits base. The hole is left to dry for 2 days and isthen reopened to collect the sap in a gourd. Thetapping of a tree is continued for 2 to 3 weeks,the hole being reopened twice a day while thecollected sap is removed. (iii) Martin (109) de-scribed the tapping of Borassus flabellifer L.in Cambodia. (iv) In India, Sri Lanka, Indo-nesia, and the Philippines the male inflores-cence ofCocos nucifera L. is beaten, and the sapfrom the wounded flowers is collected. (v) Thetree is cleared, and the sap is tapped througha hole under the terminal bud. (vi) The termi-nal bud of Raphia trees is cut, and the sap istapped from the wound. This method finds ap-plication in different parts of Zaire. A detaileddescription of the tapping methods used for the

TABLz 2. Palms from which palm wine are obtaineda

Name of palmAcromia vinifera OerstArenga pinnata (Wurmb.) Merr. (Syn.'A. saccharifera Labill.)Attalea speciosa Mart.Borassus aethiopum Mart.Borassus flabellifer Linn.Caryota urens Linn.Cocos nucifera Linn.Corypha umbraculifera L.Elaeis guineensis Jacq.Hyospathe elegans Mart.Hyphaenae guineensis Thonn.Jubaea chilensis (Molina) BaillonMauritiella aculeata (H. B. and K.) Burret (Syn. Lepidococcus

aculeatus H. Wendl and Drude)Morenia montana (Humb. and Bonpl.) Burret (Syn. Kunthiamontana Humb. and Bonpl.)

Nypa fruticans Wurmb.

Orbignya cohune (Mart.) Dahlgren ex Standley (Syn. Attaleacohune Mart.)

Phoenix dactylifera Linn.Phoenix reclinata Jacq. (Syn. Phoenix spinosa Schum. and Thonn.)Phoenix sylvestris (L.) Roxb.Raphia hookeri Mann and Wendl.Raphia sudanica A. Chev.Raphia vinifera Beauv.Scheelea princeps (Mart.) Karsten (Syn. Attalea princeps Mart.)a See references 3, 25, 30, 84, and 131.

Nicaragua, Panama, Costa-RicaFar EastBrazil, GuyanaTropical AfricaIndia, Cambodia, JavaIndiaIndia, Sri Lanka, AfricaSri LankaAfricaBrazil, GuyanaWest AfricaChiliBrazil, Venezuela

Brazil

Sri Lanka, Bay of Bengal, Philip-pines, Carolines, Salomons Is-land

Honduras, Mexico, Guatemala

North Africa, Middle EastCentral AfricaIndiaAfricaAfricaAfricaBrazil, Bolivia

Location

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oil palm and Raphia palms is given by Tuley(167, 168).The yield ofpalm sap from one palm tree may

be considerable. Simonart and Laudelout (152)mentioned a felled Elaeis guineensis tree thatproduced 150 liters of sap within 32 days. InZaire, one of us found that, from E. guineensisJacq. trees, approximately 3 liters of sap perday was collected during 14 to 21 days; from theRaphia trees 2 to 11 liters of sap per day iscollected during 3 to 6 weeks (172).As the sap oozes out in the gourd or in any

other container, it is infected by microorga-nisms colonizing the stalk of the male inflores-cense, the tap holes and the surrounding partscovered with hairy outgrowths (57, 58), the tap-ping equipment, and the gourd itself. The sap

from different gourds is pooled, and the palmwine is generally consumed within 24 h. Atthat moment the palm wine is a whitish, sug-ary, and acid alcoholic beverage (3, 166). Van-derijst (169) described it very well: "Le vin depalme frais, en pleine fermentation, est un liq-uide blanchatre, trouble, petillant, a saveur

sucree et aigrelette. II est peu alcoolise. C'estune boisson rafraichissante, nourissante, le-gerement stimulante et d'un gouft assez agrea-ble."The characteristics of palm wines depend on

many factors such as the period of tapping thepalm trees, the palm species, the storage pe-riod, the season, etc. (1, 2, 60, 61, 123, 166).Palm wine contains 0.1 to 7.1% ethanol, de-pending on the stage at which it is collected. Anormal palm wine contains approximately 4 to5% ethanol and has a pH of 3 to 4. The alcoholicfermentation of the sap starts in the collectiongourds. As the fermentation proceeds, the pH ofpalm wine drops to 3 to 4, and the presence oftartaric, malic, pyruvic, succinic, lactic, cis-aconitic, citric, and acetic acids can be demon-strated (170, 172). The hydrolyzable sugar con-tent of the sap of Arenga pinnata (Wurmb.)Merr., Borassus aethiopum Mart., Borassusflabellifer L., Cocos nucifera L., Caryota urens

L., Nypa fruticans Wurmb. ranges from 8 to16.5% (20, 25, 30, 117, 123, 131, 152, 166, 172).The main constituent is sucrose. The sap alsocontains small amounts of glucose, fructose,maltose, raffinose, and maltooligosaccharides(13, 60).The preservation of palm wines has been the

object of some reports. Faparusi (56) found thatthe sodium metabisulfite concentration neededto suppress microbial growth is at least 500 pg/ml. Okafor (122) estimated an average intake of500 mg of metabisulfite, per day and per per-son, for a daily consumption of about 600 ml oftreated palm wine. Both data show that the


acceptable maximal daily intake of 0.35 mg ofsulfite per kg of body weight (116) is largelyexceeded and not acceptable for human con-sumption. Faparusi and Bassir (59) demon-strated that the bark of Abryga gabonensisBaill. (Sacoglottis gabonensis Urb.) possessessome bacteriostatic activity and is able to re-duce the rate of souring of palm wine if thebacterial population is not too high. Okafor(123) suggested a pasteurization at 700C for 30min combined with sorbic acid as the most use-ful means of preserving palm wine. A pasteuri-zation unit for the processing of Raphia winewith a capacity of 1,000 liters per day has beenset up in Cameroon. As the preparation ofpalmwines is certainly not to be considered as amarginal activity in African countries, one cansay that the processing of palm wines opens upconsiderable technical and commercial pros-pects (65).Palm wines have rather complex microfloras

(Table 3), but it is certain that Zymomonasstrains are very important bacterial constitu-ents. They are largely responsible for the alco-hol content and for the frothing because ofC02formation. C02 and small amounts of lactic andacetic acids (see below) contribute to the acid-ity. The production of some acetaldehyde andthe characteristic fruity odor of Zymomonasprobably positively influence the odor and thetaste of palm wine.Zymomonas strains are extremely well

adapted to this ecological niche: sucrose, glu-cose, fructose, amino acids, and growth factorsare present in the palm sap. The organisms areresistant to ethanol and grow at low pH and inanaerobic conditions. Whether Zymomonas oc-curs in all palm wines, whether antagonism orsynergism with other organisms occurs, whatdegree ofcompetition for the sugar there is withyeasts, and at which stage maximum Zymo-monas density is reached remain unansweredquestions. Zymomonas has been isolated frompalm wines on many occasions. Roelofsen (139)was the first to isolate these bacteria from fer-menting Arenga pinnata (Wurmb.) Merr. sapin Java (Indonesia). His strain was unfortu-nately lost and new isolates were made at therequest of A. J. Kluyver by G. Derx and C. 0.Schaeffer in 1949 from Arenga sap, respec-tively, in Bogor and in Bandung, Java, Indone-sia. One of us (J.S.) isolated a number ofZymo-monas strains from Elaeis and Raphia palmwines in Western and North-Western Zaire.These strain will be extensively discussed be-low. Also, Faparusi (58) and Okafor (122) re-ported on the isolation ofZymomonas from Ni-gerian Elaeis and Raphia palm wines, respec-tively.


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TABLE 3. Microorganisms in palm wines

Organism SourceBacteriaAcetobacterAcetobacter rancens var. viniA. roseusEnterobacter aerogenesBacillusBrevibacteriumCorynebacteriumKlebsiellaLactobacillus brevisL. leichmanniiL. pastorianusL. plantarumLeuconostoc mesenteroidesMicrococcusPediococcusSarcinaSarcina luteaS. marcescensStreptococcusZymomonas

FungiAspergillusAspergillus flavusA. nigerCandidaCandida mycodermaC. tropicalisEndomycopsis viniKloeckera apiculataMucor mucedoPichiaPichia pastorisP. membranaefaciensRhizopus nigricansRhodotorulaSaccharomycesS. cerevisiae

S. chevalieriS. ellipsoideusS. exiguusS. florentinusS. markiiS. pastorianusS. roseiSaccharomycodes ludwigiiSchizosaccharomycesSchizosaccharomyces pombe

56, 59, 60, 122152585860, 1221225613, 12258, 12215212213, 56, 6013, 56, 58, 6013, 58, 60, 1226056, 581225856, 60, 12258, 122, 139, 170

59, 60585860, 1215960121121, 152, 170581358585815212113, 56, 59, 60,

152, 1704, 17017012112112117012141524, 13, 60

The repeated isolation of Saccharomyces,Zymomonas, lactobacilli, and Acetobactertends to confirm the hypothesis of Simonartand Laudelout (152) that palm wine may be theresult of mixed alcoholic, acetic, and lactic fer-mentations. The role of several organisms iso-lated is not understood and may not be essen-tial. Little investigation has been done on thesuccession of some microorganisms in palm

wine. Okafor (122) counted yeasts, Streptococ-cus, Lactobacillus, Acetobacter, and Micrococ-cus spp. in three different palm wines at 24 hintervals for several days; he found considera-ble differences. His conclusions consisted of thefollowing general trends (Fig. 2). (i) The yeaststended to remain constant in numbers, i.e., 106to 109/ml. (ii) Micrococcus occurred most con-sistently in the samples. (iii) Lactic acid bacte-ria were important components of the micro-flora. (iv) Certain gram-negative bacteria, e.g.,Serratia and Klebsiella spp., were only presentduring the first 3 days. (v) Acetobacter devel-oped after day 3. Unfortunately, the author didnot count Zymomonas cells present.

Fermenting Sugarcane Sap

Gongalves de Lima et al. (70) isolated Zymo-monas strains from fermented sugarcane juice(caldo-de-cana-picado) from Northeast Brazil.The bacteria were gram-negative rods, 2 to 6 by1 to 2 Ium, small and large cylinders, single orin pairs, rarely forming chains of a few cells.They were very motile with mono- or lophotri-chous flagella. The colonies were very typicallymucoid. In liquid media, the bacteria developedabundantly with a flocculent deposit. Fermen-tation still occurred at 4200. Growth occurredafter 15 to 18 h of incubation both in aerobiosis

7

5 pH

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Ea

0

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(n

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0U.0

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FIG. 2. Sequence ofsome microorganisms inpalmwine (schematically from Okafor [122D]. Symbols:( ) yeasts, (------) Micrococcus, (. ) Lacto-bacillus, (- - - - -) Acetobacter, and (- - *-) Entero-bacter.

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and in anaerobiosis. Glucose, fructose, and sac-charose were fermented with gas production.Lactose, maltose, xylose, raffinose, rhamnose,inositol, arabinose, galactose, mannitol andmannose were not metabolized. Acetylmethyl-carbinol, indole, urease, and H2S were not pro-duced. Nitrate was not reduced. Catalase waspresent. The antagonistic action against 39strains of bacteria and fungi has been estab-lished. In another publication (72), it was dem-onstrated that this antagonistic action was notcaused by the pH drop per se or by the lactic,succinic, or acetic acids produced by Z. mobilis:"The antagonistic activity proved to be a spe-cific activity but at some degree related withthe increased acidity produced by the metabolicactivity." A new taxon Z. mobilis var. recifen-sis was created for these organisms (see below).

Ripening HoneyRuiz-Argueso and Rodriguez-Navarro (141)

have isolated Z. mobilis from ripening honeyand, occasionally, also from the bee. The twomain microbial genera present in ripeninghoney were Gluconobacter and Lactobacillus.

Some Technological ApplicationsThe two properties ofZymomonas, quick con-

sumption of glucose by an alcoholic fermenta-tion and the lowering of pH of the medium,provoked much interest in the early 1930samong German microbiologists and suggesteda number of promising applications for thesebacteria. Lindner (104) used them to preserveextracted cuttings of sugar beets. After 80 daysof anaerobic incubation, the taste, aroma, andcolor were good, and the beet flavor had disap-peared. The pH fell to 4.4. Z. mobilis had noharmful effects on man or cattle. The preserva-tion of the leaves of sugar beets with thesebacteria was also tried successfully, and a winyaroma developed (104). Beer draff, treated withZ. mobilis for preservation (103), and potatoestreated in the same way (104) were both eagerlyaccepted by cattle.

Lindner (103, 104) used Z. mobilis to fermentdiluted molasses and milk enriched with fer-mentable sugars. He obtained beverages withpleasant flavors.

Schreder (107) used the property ofZ. mobilisto ferment glucose and fructose, but not mal-tose, to prepare from beer wort a near beercontaining approximately 0.7% ethanol. A maltwith low protein content was needed; it wasmashed and inoculated with the bacteria. After3 days, a nutritive beer was ready for consump-tion. It still contained maltose, dextrin, andpeptone and had a characteristic taste and a

stable foam. "Das Getrank ist erfrischend unddurstanregend, 'smeckt nach mehr' und gibtbei dem Genusz schon das Gefuhl, das es nichtmude macht," said Luers (107). Another bever-age of low alcohol content was prepared byLindner from a malt-coffee-milk mixture. Weare not aware that these preparations and bev-erages became widely appreciated.

In several countries of the Third World, someresearch is being conducted in order to promotethe valorization of the cheap and abundantlyoccurring sugar contained in fruit and plantsaps, and also of the plant starch, in order toprepare alcoholic beverages. We are convincedthat Zymomonas may be of considerable inter-est for the development of new beverages andfor the industrialization of traditional bever-ages. Sanchez-Marroquin et al. (142, 143) devel-oped a new technological method for the pro-duction of pulque. It is based on the use ofselected cultures of Saccharomyces carbajaliandZ. mobilis as alcohol producers, Lactobacil-lus spp. for the lactic acid fermentation, andLeuconostoc mesenteroides to obtain the de-sired degree of viscosity. In an industrial plant,a production of 50,000 liters per day was at-tained. The beverage obtained was comparableto the pulque prepared traditionally by sponta-neous fermentation.The alcoholic beverages of the world can arbi-

trarily be subdivided in two groups: (i) thoseobtained by fermentation of the simple sugars(mono- and disaccharides) present in plantjuices or extracts from, e.g., apple, pear,cherry, prune, grape, currant, raspberry, elder-berry, strawberry, rhubarb, orange, pineapple,palm, sugarcane, cocoa-usually called wines;and (ii) those obtained by fermentation aftersaccharification of the starch present in suchmaterials as corn, sorghum, soybean, millet,plantain, rice, barley, wheat-usually calledbeers. The empirical use ofZymomonas in thepreparation of palm, Agave, and sugarcanewines suggests that these bacteria might beresponsible for a large spectrum of alcoholicbeverages from a great variety ofplant juices intropical areas around the world. It would bevery important to examine a great variety ofthese beverages for the presence of Zymo-monas.Another possible technological application of

Zymomonas is in the large-scale production ofethanol. All fermentation equations reported inthe literature (see below) and our own experi-ence with some 40 strains show that all Zymo-monas strains produce more than 1.5 mol ofethanol per mol of glucose added. The mostsuitable strains produce about 1.9 mol ofethanol from each mol of glucose, which is

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about as much as S. cerevisiae. The conditionsmust be strictly anaerobic; otherwise, the yieldof ethanol is smaller. In aerobic conditions,only about 1 mol of ethanol is produced from 1mol of glucose. Zymomonas grows and fer-ments very fast. Its preference for low pH pre-vents foreign contamination. These bacteriastill grow in high glucose and ethanol concen-trations. Kluyver and Hoppenbrouwers (89) re-ported up to 10% (wt/vol) ethanol from 25%glucose solutions. Almost half of our strainsgrew in media with 40% glucose; how muchethanol is produced still remains to be deter-mined. These organisms seem to be very prom-ising agents for the industrial production ofethanol from molasses or other sugary juices.As far as we know, however, this possible appli-cation has not yet been pursued.

Therapeutic Use"Aguamiel" seems to have been used for ther-

apeutic purposes already in the Aztec tradition(Clavigero, cited in [73]). Lindner (101) men-tioned the therapeutic use of fresh or concen-trated juice ofAgave in cases ofrenal and meta-bolic diseases, and he considered aguamiel andthe beverages made with Zymomonas as thetropical counterparts of yogurt and kefir.On several occasions, Lindner (103, 106) reco-

mended the use ofZymomonas as a competitorto avoid the disagreeable effects of a putrefac-tive flora. The harmlessness ofZymomonas to-wards man was demonstrated by Lindner (106):".... habe ich ruhig einen biologischen Selbst-mord riskiert, indem ich in Gegenwart meinerMitarbeiter einen vollen Suppenloffel einerausgeschleuderten Masse dieses Tm (Termo-bacterium mobile) verschluckt habe." He re-ported further on the successful application ofZymomonas against foot-and-mouth andBang's diseases in cattle and against purulentfuruncles and wounds in man.

In vitro, the antagonistic effect of Zymo-monas against a number of bacteria and fila-mentous fungi has been examined by Gon-galves de Lima et al. (70, 72, 73). There are fourreports on the therapeutic applications of Zy-momonas cultures in cases of chronic entericand gynecological infections. The data are sum-marized in Table 4.

History of Individual StrainsUntil recently, few Zymomonas strains were

available. Most of the biochemical and physio-logical studies have been carried out withZym-omonas strains ATCC 10988 and NCIB 8227(see below). The history of the isolates is sum-marized in Table 5. Several strains are no

longer available. Exact and extensive informa-tion on some isolates remains scarce, despiteintensive searching.

DETECTION, ISOLATION, ANDIDENTIFICATION OF THE GENUS

ZYMOMONASDetection

A suitable detection (32) medium contains(all in percent, weight/volume) malt extract,0.3; yeast extract, 0.3; glucose, 2; peptone, 0.5;and actidione, 0.002. The pH is adjusted to 4.0.The medium is dispensed in screw-capped bot-tles (capacity, 25 ml) with Durham tubes, 20ml/bottle, and sterilized. Ethanol is added to afinal concentration of approximately 3% (vol/vol). The presence of Zymomonas is indicatedby abundant gas production at 25 or 300C after 2to 6 days. False-positive results may occur dueto lactobacilli or some wild yeasts; a Gram stainis recommended. Positive tests should be com-pleted by the final identification ofZymomonasas described in Identification, below. The sensi-tivity of the method is estimated to be 1 to 5infection units per ml of inoculum.A detection method using immunofluores-

cent staining has a sensitivity of 160 to 2,500cells per ml (35). Harrison et al. (81) developeda rapid nonspecific method for the detection oflow concentrations of viable brewery organismssuch as yeasts, Zymomonas, Lactobacillus, andAcetobacter as a quality control for pasteurizedor sterilized products. It is based on the changein pH of a medium containing 2% glucose, 0.3%yeast extract (Difco), and 0.01% Tween 80 at pH7, inoculated with the sample to be tested.

IsolationBarker and Hillier (12) isolated the cider

sickness organisms on beer wort gelatine asminute dotlike colonies after 11 days at 220C.Shimwell (150) enriched the sample anaerobi-cally in sterile beer with 2% glucose and subse-quently plated out anaerobically on glucosebeer agar. Barker (11) isolated the cider sick-ness bacteria on malt extract gelatin plates. Anisolation medium with apple juice has beenused by Millis (114, 115): applejuice was treatedovernight with pectozyme and filtered, andthen 1% yeast extract (Difco) and 0.001% acti-dione were added. The pH was adjusted to 4.5.A 10-ml sample of the infected cider was centri-fuged, and the deposit was streaked on plateswith the above medium. The plates were incu-bated anaerobically in the McIntosh and Fildesjar at 250C.One of us (162) described the isolation of

Zymomonas from fresh Zairese palm wines in

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TABLE 4. Therapeutic uses ofZymomonasNo. of cases Sri

Disorder treated Treatment Stran SourceTreated Cured

Chronic enterocolitis 6 6 Liquid cultures, 300 Ag-11 De Paula GomesChronic cystitis 1 1 ml a day between (51)

the mealsVaginitis caused by Candida albi- 20 20 Tampons impreg- Ag-11 Wanick et al. (177)

cans, Trichomonas vaginalis, nated with Zymo-and Neisseria gonorrhoeae monas, changed

dailyGynecological infections by Can- 40 40 Liquid cultures and IA-CP1 Wanick and Caval-dida albicans, T. vaginalis, and agar jellies canti Da SilvaN. gonorrhoeae (176)

Colpitis and vulvovaginitis 95 82 Tampons IA-CP1 De Souza and Decaused by E. coli, Haemophilus Souza (53)vaginalis, N. gonorrhoeae, T.vaginalis, and Staphylococcusspp.

WL differential medium (Difco). Three meth-ods can be used: (i) different dilutions of thesample are mixed with the WL differential me-dium and poured in thick layers in petri dishesbefore solidifying; (ii) samples are plated andcovered by a top layer of medium; (iii) the sam-ples are streaked on the medium in petri dishesand incubated in the GasPak anaerobic system.The last procedure seems most convenient tous. In the WL differential medium the coloniesare lenticular, 1 to 4 mm after 4 to 5 days at300C, and deep green. For the isolation ofZym-omonas from palm wines it is very important touse a young wine, about 24 h old, as we havenever been able to isolate these bacteria frompalm wines over 48 h old. We have not foundZymomonas in fermenting sugarcane juice orin fermenting cocoa in Zaire, where we ex-

pected these bacteria to occur.Richards and Corbey (137) proposed a two-

step isolation procedure. (i) The deposit from 25ml of centrifuged spoiled beer was enriched at300C in beer at pH 4.0 with 4% glucose and 0.3%yeast extract. (ii) Cultures were. streaked onthe same solidified medium and incubated an-aerobically at 300C.

Identification

The following features permit the identifica-tion ofZymomonas (i) rods 2 to 6 ,m long withan unusual cell width of 1 to 1.4 ,um, (ii) gramnegative, (iii) no spores, (iv) if motile, by 1 to 4lophotrichous flagella, (v) no growth on nutri-ent agar or in nutrient broth, (vi) anaerobic,but tolerates some oxygen, (vii) fermentation ofglucose and of (viii) fructose to (ix) nearly equi-molar amounts of ethanol and CO2, (x) oxidasenegative, (xi) 47.5 to 49.5% of guanine plus cy-

tosine (G+C). These 11 features may be consid-

ered as the minimal description of the genusZymomonas. It should be noted that the com-mon generic description ofZymomonas (88, 90,151) comprises fewer features.The key for the identification of pseudomo-

nads proposed by Hendrie and Shewan (82) hasto be improved with respect to the identificationof Zymomonas. They characterized it as (i)gram-negative rods, (ii) motile with polar fla-gella, and (iii) growing anaerobically. Motilityis not an essential feature for Zymomonas,since only 30% of the strains were motile (seeCell morphology). On the other hand, the al-most quantitative fermentation of glucose toethanol and C02 should be included as a basicfeature.

In a tentative key for the identification ofspoilage bacteria in brewing, Ault (9) charac-terized Zymomonas as (i) gram-negative orga-nisms, (ii) forming no acid from ethanol, and(iii) fermenting glucose to ethanol.

Some Commonly Used Media and GrowthConditions

Standard media. The liquid standard me-dium we use most often contains 0.5% yeastextract (Difco) and 2% glucose, 9 ml per testtube. For the solid standard medium, 2% agaris added. Kluyver and Hoppenbrouwers (89)recommended the addition of 2% CaCO3 to thismedium; however, it is not necessary. Zymo-monas cultures should be transferred every 2 to3 weeks.Apple juice medium. This contains four-

times-diluted apple juice, 1% yeast extract(Difco), and 10% gelatin, pH 5.5, in screw-capped bottles. In this medium the cider sick-ness bacteria remain viable for 17 weeks (115).Beer media. Shimwell used media contain-

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ing beer with 2% glucose. He recommendedweekly transfers. For maintenance, he pre-ferred a glucose-beer gelatin stab culture. Abeer medium made of a primed ale with 2%glucose and 0.1% yeast extract has been used byDadds et al. (36) for the detection of volatilesulfur compounds. Several commercial beersunfortunately contain disinfectants. Their usein media cannot be recommended.MYPG medium. This medium contains 0.3%

malt extract, 0.3% yeast extract, 0.5% peptone,2% glucose, and 2% agar (179).

Synthetic media. The minimal synthetic me-dium of Kluyver and Hoppenbrouwers (89) con-tains 2% glucose, 0.1% K2HPO4, 0.1%(NH4)2S50, and 0.05% MgSO4 * 7H20 in tap wa-ter.A synthetic medium for the determination of

amino acid and growth factor requirements wasdescribed by Belaich and Senez (17). Van Pee etal. (174) added the following vitamins: p-ami-nobenzoic acid, biotin, folic acid, cyanocobala-min, lipoic acid, nicotinic acid, pyridoxine,thiamine, riboflavin (1 ,ug each per ml), as wellas guanine, adenine, hypoxanthine, cytosine,and uracil (60 ug each per ml).The medium of Gibbs and DeMoss (67). It

contains 1% glucose, 1% tryptone, 1% yeast ex-tract (Difco), 0.5% KH2PO4 in 98 ml, plus 2 mlof a salt solution with 0.8% MgSO, * 7H20,0.16% MnSO4 4H20, 0.04% NaCl, and 0.04%FeSO4 * 7H20.Zymomonas can be grown at 25 to 300C. Most

authors prefer to incubate Zymomonas anaero-bically, but this is only necessary for surfacegrowth on slants or petri dishes.

TAXONOMY OF ZYMOMONASIn the present section we shall critically re-

view several proposals made in the literatureon the nomenclature and classification of thisgenus. A good modem taxonomy of a bacterialtaxon requires a numerical analysis of manyand diverse phenotypic data, the DNA basecomposition (percent G+C), the degree of ge-nome DNA relatedness (percent D, DNA "ho-mology") determined by DNA-DNA hybridiza-tions, a comparison of protein electrophero-grams, etc. We shall summarize and discussthe data that led us (50) to propose an improvedtaxonomy of this genus.

Numerical Analysis of the PhenotypeThe original description of Z. mobilis by

Kluyver and Hoppenbrouwers (89) and of Z.anaerobia by Shimwell (151) comprised some 35phenotypic features. Millis used some 40 fea-tures in comparing six Zymomonas strains (see

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below, Classification and Nomenclature). Nu-mercial analysis of phenotypical informationrequires a larger number of strains and of tests;therefore we (50) used 38 Zymomonas strainsfrom diverse origins (Zairese fermenting palmsap, Mexican pulque, British spoiled beer, andsick cider). We determined 138 phenotypic fea-tures for these strains. The extensive descrip-tion of these features will be given in Phenotyp-ical Description, below. We shall limit our-selves here to their numerical analysis. Themain new features we added were: several Csources for growth, antibiotic sensitivity,growth factor requirements, stimulation ofgrowth by amino acids, and the susceptibility todyes and some other compounds. We calculatedthe simple matching coefficients SSM (156). Thesevalues were clustered both by single linkage(154) and by unweighted average linkage (156).The dendrogram has been published (50).Twenty-eight features tested are present in

every strain. These features thus constitute avaluable description of Zymomonas and con-siderably enlarge the minimal descriptiongiven in the section, Identification. They can besummarized as follows. Zymomonas cells aregram-negative rods, 2 to 6 unm long, 1 to 1.4 jumwide, usually occurring in pairs. If motile, theyhave one to four polar flagella; motility may belost spontaneously. Colonies in standard me-dium (see above) are glistening, white, orcream colored with a diameter of 1 to 2 mmafter 2 days at 30'C. The colony edge is regular.A fruity odor of variable intensity, according tothe strain, was observed. They grow on andferment glucose and fructose, mainly to almost2 mol of ethanol and 2 mol of CO2 (gas) per molof hexose fermented. The ratio, moles of etha-nol produced to moles of glucose fermented, isat least 1.5. Some lactic acid and traces of ace-tylmethylcarbinol are formed. All strains cangrow in a medium with 2% yeast extract and20% glucose. The optimal pH for growth is 7.3.The final pH after 3 days at 30'C in standardmedium is 4.8 to 5.2. The cells can grow instandard medium in the presence of 5% etha-nol. Catalase is present. The cells require pan-tothenate and biotin for growth. They can growin standard medium in the presence of 0.1%neutral red. They reduce 2,3,5-triphenyltetra-zolium chloride and the dyes methylene blueand thionin. Their growth is sensitive toeither 500 ,4g of sulfafurazole, 30 ug of novobio-cine, 10 gg of tetracycline, or 10 /.tg of fusidicacid on disks.

All Zymomonas strains tested by us lack thefollowing 74 features. They form no spores orcapsules; they have no detectable lipids in the

cells. They do not grow in 0.5% yeast extractbroth, in 1% peptone broth, in liquid standardmedium with 2% NaCl, in liquid standard me-dium at pH 3.05, or, after three transfers, inthe synthetic medium of Kluyver and Hoppen-brouwers. Aerobically, no or poor growth wasobtained on standard medium agar. They donot grow on nutrient agar. They do not grow onor ferment starch, dextrin, raffinose, D-treha-lose, maltose, lactose, D-cellobiose, D-galactose,n-mannose, L-sorbose, salicin, L-rhamnose, D-and L-arabinose, D-xylose, D-ribose, D-sorbitol,dulcitol, D-mannitol, adonitol, erythritol, glyc-erol, ethanol, Na D-galacturonate, Na DL-mal-ate, Na succinate, Na pyruvate, Na DL-lactate,Na tartrate, or Na citrate. Their growth is notstimulated by L-cysteine, L-glutamate, glycine,L-histidine, L-lysine, DL-methionine, L-orni-thine, DL-serine, L-threonine, or L-tyrosine. Forgrowth, they require neither lipoic acid, folicacid, nicotinic acid, nor p-aminobenzoic acid.They are oxidase negative. They do not formindole. They reduce neither nitrate, neutralred, nor safranin. They hydrolyze neither gela-tin nor Tween 60 or 80. They are not sensitive to0.01% actidione, 5 U of bacitracin, 10 ,ug ofgentamycin, 10 ,ug of kanamycin, 10 jug of lin-comycin, 30 ,g of nalidixic acid, 10 ,g of neo-mycin, 5 U of penicillin, 300 U of polymyxin, 10,ug of streptomycin, or to the vibriostatic com-pound 0/129.

Thirty-seven features occur in some strainsbut not in others. They are (percentage ofstrains with features present are given in pa-rentheses): motility (26), formation of rosettes(50), pleomorphism (63), the nature of the celldeposit (compact [65], granular to flocculent[34]), the growth on and the fermentation ofsucrose (50), the formation of H2S (11), thepresence of the decarboxylases for L-ornithine(11), L-arginine (82), and L-lysine (47), the pres-ence of urease (24), growth in standard mediumwith 0.5% NaCl (97) or 1% NaCl (71), growth instandard medium at pH 3.5 (45) or 7.55 (84),growth in standard medium at 34°C (97) or at38°C (74), growth in 2% yeast extract with 40%glucose (55), a pH <4.8 after growth in stan-dard medium at 34°C for 7 days (2), growthafter a second transfer in the synthetic mediumof Kluyver and Hoppenbrouwers (11), growthdecrease by omission of one or more of thefollowing amino acids: L-alanine (8), L-arginine(11), L-asparagine (5), L-hydroxyproline (11), L-isoleucine (5), L-leucine (21), DL-phenylalanine(2), DL-proline (5), DL-tryptophan (2), or L-va-line (47), the requirement of either riboflavin(8), thiamine (2) or cyanocobalamin (8), theresistance to 10 ,g of ampicillin (39), 10 ,ug of

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cephaloridine (29), 30 ,tg of chloramphenicol(2), 10 tkg of erythromycin (89), 10 gg of methi-cillin (97), or 30 gg of vancomycin (53).The phenotypic similarity between all the

strains is very high: they all form one clusterabove 88% SSM. The strain Z6 from Zairesepalm wine was calculated to be the phenotypiccentrotype, the most representative strain ofZ.mobilis subsp. mobilis. It was deposited as

NCIB 11199 and ATCC 29191. Within this sub-species, the fermentation of sucrose and themotility are not correlated with other features.To maintain the separate species anaerobiaand its variety immobilis is not justified (seebelow).Three strains in our collection are slightly

aberrant. One was isolated in Bristol from sickcider. We deposited it as Z. mobilis subsp. po-

maceae ATCC 29192 and NCIB 11200. Thisstrain forms the border of the group, with an

average difference of 17 features and six corre-lated features, differentiating this strain fromall the other zymomonads (Table 6). Two otherZymomonas strains, NCIB 8777 and NCIB10565, have been investigated less extensively,but they are almost identical to ATCC 29192.

DNA Base Composition and DNA GenomeSize

We determined the DNA base composition of41 Zymomonas strains by thermal denatura-tion (163). All these strains have a midpoint ofthermal denaturation (Tm) between 88.9 and89.70C, with an average Tm of 89.3 + 0.20C(standard deviation). DNA base compositionvaries within the narrow range of 48.5 1.0%G+C. The cider sickness organism Z. mobilissubsp. pomaceae ATCC 29192 is near the bot-tom of the group, with 47.7% G+C.A few other percent G+C values have been

published; on the whole, they fit well within the

range we established. Dadds et al. (36) reported48.3% for Zymomonas NCIB 8938, Kiehn andPacha (87) found 48.6%, presumably for thesame strain, and we found 49.1 and 48.6% fortwo subcultures. For Zymomonas NCIB 8227,we found 49.0% G+C; Dadds et al. (36) reported47.6% G+C, slightly too low. All the abovepercent G+C values are close and support theidea that there is great similarity within thisgenus.Dadds et al. (36) reported 46.4% G+C for

Zymomonas NCIB 10565 and 45.1% G+C forZymomonas B70. In our experience, the latterstrain is genotypically and phenotypically in-distinguishable from all the other Z. mobilissubsp. mobilis strains. The low percent G+Cvalues of the above two strains strongly suggestthat they should be redetermined.The genome size of some 40 Zymomonas

strains was determined with the initial rena-

turation rate method (68, 69). The DNA ge-nome size, expressed as molecular weight, ofZymomonas strain 5.3 is 1.53 (± 0.19) X 109,and those of the other Zymomonas strains arenot significantly different. This genome size israther small, about 56% of the Escherichia coligenome. It can accommodate some 1,500 cis-trons.

Genome-DNA RelatednessWe determined the degree of genome-DNA

relatedness (percent D; DNA "homology") (163)with the initial renaturation rate method (48)in stringent conditions, i.e., those that allowduplex formation to occur only between veryclosely related or identical polynucleotide se-quences.

All strains except one were found to be genet-ically very similar. The degree of DNA related-ness in the main group is at least 76% D. Thismain group consists of two subgroups of strainsthat are almost identical. One subgroup con-

TABLE 6. Differential features of the cider sickness organism Z. mobilis subsp. pomaceae ATCC 29192 (=NCIB 11200)a

Z. mobilis subspeciesFeatures

mobilis pomaceae

Growth in standard medium + 0.5% NaCl Growth after 1-3 days No growthGrowth in standard medium at 360C Growth after 1 day Scant growth after

2 daysGrowth in standard medium + 0.0075% KCN Growth after 1-5 days No growthFinal pH after 7 days of growth at 340C in stan- 4.9-5.4 4.7dard medium

No. of amino acids stimulating growth 0-5 (exact number depends on 6the individual strain)

Tolerance towards 02 Various degrees of microaero- Most anaerobicphily strain

a Modified from reference 50.

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sists of 28 Zairese palm wine strains Z1, Z2, Z3,Z5, Z6, Z7, Z8, 7.4, 17.1, 17.3, 17.4, VP1, VP2,VP3, 42.1 42.2, 42.3, 42.4, 70.1, 70.2, 70.3, 70.7,70.9, 70.11, 70.14, 5.1, 5.4 and 5.5. Their averageDNA relatedness is 94% D, with a minimum of88%. The second subgroup consists of theZairese palm wine strains 17.2, 70.10, 70.12,strains 2.1, 2.2, 409, and NCIB 8227 from in-fected British beer, and strains 410 and ATCC10988 from Mexican pulque. Their average per-cent D is 96. Both subgroups are linked at anaverage of 82% D. Both subgroups do not repre-sent taxonomic units, since they have no char-acteristic phenotypic features, protein electro-pherograms, or percent G+C.There are no genetic differences between

named Z. mobilis and Z. anaerobia strains,between sucrose-fermenting and sucrose non-fermenting strains, or between motile and non-motile strains.The pomaceae organism ATCC 29192 has a

distinctly different genome: it has less than 32%D with all subspecies mobilis strains.

Similarity of Protein ElectropherogramsKersters and De Ley (86) pointed out that the

cells of each bacterial strain, grown in stand-ardized conditions, should always produce thesame set of protein molecules, within experi-mental error. The protein gel electrophero-grams, prepared in rigorously reproducible con-ditions, were indeed found to be specific foreach strain. Kersters and De Ley (86) developedtechniques for preparing these electrophero-grams and comparing them by computer-as-sisted techniques.We applied these techniques to our battery of

strains; the dendrogram of quantitative corre-lations has been published (164). A visual com-parison of the stained gels of the Zymononasstrains reveals a very similar pattern, com-posed of 15 bands (Fig. 3). The three strains ofZ. mobilis subsp. pomaceae (NCIB 10565,NCIB 8777, ATCC 29192) have a different pro-tein pattern, which makes them easily recog-nizable and stresses again their separate taxo-nomic position. Strain identification is possibleupon visual inspection in three other cases, i.e.,the strains 7.4 and 70.7, both lacking band H,and strain Z. anaerobia 1, showing a verystrong P band. However, all our Zymomonasstrains, except three, have a very similar, andsometimes identical, soluble-protein composi-tion, whether the strains were isolated from avariety of Zairese palm wines, Mexican fer-menting Agave juice, Indonesian Arenga sap,or British deteriorated beers. The highly corre-lated protein patterns of the strains reflect the

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close genetic resemblance shown by DNA-DNArelatedness, percent G+C, and numerical anal-ysis of the phenotype. The strain Z6 = ATCC29191 is the electrophoretic centroid strain, i.e.,the most representative strain of the cluster.We found no noticeable differences of the pro-tein patterns between strains named mobilisand anaerobia, between sucrose-fermentingand sucrose-nonfermenting strains, and be-tween motile and nonmotile strains.

Infrared Spectra of Intact Cells

In the past, several authors have tried to usethe infrared (IR) spectra of intact bacterial cellsas an aid in differentiation and identification(7, 26, 76, 92, 94, 120, 128, 138, 148).The broad, diffuse absorption bands in the IR

spectra of intact Zymomonas cells (Fig. 4) aredue to components common to all bacterialcells, i.e., lipids, proteins, and polysaccharides.The IR spectra of various genera, species, andtypes are therefore very similar. This clearlyreduces the usefulness of IR spectra for bacte-rial classification.

Reproducible differences, which are not al-ways correlated with other classifications, oc-cur, e.g., in Acetobacter and Gluconobacter(148), lactobacilli (75), Desulfovibrio and Desul-fotomaculum (26), and Actinomycetales (7, 92,94, 128). Minute, but reproducible, differencesoccur in the shape and intensity of the absorp-tion regions ofZymomonas spectra from 800 to1,000 cm-' and from 1,000 to 1,200 cm-'. StrainsZ2, 70.7, and VP2 have a typical uneven absorp-tion maximum between 1,000 and 1,200 cm-1.Z. mobilis subsp. pomaceae ATCC 29192 has ashoulder at 960 cm-', whereas all the otherstrains have a distinct peak (162).Thus, the IR spectra of Zymomonas strains

are too similar to allow taxonomic conclusions.In some strains, specific IR details occur.

SerologyThere seem to be at least two serological

groups in Zymomonas (36; P. A. Martin, per-sonal communication). The following strainsbelong in serotype 1: NCIB 8227, NCIB 8938,B72, Z2, and 42.1. In serotype 2 belong strainsNCIB 10565, B70, and 42.1. In his Ph.D. thesis,Dadds (33; no permission for quoting data wasobtained) lists a few more strains. By compari-son with our own phenotypic data we found nocorrelation between the serotypes and otherfeatures of all these strains. The serologicaldifferences seem to be of no validity for speciesseparation in Zymomonas, but could have someuse in strain identification.


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BIOLOGY OF ZYMOMONAS

F

A (Top)

a

D

L-

b

L

H

K

L

ovalbumin

550 100

E

K

p

M

ovalbumin

50 100

FIG. 3. Protein electropherograms and densitometer scannings ofZymomonasATCC 29191 (a) andATCC29192 (b).

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1600 1400 1200 1000 800 600 crn,

Z. MOBILIS

SUBSP. MOBILIS VP2

Z. MOBILIS

SUBSP MOBILIS 42.1

Z.MOBILIS

SUBSP POMACEAE ATCC 29192

FIG. 4. IR spectra ofZymomonas.

Classification and NomenclatureSince these bacteria were first named in

1928, they have been moved into five differentgenera, but, since 1936, they have been appar-ently securely fixed in Zymomonas. They havereceived some 20 different names, which arelisted in Table 7.One name requires immediate comment.

Shimwell (150) named his new strainAchromo-bacter anaerobium. In a footnote he suggestedalso a new name Saccharobacter as suitable, "if... it should be considered that the character-istics of this organism justify its inclusion in a

new genus." However, the name Saccharobac-ter has no nomenclatural value, since it is incontradiction with Principle 8 of the Interna-tional Code ,of Nomenclature of Bacteria (96)and it is not validly published, according toRule 28b.Barker and Hillier (12) were the first to de-

scribe the cider sickness organism, but they didnot give it a latin name. It was the real discov-ery of the genus Zymomonas. Lindner de-scribed in 1905 (100) a variety of fermentativebacteria that deteriorated beer wort; he as-

signed them to Termobacterium, an ill-definedgenus comprising diverse small nonsporingrods, as T. fuscescens, T. album, etc. It istherefore not surprising that he assigned thefermentative pulque bacteria to the same genusas T. mobile (102). Termobacterium is no

longer accepted as a genus.Kluyver and Hoppenbrouwers (89) renamed

Lindner's isolate Pseudomonas lindneri accord-ing to the scheme of Lehmann and Neumann(98). The genus Zymomonas was created byKluyver and van Niel (90), "for polarly flagel-lated bacteria causing alcoholic fermentation."They designated Z. mobilis (Lindner) as thetype species.Shimwell (150) isolated a strain from beer

causing dense turbidity with unpleasant odorand flavor. Although the organism was pre-sented as a powerful and dangerous cause of a

beer disease, the nature and the economic im-

portance of this disorder remained obscure andundetailed. The organism was described. Itsanaerobic nature, approaching a microaero-phile, was stressed by the new species nameAchromobacter anaerobium. The same authorproposed, in 1950 (151), a new genus Saccharo-monas, "to indicate the physiological resem-blance to Saccharomyces and the morphologi-cal kinship with Pseudomonas" for bacteriaproducing a quantitative alcoholic fermenta-tion of glucose. Two species were proposed:

Saccharomonas anaerobia ShimwellSaccharomonas anaerobia var. immobilis

subsp. novaSaccharomonas lindneri (Kluyver and Hop-

penbrouwers) Shimwell.

S. anaerobia was advanced as the type species.Its inability to ferment sucrose (see below) wasstressed in italics; the organism was describedas, "anaerobic but microaeroduric (not mi-croaerophilic)." Shimwell made no experimen-tal comparison between both species anaerobiaand lindneri; for the latter, he extracted litera-ture data from Bergey's Manual, 6th ed., fromKluyver and Hoppenbrouwers (89), and fromSchreder et al. (144-147). Therefore, the greatsimilarity between both presumed species didnot become apparent. Shimwell (151) deservescredit for relating Pseudomonas lindneri, Ach-romobacter anaerobium, and Barker and Hil-lier's cider sickness bacteria. However, thename Zymomonas has clear precedence overthe name Saccharomonas Shimwell 1950, thelatter being illegitimate [Rule 51b (2), Interna-tional Code (96)].

Kluyver (88) introduced the genus nameZymomonas in Bergey's Manual, 7th ed. Heproposed two species:

Z. mobilis (Lindner 1928) Kluyver and vanNiel 1936

Z. anaerobia (Shimwell 1937) Kluyver 1957.

Barker and Hillier's cider sickness organismswere mentioned but remained unnamed. Kluy-ver mentioned an unpublished comparativestudy of cultures of Z. mobilis, Z. anaerobia,and the cider sickness organism, carried out in1951, showing that all these organisms areclosely related, but data were not presented.

Millis (115) was the first to give the scientificname Zymomonas anaerobia var. pomaceae tothe perry and cider sickness organisms. Milliscompared two strains, 1 and 2, (isolated by herfrom ciders and perries) with Z. anaerobiaNCIB 8227 (from "bad" beer, United Kingdom)and with three strains of Z. mobilis (Lindner's

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TABLE 7. List of names given to representatives of the present genus Zymomonas

Zymomonas mobilis subsp. mobilis (Lindner) De Ley and Swings, 1976SynonymsTermobacterium mobile Lindner 1928, 253Pseudomonas lindneri Kluyver and Hoppenbrouwers 1931, 259Saccharomonas lindneri (Kluyver and Hoppenbrouwers) Shimwell 1950, 182Zymomonas mobile (sic) (Lindner) Kluyver and van Niel 1936, 399Zymomonas mobilis (Lindner) Kluyver and van Niel 1936, 399Zymomonas mobilis var. anaerobia Richards and Corbey 1974, 243Zymomonas congolensis Van Pee and Swings 1971, 311Achromobacter anaerobium Shimwell 1937, 509Saccharobacter Shimwell 1937, 509Saccharomonas anaerobia Shimwell (Shimwell) 1950, 181Zymomonas anaerobia Shimwell (Kluyver) 1957, 199Zymomonas anaerobia var. anaerobia (Shimwell) Carr 1974, 353Saccharomonas anaerobia var. immobilis Shimwell 1950, 182Zymomonas anaerobia var immobilis (Shimwell) Carr 1974, 353Zymomonas mobilis var. recifensis Gongalves de Lima, De Arafijo, Schumacher and Cavalcanti Da Silva,

1970, 3Zymomonas mobilis subsp. pomaceae (Millis) De Ley and Swings, 1976Synonyms

Cider sickness organism Barker and Hillier 1912, 78Zymomonas anaerobia var. pomaceae Millis 1956, 527

original pulque organism; Derx's strain iso-lated from Arenga sap in Bogor, Java; Schaef-fer's strain isolated from Arenga sap in Ban-dung, Java) (see also Table 1). These six orga-

nisms were identical in 20 features. The cidersickness organisms resembled Z. anaerobiaNCIB 8227 and differed from the Z. mobilisstrains in two features only (lack of sucrose fer-mentation; no gas from sorbitol). The main dif-ference between Z. anaerobia NCIB 8227 andthe cider sickness organisms resided in thechanges in aroma and flavor produced in cider.

In Bergey's Manual, 8th ed. (28), Zymo-monas is divided into:

Z. mobilis (Lindner) Kluyver and van NielZ. anaerobia (Shimwell) KluyverZ. anaerobia var. anaerobia (Shimwell) CarrZ. anaerobia var. immobilis (Shimwell) CarrZ. anaerobia var. pomaceae Millis.

This classification showed that no progresshad been made in the taxonomy ofZymomonassince 1956. It introduced some unfortunate er-rors: (i) both Kluyver (88, 89) and Shimwell(151) knew that Z. mobilis strains can lose andregain the ability to metabolize sucrose; thisimportant feature was not mentioned in Ber-gey's Manual, 8th ed. (ii) The "cider sicknessorganism" is Z. anaerobia var. pomaceae (Mil-lis) and not Z. anaerobia (Shimwell) Kluyver.(iii) There is no proof that Z. anaerobia is thecause of the unpleasant flavor of the "fram-bois6" in France.Our extensive reexamination ofmany strains

of Zymomonas with modern methods (50, 163,

164) shows that a more realistic classification ofthis genus is now possible. We summarize ourarguments herewith.

(i) The main reason to maintain two species,mobilis and anaerobia, was the supposed differ-ence in the ability to ferment sucrose (28). Thisconclusion was based on work with a fewstrains only. However, this difference is illu-sive. From the observations of Kluyver andHoppenbrouwers (89), Dadds et al. (36), andRichards and Corbey (137), it follows that theability to ferment sucrose is an inducible phe-nomenon. We found no correlation whatsoeverbetween growth on, and fermentation of, su-crose with other features of Zymomonas (seeabove). The enzymic difference between su-crose-fermenting and sucrose-nonfermentingstrains is probably one or at most two enzymes(levansucrase [EC 2.4.1.10] and perhaps inver-tase [EC 3.2.1.26]). It is not justifiable to main-tain a separate species status because of such asmall difference.

In the past, some authors have alreadystressed the resemblance between Z. mobilisand Z. anaerobia. Kluyver (88) wrote that Z.mobilis and Z. anaerobia are closely relatedorganisms. De Ley (47) stated that Z. anaero-bia was probably only a variety of Z. mobilis.Richards and Corbey (137) came to the sameconclusion and proposed, "that the genus Zymo-monas be represented by one species only, viz.Z. mobilis with the second species relegated tovariety level as Z. mobilis var. anaerobia."Van Pee and Swings (171) thought that theexistence of both species designations was notjustified by morphological and biochemical dif-

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ferences. Dadds et al. (36) studied five Zymo-monas strains and concluded that their work,"casts grave doubts on the necessity for main-taining two species within the genus Zymo-monas."

(ii) Shimwell (151) introduced the name Sac-charomonas anaerobia var. immobilis. The dif-ferences between Z. anaerobia and its varietyimmobilis are really minor: the latter varietydisplays (i) no flagella and no motility, (ii) norosette-like clusters, and (iii) a slightly differ-ent colony shape; physiologically both taxa areotherwise identical. This proposal was contin-ued by Carr (28) as Z. anaerobia var. immobilis(Shimwell) Carr. However, motility is not astable feature and may be readily lost. We es-tablished that motility is randomly distributedamong the strains ofZymomonas and that mo-tile and nonmotile strains do not cluster inseparate groups (see above). We conclude that aseparate taxon for nonmotile strains cannot bemaintained.

(iii) The name Z. congolensis was proposedby Van Pee and Swings (170). However, it waspublished as a nomen nudum, without typestrain. We established (50) that these strainsare indistinguishable from our other Z. mobilisstrains and that a separate species status is notjustified.

(iv) Gongalves de Lima et al. (70) studiedstrains isolated from caldo-de-cana-picado (fer-menting sugarcane juice) and named them Z.mobilis var. recifensis. They are characterizedby very mucoid colonies, a high growth yield inliquid medium at 25 to 420C, the capacity toferment at 420C, a great tolerance towards oxy-gen, the absence of H2S formation, and a typi-cal antagonistic spectrum against a number ofbacteria and fungi. This taxon was not men-tioned in Bergey's Manual, 8th ed. (28). Werecently examined two strains labeled Z. mobi-lis var. recifensis CP3 and CP4 (kindly suppliedby 0. Gongalves de Lima) by phenotypical andprotein electrophoretic techniques. Both are al-most identical with the strains ofthe subspeciesmobilis. We propose to regard the variety reci-fensis as a synonym of the subspecies mobilis(Table 7). Full details will be published else-where.

(v) From the evidence we submitted in thesections, Numerical Analysis of the Phenotype,DNA Base Composition and DNA GenomeSize, Genome-DNA Relatedness, and Similar-ity of Protein Electropherograms (50, 163, 164),it follows that nearly all existing strains ofZymomonas are so similar, in spite of theirgreatly diverse origin, that they deserve to beunited in one taxon, Z. mobilis subsp. mobilis(50).

(vi) Three Zymomonas strains of our collec-tion were distinctly different. Strain ATCC29192 was isolated in Bristol from sick cider. Itwas sent in 1951 by B. T. P. Barker as Sacchar-omonas pomaceae strain I to the collection ofthe Laboratory of Microbiology, Delft, theNetherlands, where it was renamed Z. anaero-bia subsp. pomaceae. We believe that it is verylikely either strain B from Barker or strain 1from Millis (115). This strain has a low DNAhybridization with all the other strains (163),its protein electropherogram is quite separate(164), and it differs in six phenotypic featuresfrom all the other strains in our collection.Strains NCIB 8777 and NCIB 10565 are almostidentical with ATCC 29192. The separate na-ture of these organisms requires its recognitionin a separate taxon, Z. mobilis subsp. poma-ceae (50). Some other strains isolated by Barkerand Hillier (12), Barker (11), and by Millis (114)from sick cider possibly belong in the sametaxon.

(vii) We formally proposed the following newclassification (50).

Genus: Zymomonas Kluyver and van Niel1936, 399

Type species: Zymomonas mobilis (Lindner)Kluyver and van Niel 1936, 399

1. Zymomonas mobilis subsp. mobilis (Kluy-ver and van Niel) De Ley and Swings, 1976

Lectotype strain: ATCC 10988 = NCIB 8938= NNRL B-806 = DSM 424 = IMG 1655 =L192 (Lab. Microbiol., Techn. Univ. Delft)= strain 1 (ibid.) = Ampoule no. 410 (Dept.Microbiol., Fac. Med., Univ. Queensland,Australia)

Phenotypic centrotype: strain Z6 = ATCC29191 = NCIB 11199

2. Zymomonas mobilis subsp. pomaceae (Mil-lis) De Ley and Swings, 1976

Lectotype strain: ATCC 29192 = NCIB 11200= strain I (Barker).

The synonyms ofboth subspecies are listed inTable 7.

Relationship Between Zymomonas and OtherGenera

Opinions on the possible relationship be-tween Zymomonas and other genera have var-ied considerably. When Kluyver and van Nielcreated the genus Zymononas in 1936, theyplaced it together with 14 other genera such asPseudomonas, Acetobacter, Rhizobium, etc.,in the tribe Pseudomonadeae of the familyPseudomonadaceae. The genus Zymomonaswas not recognized by R. S. Breed in Bergey'sManual, 6th ed., in 1948. The organism from

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pulque was still referred to as Pseudomonaslindneri Kluyver and Hoppenbrouwers. Thegenus Zymomonas was finally accepted in Ber-gey's Manual, 7th ed. (1957), and described byA. J. Kluyver. Together with the genera Pseu-domonas, Acetobacter, Xanthomonas, Photo-bacterium etc., it constituted the family Pseu-domonadaceae. In Bergey's Manual, 8th ed.(28), Zymomonas has been moved, as a genus ofuncertain affiliation, to Part 8, together withthe Enterobacteriaceae and the Vibrionaceae,the gram-negative facultatively anaerobic rods.The physiological properties (facultative anaer-

obiosis, fermentation of glucose to ethanol andCO2, percent G+C) were considered to be asimportant as the morphological ones (polarlyflagellated rods). However, Staley and Colwell(157) found no reassociation between DNA fromVibrio and Zymomonas. Zymomonas is notmentioned in Krassilnikov's "Diagnostik" (91).He mentions that the species P. mobilis and B.lindneri are unrelated to Zymomonas. Pr6votet al. (129) accommodate Zymomonas, as a ge-nus incertae sedis, in the family ofthe Sphaero-phoraceae. This diversity of proposals showsthat the exact taxonomic position of the genusZymomonas was obscure.

In our laboratory, the similarities betweenthe ribosomal ribonucleic acid (rRNA) cistronsfrom several hundred species of gram-negative,heterotrophic, rodlike bacteria were examined(J. De Ley et al., to be published). It was foundthat there is a good correlation between thedegree of rRNA similarity and the overall phe-notypic similarity of bacterial genera and/orsubgenera. Zymomonas belongs in a constella-tion of genera, represented in Fig. 5. A common

feature to most of them is the presence of theEntner-Doudoroff pathway (85). The rRNA cis-trons ofZymomonas, Acetobacter, Gluconobac-ter, Agrobacterium, Rhizobium, Phyllobacter-ium, and a few other genera (on which one of uswill report later) have much in common. It isvery striking that all these genera have relatedecological niches: in, on, or around, plants. Wesuggest that all these genera are of a common

evolutionary origin and may later be united bytaxonomists into one family.

Phenotypically, Zymomonas resembles mostclosely the acetic acid bacteria; perhaps theyare more like Gluconobacter more than Aceto-bacter, because of the polar flagellation, theincomplete Krebs cycle, the occurrence of theEntner-Doudoroff mechanism (which is more

widespread in Gluconobacter than in Acetobac-ter), and the ready consumption of glucose.Zymomonas and acetic acid bacteria both occur

on plants and prefer to grow in niches rich inplant juices. They complement each other,

since Zymomonas produces ethanol, which theacetic acid bacteria in turn, oxidize. In ferment-ing tropical plant juices, beer, etc., the aceticacid bacteria not infrequently occur simultane-ously with or subsequent to Zymomonas.Zymomonas tolerates low pH; many strains

grow at pH 3.5; most of them grow at pH 4.Acetic acid bacteria are likewise able to grow atpH 4 to 4.5. This pronounced tolerance to lowpH is not common in the bacterial world.Many Zymomonas strains are ethanol toler-

ant. At 7.7 and 10% ethanol concentrations, 73and 47%, respectively, of our strains are stillable to grow. Many acetic acid bacteria likewisegrow readily in 10 to 13% ethanol.

Tolerance of high glucose concentration isanother common feature. All our Zymomonasstrains and most acetic acid bacteria are able togrow on media with 20% glucose. About half ofour Zymomonas collection is able to grow onmedia with 40% glucose. Many acetic acid bac-teria grow readily in media with 50% glucose.The outstanding feature of all the acetic acid

bacteria is their ability to oxidize ethanol toacetic acid. It is interesting to point out thatZymomonas NCIB 8938 is likewise able to oxi-dize ethanol to acetic acid (17, 38).Zymomonas assimilates but a small part of

its glucose substrate as cell material (17): 98%is fermented and about 2% is used as carbonsource. The acetic acid bacteria are likewiseinefficient: 85% of glucose is oxidized to glucon-ate and only 15% is consumed by way of theshunt (49).There are strong indications of a correlation

between the taxonomic grouping of organismsand the regulatory properties of their citratesynthase (178). Gram-negative strictly aerobicbacteria, such as Pseudomonas, have a large-molecular-weight citrate synthase, inhibited byreduced nicotinamide adenine dinucleotide(NADH) and reactivated by adenosine 5'-mono-phosphate (AMP). Gram-negative fermenta-tive bacteria, such as E. coli, have large-molec-ular-weight citrate synthase inhibited byNADH but not reactivated by AMP. Gram-positive bacteria have a small-molecular-weight citrate synthase that is not inhibited byNADH. Zymomonas NCIB 8938 and some Ace-tobacter strains have a large-molecular-weightcitrate synthase, as expected from gram-nega-tive bacteria; however, the enzyme is not in-hibited by NADH (D. Jones, personal commu-nication).The DNA genome sizes of the 40-odd Zymo-

monas strains which we determined are rathersmall; the molecular weight is about 1.5 x 109.The DNA genome sizes of the acetic acid bacte-ria are more diversified: a number of them are

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°P CD °-<. Tm(e). 0 ity of some 30% is motile with 1 to 4 polarZ momonas flagella. Motility is lost in some strains. Forty-

five percent of our strains form rosettes, 33%Acetobacter form chains, and 62% have filamentous cells;

Auluconobacter the latter are sometimes over 28 jtm long.Agrobacterium These three features are quite characteristic for

some individual strains. The strains 17.1 andRhizobium 17.3 are the only ones with U-shaped cells. NoS~hizobiumn spores, capsules, intracellular lipids, or glyco-

(subpolar) gen have been found.Chr. lividum Macromorphology. In solid standard me-

Chr. violaceum dium, deep colonies are lenticular, regular, en-Ps. acidovorans tire edged, butyrous, white or cream colored,

group and 1 to 2 mm in diameter after 2 days. Anaero-Ps. solanacearum bic surface colonies are spreading, entire edged,groupAlcaligenes convex or umbonate, and 3 to 4 mm in diame-

ter. Strain Zymomonas 6 TH Delft has an irreg-Xanthomonas ular colony edge.

Ps. fluorescens Cellular composition. The main componentsgroup of the Zymomonas cell are given in Table 8. A

Enterobacterioceae comparison with some other bacteria is desira-Vibrionaceae ble. The dry weight of many bacteria contains

50 to 90% protein. The RNA content of someFIG. 5. Similarities of rRNA cistrons between gram-negative bacteria (e.g. E. coli) is about

several taxa. The results are expressed as the thermal gramnegtie bateria (eg aenoli) is aboustability of the hybrid (melting point of the hybrid in 20% of the dry welght. The adenoslneiZ-tri-degrees centigrade) (unpublished results of J. De posphate tP) lof 1 to 5smg/ml in Zymo-Ley, M. Gillis, J. De Smedt, P. De Vos, P. Segers, monas durngthe log phase is similar to that ofand R. Tytgat). other bacteria, shown to have 1 to 7.1 ,ug of ATP

per mg (dry weight) (83). Zymomonas cells are

as small as the Zymomonas genome: A. pasteu- thus not abnormal in those respects. The DNArianus subsp. pasteurianus 23 kl+ (1.81 x 109) content of the bacterial cell depends on the(68), G. oxydans subsp. suboxydans NCIB 7069 molecular weight of the genome, its state of(1.6 x 109), G. oxydans subsp. suboxydans replication, and the number of nucleoids pres-NCIB 6723, (1.5 x 109), G. oxydans subsp. sub- ent. The amount of 2.7% in Zymomonas is alsooxydans NCIB 9119 (1.9 x 109); other strains, in the normal range (about 3.3% in E. coli).

According to different authors, 12 to 30% oflike A. aceti subsp. aceti NCIB 8627 and A. the dry weight of many bacteria consists of car-aceti subsp. liquefaciens NCIB 9136, have a bohydrates. There is very little reserve mate-:vAL>n^ ^A^ NQ [ ^I; t9 r44 vgenome size OI tne same oraer as r . COllt kDD x109) (all molecular weights from M. Gillis andJ. De Ley, unpublished).

PHENOTYPICAL DESCRIPTION:MORPHOLOGY, GROWTH,

PHYSIOLOGY AND BIOCHEMISTRY

In this section, we shall summarize and dis-cuss the present knowledge on the phenotype ofZymomonas, including both our own data andthe information available in the literature.

The CellCell morphology. Zymomonas are gram-neg-

ative rods, 2 to 6 ,um long and 1 to 1.5 ,um wide,occurring singly but mostly in pairs. The largecell-width of Zymomonas is very striking uponmicroscopic observation; most other bacteriaare only 0.5 to 0.75 ,m wide. Most of the strains(70% of our collection) are nonmotile, a minor-

TABLE 8. Cellular constituents ofZymomonas a

Component Content (dry wt)

ProteinsGrowth phase 65-69%Stationary phase 54%

RNA 17-22%DNA 2.7%Carbohydrates 4-5%Poly-/3-hydroxybutyrate 0%Polyphosphate 0%Sulfur 0.5%Ammonia 0.1-0.5,mol/mgAmino acids 0.02-0.2 ,umol/mgATP

Exponential phase 1-5 ,ug/mgStarvation 0-0.4 pg/mg

a Most data are from Dawes and Large (41); othersources include: sulfur content, from Anderson andHoward (5); and ATP pools, Lazdunski and Belaich(97). The data on ATP pools concern strain NCIB8938; all other data concern strain NCIB 8227.

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rial present in Zymomonas. The amount of pol-ysaccharides is low, if any. Polyphosphates(volutin) and poly-fl-hydroxybutyrate, whichare common in many bacteria, are reported tobe absent in Zymomonas. However, we ob-served metachromatic granules in four strains,i.e., Z1, Z6, 7.4, and ATCC 10988. If the aminoacids (Table 8) represent the free pool in thecytoplasm, then it can be calculated that eachcell contains between 8 and 80 million mole-cules of them.

Resting cells and starvation. Resting cellsare cells that are not growing and dividing.They are usually suspended in buffer, withoutnutrients for growth, and are frequentlystarved for a few hours up to a few days. Thetechnique of resting cell preparations is widelyused in bacterial physiology and biochemistry.Some properties of resting and starving Zymo-monas were studied by Dawes and collabora-tors (38-43).When Zymomonas strain NCIB 8227 is

starved, it dies off very quickly. During day 1(41), about 90% of the cells die. MgCl2 protects,in its presence, about 60% of the cells, initially,from death. From then on the cells continueto die off slowly. During the death and starva-tion period, most polymeric cell componentshardly change. For 118 h, under N2, the carbo-hydrate content remains constant at about4%, and DNA at 2.7%, whereas the protein con-

tent falls from about 67% to about 57%. Theconcentration of ammonia (0.1 to 1 ,umol/mg[dry weight]) and of amino acids (0.02 to 0.2,umol/mg [dry weight]) does not change insidethe cell in starvation periods of up to 2 days.Nevertheless, these compounds leak out in themedium. In both strains NCIB 8227 and NCIB8938, RNA is the only constituent that is sig-nificantly broken down in starvation; its con-tent drops drastically to 3 and 5%, respectively.Degradation of RNA is accompanied by releaseinto the medium of material absorbing at 260nm. This material accounts for all the RNAdegraded. The degradation of RNA is sup-pressed in the presence of 33 mM MgCl2. TheRNA is lost more rapidly from strain mobilisNCIB 8938 than from strain anaerobia NCIB8227. From this isolated case, however, onemay not conclude that differences in RNAbreakdown constitute a significant species dif-ferentiation; they are only strain differences.Upon starvation, the ATP content falls rapidlyand levels off to a small value. Cells starvedfor up to 7 days still contain the enzyme systemfor producing ATP from glucose and ethanol(40, 41). There seems to exist no correlationbetween the intracellular ATP concentrationsand the viability of the cells.

Growth Response to Different Conditions

Growth in some ordinary media. Zymomonasneeds a fermentable sugar in its medium forgrowth, i.e., glucose or fructose, or (for somestrains) sucrose. In the liquid standard medium(see Some Commonly Used Media and GrowthConditions), good growth and gas is seen after24 h. In peptone broth plus 2% glucose, in beerplus 2% glucose, in nutrient broth or nutrientagar plus 2% glucose or in palm juice, goodgrowth occurs. From the strains of our collec-tion, 67% form a compact, and 33% form aslimy-flocculent to granular deposit in liquidmedia. The type of deposit is strain specific.Zymomonas does not grow in 0.5% yeast ex-tract, beer, beer with 0.5% yeast extract, pep-tone broth, peptone broth with 0.5% yeast ex-tract, nutrient broth or agar, or nutrient agarwith 0.5% yeast extract.

In apple juice liquid medium (see Some Com-monly Used Media), two cider sickness strains(115) grew with a dense turbidity within 2 to 3days; afterwards, the medium became clear anda compact (strain 1) or flocculent (strain 2)deposit was formed, darkening with age.Growth in the liquid synthetic medium of

Kluyver and Hoppenbrouwers. All our strainstested were transferred from the standard me-dium with 1 drop of a Pasteur pipette. Thirty-eight strains develop within 3 days on this syn-thetic medium (see Some Commonly Used Me-dia and [89]). On a second transfer, only Zymo-monas strains NCIB 8227, 409, ATCC 10988,and 410 develop. On a third transfer no straingrows. Zymomonas is thus not able to grow onthis simple synthetic medium, demonstratingthe need for a source of organic nitrogen orsome growth factors or both. Growth in the firstand second transfer is due to carry-over. Thissynthetic medium has been used in fermenta-tion studies by Kluyver and Hoppenbrouwers(89).Growth at different pH values. Shimwell

(150) found that his Z. anaerobia strain grewwithin the pH range 3.4 to 7.5. Millis (115)determined that two Zymomonas isolates fromsick cider grew in the pH range 3.5 to 7.9; in thepH range 2.5 to 3.4, there was no growth; at pH3.5 and 3.6, growth occurred sometimes. Theresults of the growth tests at different pH val-ues with our collection are summarized in Ta-ble 9. The wide pH range for growth, from 3.5 to7.5, and the acid tolerance are both quite typi-cal for Zymomonas. This is not surprising,since in nature they live in acid palm wines andciders below pH 4. These facts disagree with theassertion of Masschelein (110) that Z. anaero-bia is acid intolerant and does not develop be-

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low pH 4.2.Growth at different temperatures. The re-

sults from the strains of our collection are givenin Table 10. Above 30'C, some strains do notgrow, and growth at 400C is rarely observed.Strains from the subspecies pomaceae are par-ticularly sensitive, as they do not grow above34CC. The best test we can recommend to distin-guish between both subspecies mobilis and po-maceae is growth at 360C.The cider sickness organisms of Millis (114)

did not show any sign of fermentation whenincubated for 28 days in the refrigerator; atcellar temperatures (14 to 18'C), the onset offermentation occurred after 4 days, and, at roomtemperatures (16 to 260C), fermentation beganafter 2 days. The same author (114) found theoptimum temperature range to be 25 to 310C.Dadds et al. (36) noticed a slow growth at 150Cfor the strains Zymomonas B70 and NCIB10565. According to Gongalves de Lima et al.(70), Z. mobilis var. recifensis strains CP1,CP2, and CP3 grew well between 25 and 42CC.However, we found that strains CP3 and CP4still grew at 38°C but not at 40°C.Thermal death point. Z. anaerobia is killed

by exposure at 60°C for 5 min (150). We testedstrains Z1 to Z8 from our collection; they werealso killed in these conditions. Grove (78) foundthat the cider sickness bacillus was killed after5 min at 55°C.Growth in the presence of ethanol. It can be

expected that Zymomonas is alcohol tolerant, asit is isolated from alcoholic beverages contain-ing from 2 to 10% ethanol. A concentration of5.5% ethanol in liquid standard medium is

TABLE 9. Growth ofZymomonas strains in liquidstandard medium at different pH valuesa

Initial pH % of strains growing

3.05 03.5 433.7 713.85 905-7 1007.50 878.0 0

a Our data.

TABLE 10. Growth ofZymomonas strains in liquidstandard medium at different temperaturesa

Incubation temp % of strains growing(,0C)30 10034 9736 9738 7440 5

a Our data.

quite harmless to Zymomonas, since all strainsof our collection grow. At 7.7 and 10% ethanol,respectively, 73 and 47% of our strains stilldevelop. Growth of the cider sickness bacilluswas prevented by 6.5% ethanol (79).Growth in high glucose concentrations.

Kluyver and Hoppenbrouwers (89) observedthat the pulque strain was able to grow in andferment media with 25% glucose.We tested several tyndallized liquid media

containing 2% yeast extract (Difco) and variousconcentrations of glucose. With 20% glucose, allof our strains grow within 34 h; with 30% glu-cose, 88% of our strains develop after 2 to 5 days(strains Z1 to Z8 develop within 1 day); with40% glucose, 54% of our strains grow after lagphases of 4 to 20 days.Growth in the presence of KCN. We used

liquid standard medium with 0.0075% KCN.The strains ATCC 29192, NCIB 8777, NCIB10065, CP3, CP4, and Ag 11 do not grow. Zymo-monds strains ATCC 10988, 410, NCIB 8227,and 409 grow after 5 days; 12 other strains arenot inhibited by this KCN concentration anddevelop after 1 to 2 days.Growth in the presence of NaCI. In liquid

standard medium with 0.5% NaCl, only twostrains of our collection, i.e., Z. mobilis var.pomaceae ATCC 29192 and NCIB 8777, do notgrow. Seventy-one percent of our strains growin the presence of 1% NaCl. None of our strainsgrow in the presence of 2% NaCl. Grove (79)found that the cider sickness bacillus was in-hibited by 0.7% NaCl.Growth in the presence of 0.01% Acti-dione.

This antibiotic is a component of the WL differ-ential medium (Difco) we used for the isolationof Zymomonas. Millis (115) added Acti-dione(cycloheximide) to the apple juice isolation me-dium. Dadds (32) included it in a differentialmedium for Zymomonas (see Detection). Fungiare sensitive to 0.01% Acti-dione, and bacteriaare not. All our strains tested develop within 4days.Growth in the presence of oxgall. Of 17

strains tested, only Zymomonas ATCC 10988and its subculture 410 develop in 0.2% oxgall insolid medium. At 1% oxgall, both strains areinhibited. Much higher concentrations of oxgallare tolerated by Zymomonas in liquid media; in0.2% oxgall, all our strains grow except thepomaceae organism ATCC 29192; in 1% oxgall,53% of our strains develop, and in 1.5% oxgall,41% of our strains grow after 2 to 9 days.Growth in the presence of 0.1% 2,3,5-triphen-

yltetrazolium chloride (TTC). In both liquidand solid standard media all the strains of ourcollection grow and reduce ITC to the red for-mazan.

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Growth in the presence of 0.01% thalliumacetate. In solid standard medium, growth didnot occur.Growth in the presence of 0.001% cadmium

sulfate. Sixty-three percent of the strains grow.Growth in the presence of the vibriostatic

agent 0/129. The importance of this test for theidentification of gram-negative, polarly flagel-lated, and oxidase-positive bacteria (10)prompted us to check it. All Zymomonasstrains in our collection are insensitive to com-pound 0/129 (2,4-diamino-6,7-diisopropylpteri-dine) in solid standard medium.Tolerance to SO2. This feature is of practical

importance, since SO2 is the only disinfectantpermitted in English cider. Two strains of thecider sickness organism (115) grew at 0.05%,but not at 0.075%, sulfur dioxide. Zymomonasis not inhibited at the legally permitted.(UnitedKingdom) 0.02% SO2 concentration in cider.Growth in the presence of dyes. Fung and

Miller (64) examined the effect of 42 dyes insolid media on the growth of 30 bacterialstrains. They showed that gram-negative orga-nisms exhibited greater resistance to dyes thangram-positive strains. Our results show thatZymomonas is more sensitive towards dyesthan the gram-negative bacteria tested bythese authors.

In Fig. 6, the results are summarized of thegrowth responses of Zymomonas in the liquidstandard medium with different dyes. Growthinhibition of some strains by certain dye con-centrations depended on whether solid or liquidmedia were used (173). Strain VP2 is remarka-bly resistant toward dyes. On the whole, ourstrains are very sensitive to brilliant green.

Malachite green was used by Millis (114) at0.025% in the isolation medium. With this dye,we found that, even at 0.0005%, some Zymo-monas strains do not develop; thus, the use ofmalachite green in isolation media for thesebacteria cannot be recommended.Reduction of dyes and HgCl2. These com-

pounds were incorporated at 0.001% in the liq-uid standard medium. All our strains reduceNile blue, cresyl violet, thionine, methyleneblue, TTC, and HgCl2. Indigo carmine, neutralred, safranine, and Janus green are not re-duced.Growth in the presence of antibiotics. The

tests were performed on solid standard mediumusing Difco or Oxoid antibiotic disks. Figure 7represents the sensitivity-resistance pattern of38 Zymomonas strains toward 20 antibiotics.This figure speaks for itself and needs littleadditional comment.

Greatest sensitivity was shown by strain70.7, which is inhibited by 11 antibiotics. Strain

Anl.

z _.z3.0I--

rz>u-

0.01

0.001

°I-0

-j

LI)

I--

Lz4U-

UI)

X

I

LUlx

D-Jz

z

I

U-n:D

z

0

4

cL

LUJz

-J

I--

uLJ

0

0

z0U)

aD

w-J40-

z

z0

I

DYE TESTED

FIG. 6. Sensitivity ofZymomonas to dyes. The barheights indicate the dye concentrations at which allof the strains tested grew in the liquid standardmedium (data from [173D).

VP2, mentioned above for its greater resistanceagainst dyes (see Growth in the presence ofdyes), has a high resistance against antibiotics:it is inhibited by five antibiotics only. Cross-resistance was observed for the pairs ampicil-lin-penicillin, gentamycin-kanamycin, genta-mycin-streptomycin, and gentamycin-neomy-cin. The cross-resistance was not always revers-ible for all the strains with lincomycin-erythro-mycin.

Carbohydrate MetabolismMetabolism of glucose and fructose. The

almost quantitative fermentation of both hex-oses to ethanol and carbon dioxide is a distinc-tive characteristic of the genus Zymomonas.After 12 to 24 h, the liquid standard mediumbecomes densely turbid, with abundant gas for-mation.

In 40 Zymomonas strains of our collection,the ethanol produced amounts to 1.5 to 1.9 molper mol of glucose. The original pH in the liquidstandard medium is 6.1. The final pH after 3days at 30"C is 4.8 to 5.2. The acidification ismore pronounced upon incubation at 38"C; inseveral cultures, the pH drops to 4.0. At 340C,the pH ofZ. mobilis var. pomaceae ATCC 29192drops to 4.7; with all strains of the varietymobilis, the pH drops to 4.9 to 5.4.

I

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I

-z Zw ui-ui LLIIx w(D LI)

wz I-< m:. u-i <

-i0: <

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PERCENTAGE OF RESISTANT STRAINS0 50 100I I I I I .I I

o I

100 50 0PERCENTAGE OF SENSITIVE STRAINSFIG. 7. Resistance and sensitivity of Zymomonas

toward antibiotics.

In 1912, Barker and Hillier (12) were the firstto report on the fermentation by a Zymomonasorganism (a strain from sick cider). Their iso-late fermented glucose or fructose, but not su-crose, maltose, or lactose. They reported: "Thegas given off during the fermentation of dex-trose consists almost entirely of carbon dioxide....Ethyl alcohol is formed in some quantity,nearly 5 per cent being produced at times froma 10 per cent dextrose solution. A limitedamount of acid is also formed." One can thuscalculate that about 1.9 mol of ethanol was pro-duced per mol of glucose added. The formationof lactic acid was not reported.The enzymic mechanism of hexose fermenta-

tion in this genus has been thoroughly exam-ined in the strains, Z. mobilis ATCC 10988, aswell as Z. anaerobia NCIB 8227 and Z. anaero-

bia NCIB 8777.(i) Hexose catabolism inZymomonas ATCC

10988. Lindner (104) established that his Ter-mobacterium mobile was able to grow on and toferment sucrose, glucose, and fructose with theformation of ethanol, CO2, and lactic acid. Inthe 1920s, A. J. Kluyver (Delft) was very inter-ested in fermentative bacteria. Sugar fermen-tations by E. coli and coliform bacteria, severalsaccharolytic clostridia, and propionic acid bac-teria were being studied by several of his col-laborators. It is therefore not surprising that hewas interested in Zymomonas, an organismwith a yeast-type fermentation. Kluyver andHoppenbrouwers (89) confirmed that CO2 wasformed, but no H2. The number of carbonsources for fermentation was limited to glucose,fructose, and sucrose. The fermentation of glu-cose was very active after 10 h at 300C and wasover after 2 days. Glucose was fermented tocompletion. In addition to ethanol, some lacticacid was produced. Traces of acetaldehyde weredetected, but no other end products. Succinicacid was detected only when the fermentationoccurred in the presence ofyeast extract. It wasnot formed in a synthetic medium where theyeast extract was substituted by 0.1% K2HPO4,0.1% (NH4)2SO4, and 0.05% MgSO4. It is anartificial end product, arising from the reduc-tion of amino acids in the yeast extract.The molar fermentation equation, calculated

from Kluyver and Hoppenbrouwer's data, is:

1 glucose -- 1.8 ethanol + 1.9 C02 + 0.15 lacticacid.

Similar equations were later reported by otherauthors for strain NCIB 8938.

1 glucose -* 1.93 ethanol + 1.8 C02 + 0.053lactate

(Gibbs and DeMoss [67])1 glucose -- 1.58 ethanol + 1.7 C02 + 0.2

lactate(Belaich and Senez [17])

1 glucose -* 1.63 ethanol + x C02 + 0.02lactate

(Dawes et al. [44]).

Slightly less ethanol and lactic acid accumulatewhen the bacteria are growing. It is thus notsurprising that, in the early 1930s, the mecha-nism of the fermentation by Zymomonas wasthought to be similar to that ofSaccharomyces.That it was not, became obvious only manyyears later.The above fermentation balance was con-

firmed by Schreder et al. (147). They found that98% of the glucose is converted to C02 andethanol. Small amounts of acetaldehyde, ace-

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PENICILLIN 5U

POLYMYXIN 300USTREPTOMYCIN 10FtgMETHICILLIN 10|z9ERYTHROMYCIN 10|w9NITROFURANTOIN 200 U9 |VANCOMYCIN 3099 |

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I I I L ----A. .

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tylmethylcarbinol, acetic acid, lactic acid, andglycerol were found.Neuberg and Kobel (119) examined some as-

pects of the intermediary carbohydrate metabo-lism in Lindner's organism. The following con-versions were detected. Pyruvic acid is decar-boxylated to acetaldehyde. Acetaldehyde can betrapped during the fermentation of glucose.Glucose-6-phosphate (G6P) and hexose diphos-phate are dephosphorylated before fermenta-tion. Phosphorylative processes occurred. Theconclusion of Neuberg and Kobel that the fer-mentation of glucose occurs by way of the gly-colysis was experimentally unfounded.That the fermentation of glucose in Zymo-

monas does not follow the glycolytic pathwaywas discovered by Gibbs and DeMoss (66) withstrain ATCC 10988. Almost all the activity of[1-14C]glucose accumulated in CO2; about halfof the activity of [3,4-14CIglucose was releasedin C02, and the remainder was found inethanol. The breakdown by way of G6P-dehy-drogenase and a C2-C3 split (phosphoketolase),as in Leuconostoc, was postulated but laterfound to be erroneous. These authors detectedthe presence of carboxylase and an acetoin-forming cell-free enzyme system. Gibbs andDeMoss (1954) discovered the basic mechanismof glucose and fructose breakdown in Zymo-monas: it is a modification of the Entner-Dou-doroff pathway. The fate of the majority of theindividual C atoms is:

CHO

HCOH

HOCH

HCOH-

HCOH

CH2OH

demonstrate the presence of phosphohexoki-nase were negative (134). Ribbons and Dawes(135) and Dawes et al. (44) detected a number ofenzymes in Zymomonas NCIB 8938. Crude ex-tracts with added ATP converted both glucoseand gluconate into pyruvate. Hydrazine trapsequivalent amounts of pyruvate and glyceral-dehyde-3-phosphate from gluconate-6-phos-phate. The conversion of glyceraldehyde-3-phosphate to pyruvate requires NAD+, ADP,and inorganic phosphate. Indirect evidencestrongly suggested the intermediate formationof 2-keto, 3-deoxygluconate-6-phosphate fromgluconate-6-phosphate. It was, however, notisolated. The presence of hexokinase, glucono-kinase, glucose dehydrogenase and pyruvatedecarboxylase was established. G6P dehydro-genase is present and reduces either NAD+ orNADP+. Ethanol and triose phosphate dehy-drogenases are NAD+ specific. NADH oxidaseactivity is located mainly in the particulatefraction. NADPH oxidase was not detected.Neither gluconate dehydrogenase nor F1,6P al-dolase was detected. However, Raps and De-Moss (134) had previously reported aldolase inthe same strain. The presence of the latter en-zyme is thus not clear. Sly and Doelle (153)presented a detailed study of the kinetics ofG6P dehydrogenase with NADP+. The values ofoptimal pH (8.7), optimal MgCl2 concentration(10-2 M), and Km (5 x 10-4 M for G6P and 3.6 x10-5 M for NADP+) were determined. The en-

COOH COOHI

HCOH C=O

HOCH OH3

-v. HCOH oCHO

HCOH CHOHH

(;k2OP3H2 CH2OPO3H2

C02

*CH2OH

OH3

C02

* CH2OH

OH3

This assumption was tested and confirmed byStern et al. (159) with a radiorespirometricmethod. 14C, and 14C4 from glucose are rapidlyreleased as CO2. 140C and 14C6 are not convertedinto C02.The presence of the Entner-Doudoroffmecha-

nism was tested and confirmed later at theenzymic level by several authors. Cell-free ex-tracts ferment G6P to ethanol and C02. Theextracts contain phosphohexoisomerase, and anNAD-linked dehydrogenase for G6P. Fructose-1,6-diphosphate (F1,6P) is metabolized by wayof aldolase and glyceraldehyde-3-phosphateNAD+-linked dehydrogenase. All attempts to

zyme fromZymomonas was quite different fromthat ofE. coli and some other microorganisms.A number of the reactions of carbohydrate

catabolism are summarized in Fig. 8. The oxi-dation-reduction balance of glucose and fruc-tose breakdown is thus rather simple: it is prob-ably maintained through NAD+, rather thanNADP+, since NAD+-NADP+ transhydrogen-ase activity was not detected. For each mole-cule of hexose consumed, two NADH + H+ areproduced, one through G6P-dehydrogenase andone through glyceraldehyde-3-phosphate dehy-drogenase. The reduced coenzyme is reoxidizedlargely by ethanol dehydrogenase and acetalde-

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hyde, which is thereby reduced to ethanol, andto some minor extent by a weak lactate dehy-drogenase. Figure 8 also shows that the overallATP yield is one ATP per one glucose fer-mented. The complete enzymic pathway for thebreakdown of gluconate appears to be present.Nevertheless, gluconate is neither a carbon noran energy source (40). One reason may be thatonly one molecule of NADH + H+ can beformed per molecule of gluconate added (fromtriose dehydrogenase), whereas two are re-quired to reduce both molecules of pyruvateformed.

(ii) Hexose catabolism inZymomona8 NCIB8777. Strain NCIB 8777 was compared withstrain NCIB 8938 by McGill et al. (112) andMcGill and Dawes (111). The former straingrew more slowly and with a smaller yield. Themolar growth yield coefficients on glucose andfructose were 5.89 and 5.0, respectively. About2% of the glucose was incorporated into thecells. The growth of this organism is thus notvery efficient.Both hexoses are fermented as follows:

1 glucose -- 1.78 ethanol + 1.88 C02 + 3.10-5acetaldehyde

+ 0.011 (cell material CH20)

The carbon balance of 90% indicates missingproducts.

1 fructose -- 1.51 ethanol + 1.55 C02 + 0.02acetaldehyde

+ 0.054 glycerol + x dihydroxyacetone

The carbon balance is 77% without the three Ccompounds. Energetically wasteful products

are formed during fructose metabolism, e.g.,glycerol, dihydroxyacetone, and presumablyothers that remain to be identified.The following enzymes were detected, in the

order of decreasing specific activity: G6P dehy-drogenase (NAD+ or NADP+ linked), ethanoldehydrogenase (NAD+ linked), pyruvate decar-boxylase, gluconate-6-phosphate (6GP) dehy-dratase plus 2-keto, 3-deoxygluconate-6-phos-phate aldolase, triose phosphate dehydrogenase(NAD+ linked), phosphohexose isomerase, glu-cokinase, fructokinase. The following enzymesare weak to very weak: phosphofructokinase,F1,6P-aldolase, NADH oxidase, 6PG dehydro-genase (NADP+ specific), NADPH oxidase,gluconokinase, glucose dehydrogenase. The fol-lowing enzymes are absent: NADP+-NAD+transhydrogenase, transketolase, phosphoketo-lase, and transaldolase. The key intermediate,2-keto, 3-deoxygluconate-6-phosphate, was de-tected by paper chromatography, but it wasneither isolated nor identified more closely.For growth and enzyme biosynthesis, DNA

and RNA are required. The deoxyribose moietyis derived from ribose-5-phosphate (R5P). Inmost organisms, the latter compound is pro-duced by the pentose phosphate pathway. How-ever, in Zymomonas NCIB 8777, this pathwaycannot be active because transketolase andtransaldolase activities were not detected. Thefunction of the weakly active 6PG dehydrogen-ase may be to provide R5P.Zymomonas strains NCIB 8938 and NCIB

8777 are the only bacteria currently known toutilize the Entner-Doudoroff pathway anaero-bically in association with a pyruvate decarbox-ylase. This is almost certainly the case for allother strains in this genus, although they havenot yet been examined in this respect. The

gluconate

Levan ATPdehydrog NAD JATP H20ATP glucose- NADP gluconate- / 2 keto, 3-deoxy-Se-* nAD °-Ihosphate- gluconate-

gluconat6'fructose ?6-phosphate [6-phophae 6-phosphate pyhtosateASucrose.2.(I4[sicoJphsht laae

dehydrog tC0

ATP fructose-6- | NADHtructose -,CHCOC3 2Ophosphate glyceradehyde- pyruvateCH

3-phosphate I

~PfI

r#?. lructose-t16- 3-diphospHaleP4

aldolasediphosphate ~~~~~~~~~~~~~~~~~~acetoinCH3a~dofa-se-iphosphalase glyceric acid- phospho- COOH

phosphatase 1,3-diphosphate enolpyruvate|_*-ATP H20OINADH NADP , glyceric acid- glyceric acid-1NADNADPH~' 3-phosphate - 2-phosphate

sNAD+ NADPH'particulate

FIG. 8. Mechanism ofcarbohydrate catabolism in two Zymomonas strains. Symbols: for strain NCIB 8938(- ), active; ( ---- -), weakly active; for strain NCIB 8777 ( ), active; (----), weakly active.

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differences in the enzymic equipment for carbo-hydrate catabolism between both strains aresmall. They have to be considered only as dif-ferences between individual strains and not ascriteria or proof for species differentiation.Metabolism of sucrose: fermentation and

levan formation. Strains able to grow on andferment sucrose have been isolated mainlyfrom fermenting plant saps in tropical and sub-tropical areas and from infected British beer.Strains unable to metabolize sucrose have beenisolated in the United Kingdom mainly frominfected beers, sick cider and perry, and applepulp (see Table 5). The metabolism of sucrose isnearly always tested in a growth medium.Fermentation to ethanol and CO2 is accompa-nied by growth. Sixteen out of 33 Zymomonasstrains, isolated by one of us from Zairese palmwines, neither grow on nor ferment sucrose.From the numerical analysis of 138 pheno-

typical features in 38 Zymomonas strains, wefound that the ability to metabolize sucrose isnot correlated with any other feature (50). Inaddition, the fermentation of sucrose seems tobe an inducible strain-specific phenomenon (seeabove and reference 17, 36, 89, 137) withouttaxonomic value.The first step of sucrose breakdown should

almost unavoidably yield glucose or fructose orboth. Important aspects of the enzymic mecha-nism were clarified by Dawes et al. (45, 136).They discovered that Zymomonas strain NCIB8938 produced levan from sucrose, but not fromglucose or fructose or a mixture of both; 640 g ofsucrose in 32 liters of medium gave 4.5 g of drypolymer. The yield range was less than 2%. TheZymomonas levan would appear to be a typicalmember of the bacterial levan family. It has amolecular weight of about 107. It contains onlyfructose, consisting thus of about 60,000 fruc-tose units. The major linkage is 2,6; many(probably all) units are in the furanose ringstructure; 2,1 linkages are present at branch-points; the configuration is probably P3. Duringthe synthesis of levan, glucose is utilized fasterthan the fructose moiety. Levan formationstops when the sucrose concentration is 0.5 mg/ml. Only free fructose is left, and this continuesto be metabolized by the cells without levanformation. Levan is synthesized by crude ex-tracts of Zymomonas from sucrose and fromraffinose without added primer; the majority ofthe sucrose is hydrolyzed to glucose and fruc-tose. The levan sucrase differs from those previ-ously studied in Acetobacter levanicum and inBacillus subtilis.Two strains B70 and NCIB 8227 from infected

British beer also produce levan when grown ina sucrose-containing medium (36). Unfortu-

nately, the chemical proof of the identity of thismaterial was incomplete, and no quantitativedata were given on the amount of levan pro-duced and on the importance of the levan su-crase pathway in the breakdown of sucrose byboth strains. Thus, so far, three Zymomonasstrains appear to produce levan from sucrose. Itmay very well be that all sucrose-fermentingZymomonas strains behave in the same way.The results ofDawes et al. (45) show that less

than 10% of the sucrose is metabolized by alevan sucrase with the formation of levan andglucose. The major pathway of sucrose catabo-lism in Zymomonas starts after a hydrolyticsplit to glucose and fructose. Dawes et al. (45)have not established whether an independentinvertase is present. They suggested tenta-tively that a levan sucrase can account for bothlevan formation and sucrose hydrolysis by thereaction:

n sucrose + acceptor -- levan + n glucoseorn fructose

Ifthe acceptor is fructose, levan is formed; if theacceptor is water, sucrose is hydrolyzed. Phos-phorolysis by way of the reaction

sucrose + Pi glucose-i-phosphate + fructose

plays no part in the sucrose degradation byZymomonas.The average molar growth coefficient ofZym-

omonas strain NCIB 8938 on sucrose, Y8 = 7.4,is appreciably lower than those for the equiva-lent concentrations of glucose, fructose, or glu-cose plus fructose (average, about 9.2). Molargrowth yields on sucrose are erratic and low,due to the formation of levan (45).Two important problems in sucrose metabo-

lism remain to be solved: (i) Is the hydrolysis ofsucrose effected by a levan sucrase or by aseparate invertase? (ii) Is the acquisition ofsucrose catabolism due to the induction of newenzymes, eventually accompanied by levan pro-duction, or to the selection from the populationof some cells with constitutive enzymes?Other carbon sources. None of the following

C sources supported growth of the Zymomonasstrains of our collection: starch, dextrin, raffi-nose, D-trehalose, maltose, lactose, i>.cello-biose, D-galactose, P-mannose, L-rhamnose, L-

sorbose, n-arabinose, L-arabinose, D-ribose, D-xylose, D-sorbitol, D-mannitol, dulcitol, adoni-tol, erythritol, glycerol, ethanol, salicin, andthe sodium salts of -galacturonic, succinic, DL-malic, tartaric, DL-lactic, pyruvic, and citricacids.

According to Millis (115), Zymomonas strain

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L192 (= ATCC 10988) grew on raffinose butformed no gas; five other strains examined byher did not grow. Dadds et al. (36) stated thatgrowth on raffinose could be induced in two Z.anaerobia strains and in Z. mobilis B70.

Sorbitol is said by Millis (115) to be a carbonsource permitting growth without gas forma-tion in five of the six strains examined. Growthon sorbitol occurred neither in another strain ofZ. anaerobia (29) nor in the strains B70 andNCIB 8938 (36). As in the cases of raffinose andsucrose utilization, inducibility is a plausiblehypothesis.Amino acids as sole C source are not fer-

mented; they do not support growth when glu-cose is absent (15).Formation of acetaldehyde. Acetaldehyde

can be trapped during the fermentation of glu-cose. Millis (115) attributed the full fruityaroma that develops in cider sickness to theliberation of acetaldehyde. This compound, to-gether with variable amounts of hydrogen sul-fide, contributes to the unpleasant taste andsmell of sick cider. Barker (11) suggested that"the designation (cider-) sickness must be con-sidered as a general one, applying to a type offermentation distinguished by the breakdownof certain sugars to yield not only ethanol butalso aldehydes-especially acetaldehyde-insufficient quantity to justify its appellation as'aldehydic' fermentation." In cask or keg beers,Zymomonas infection produces an unpleasantodor of rotten apples due to traces of H2S andacetaldehyde (9, 151). Zymomonas produceshigh acetaldehyde concentrations in beer whenbrewing yeast or Gluconobacter is absent (34).There is a linear correlation between the num-ber of Zymomonas cells present and theamount of acetaldehyde produced: 1010 cells perliter of beer produce about 80 mg of acetalde-hyde. The results of a laboratory experiment onthe production of acetaldehyde by Zymomonasstrain NCIB 8227 in a pale-ale in the presenceofSaccharomyces cerevisiae and Gluconobacteroxydans NCIB 8131 are summarized in Table11.Acetaldehyde flavors have only been noted in

beers containing glucose and fructose. High ac-etaldehyde levels and Zymomonas infectionhave never been observed in lager beers (34).Schreder et al. (147) made the interesting ob-servation that limited aeration of the ferment-ing mass increased the acetaldehyde formation.Formation of acetylmethylcarbinol. Kluy-

ver and Hoppenbrouwers (89) detected traces ofacetylmethylcarbinol in Zymomonas cultures.Tanko (165) artificially increased the produc-tion of acetoin by this strain by carrying out

TABLE 11. Production of acetaldehyde byZymomonas NCIB 8227 in the presence of

Saccharomyces cerevisiae or Gluconobacter oxydansNCIB 8131 or botha

Aldehyde production (mg/li-ter) with initial Zymomonas

Initial concn (cells/ml) concn (cells/ml) of:

0 2x106

Yeast -oGluconobacter added:

0 3.0 78.32 x 106 5.8 61.4

Yeast-2 x 106Gluconobacter added:

o 10.1 17.72 x 106 6.4 24.5

a Data from reference 34.

fermentations of glucose or sucrose in the pres-ence of added acetaldehyde. Twenty to 35% ofthis substrate was converted into acetoin. No2,3-butyleneglycol was detected. Schreder et al.(147) showed an increased formation of acetal-dehyde and acetylmethylcarbinol by increasedaeration. We found that the 42 strains of ourcollection all produce traces of acetylmethylcar-binol. Shimwell (150) and Millis (115) did notdetect this compound. These negative resultsare probably due to different test conditions orsensitivity or both.The enzymic mechanism of acetoin formation

inZymomonas is not known. In Enterobacter itis:

pyruvate + {aldehyde}:TPP + H+ -- a-acetolac-tate + TPP+

anda-acetolactate -- acetoin + CO2

In yeast, plants and animals it is:

acetaldehyde + {aldehyde}:TPP + H+ -- ace-toin + TPP+

It is interesting to recall that many acetic acidbacteria produce acetoin both by the a-acetolac-tate and by the acetaldehyde pathways (46).Molar growth yield. Bauchop and Elsden (14)

determined the quantitative relation betweenthe growth yield ofsome anaerobic bacteria andthe amount of energy supply. As long as thelatter is the growth-limiting factor, the dryweight of cells produced is proportional to theamount of energy source added, or better, to theATP yield of the catabolic reactions. Thegrowth yield coefficient Ysb~trate is the gram(dry weight) produced per mole of substrateadded. The ratio YATP is the dry weight of cells,

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expressed in grams, produced per mole of ATPformed. For Streptococcus faecalis and S. cere-visiae, the average YATP is 10.8 (14). Untilrather recently it has been assumed that allanaerobic microorganisms have about the sameefficiency at converting the catabolic energyinto cellular material. This appears not to bethe case, as several higher YATP values are nowknown, e.g., YATP = 18 to 20 for LactobaciUuscasei (54).With Zymomonas, Ygl..roae = YATP because of

the mechanism of glucose catabolism. The mo-lar growth yields of Zymomonas with glucoseor fructose as energy source have been deter-mined by several authors and found to varyconsiderably, from 3.5 to 9.3 (Table 12). Growthyields and growth rates of anaerobic cultures ofstrain NCIB 8938 were determined in threemedia (17) (see Table 15). Aeration of.the cul-tures did not modify either the yield or thedivision time. The growth yield ofZymomonasis thus much less efficient, as with most otheranaerobes, even in rich media where the en-ergy substrate is certainly the limiting factor.There is no significant difference in YATP be-tween strains labeled mobilis and anaerobia.The low values ofYATP in batch cultures may bea function of the specific growth rate g, themaintenance energy me, the cell composition,etc. (54, 160, 161). The differences in YATP val-ues observed for one organism by several au-thors (Table 12) may be due to differences inspecific growth rate caused by different growthconditions and medium composition (161).The growth of Zymomonas NCIB 8938 was

studied calorimetrically by Senez and collabo-rators (15, 18, 149). The rate of heat evolutiondQldt was measured with an isothermic calo-rimeter. This strain, inoculated in 0.4% yeastextract, 0.4% peptone, and 0.05 to 0.2% glucosein 0.025 M tris(hydroxymethyl)aminomethane-maleate buffer, pH 6.8, grows quickly and expo-nentially until only 0.4 mg of glucose per ml(0.04%) remains; then growth and heat produc-tion slow down and stop when all glucose iscompletely exhausted. The complete reactionsof glucose decomposition are:

Glucose + ADP + Pi -- 1.8 ethanol + 1.8 CO2 +0.2 lactate + ATP + H20

and

ATP + H20 -- ADP + Pi

The molar growth yield ygltcose is a constantduring the entire growth period. The change inenthalpy of the sum of the above reactions is

TABLE 12. Molar growth yields ofZymomonasa

Strain Growth yield coeffi- Referencescients, Y

ATCC 10988 YdOU = 4.32 - 9.32 14,16,17,18, 21,45, 62, 97

Y.,,O,= 5.35 - 8.0 45NCIB 8777 Y,,,,,e,, = 5.89 111

Yf.to,, = 5.0 111NCIB 8227 Y,,,, = 3.4 - 3.88 21, 42Z1-Z8 Ydeose = 3.48 - 4.12 21

a For definition of Y, see text.

-32.62 kcal/mol of glucose consumed. The heatreleased from glucose during growth agreesperfectly with the theoretical enthalpy calcu-lated for the equation of Kluyver and Hoppen-brouwers (89) from the standard enthalpies offormation for aqueous glucose (-302.03),ethanol (-68.85), C02 (-98.69), and lactic acid(-164.02; -0.85 heat of neutralization). Theamount of cells produced and of heat evolveddepend linearly on the quantity of glucose me-tabolized:

1 mol glucose consumed -- 6.5 g dry cells + 32.7kcal

If the dry, living cell material is roughly repre-sented by (CH20) the total equation of growthbecomes:

Glucose + (carbon source) -- 1.8 ethanol + 1.8C02 + 0.2 lactic acid + 0.22 (CH20) + 32.7 kcal

When cultivated in the usual complex mediumcontaining large quantities of peptone andyeast extract, Zymomonas utilizes glucose asthe exclusive energy source for growth and hasno detectable endogenous metabolism. Thebacteria do not accumulate appreciableamounts of intermediates such as glycogen orphosphorylated polymers from glucose.One of the important conclusions on the car-

bohydrate metabolism of Zymomonas is that,when growing in a complex medium, 98% of theglucose consumed is converted in ethanol, C02,ATP, and heat, and only 2% is used as buildingmaterial for the cell. This situation is rathersimilar to what happens in the acetic acid bac-teria, where 85% of the glucose is oxidized togluconate, 15% by way ofthe pentose phosphatepathway (49). Nevertheless, the 2% glucose in-corporated in Zymomonas produces 48% of thecellular carbon (17); the rest is derived fromyeast extract components or peptone or both.Amino acids and/or the various organic constit-uents of peptone and/or yeast extract are notutilized as an energy source for growth, butonly as building blocks for biosynthesis.

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Uncoupled growth. In the previous section,we have seen that not all ATP produced fromsugar catabolism by Zymomonas is used forgrowth processes. This phenomenon is but oneaspect of an uncoupled growth. One of its defi-nitions is that the rate of substrate dissimila-tion per unit weight of organism is independentof the growth rate.

Several aspects of uncoupled growth in Zym-amonas NCIB 8938 were studied by Belaich etal. The discussion below is based mainly ontheir results (16, 19, 97). One of the factorsnecessary for uncoupling growth is lack of pan-tothenate. This compound apparently can notbe synthesized by Zymomonas strains. As acomponent of coenzyme A (CoA), it is requiredfor the activity of various transferases and isthus of very great importance in biosynthesis.With limiting pantothenate concentrations (5X 10-7 mg/ml) in defined media, the molargrowth yield on glucose falls to Yglucose = 2.5.The amount of glucose metabolized is constantat 0.991 mmol of glucose per min per g (dryweight) and independent of the rate of growth,the nature of the medium, and the concentra-tion of the growth factor pantothenate.The explanation of uncoupled growth pivots

upon the fate ofATP produced by fermentation.Lazdunski and Belaich (97) measured changesin the ATP pool. The ATP content of the cellgoes through a weak maximum at the onset ofgrowth and remains constant during the expo-nential phase; depending on the growth me-dium used there is between 1 and 4 ,ug of ATP/mg (dry weight). During this growth phase,energy production and consumption seem to bewell balanced. When the last generation of cellsis formed, the ATP content drops to about 0.4pug/mg (dry weight). The constancy of the ATPpool during exponential growth has been ob-served in other bacteria, e.g., in E. coli (83).

In conditions of poor biosynthesis andgrowth, e.g., in synthetic and minimal mediapoor in pantothenate, the rate of glucose break-down remains the same, and the ATP pool inthe cell is twice as large. When growth isstopped suddenly by chloramphenicol, glucosebreakdown continues, the rate of heat produc-tion remains the same, and the ATP pool al-most doubles. The level of ATP in the cell de-pends on (i) its rate offormation and (ii) its rateof disappearance. According to Belaich's data,the rate of fermentation, and thus of ATP for-mation, is rather constant. ATP could disap-pear in several ways, by biosynthesis, by ATP-ase action, by leakage, etc. There may be stillother reasons for apparent growth uncoupling,such as extra energy required to maintain anorganism at low growth rates (126) or leakage

of important metabolic intermediates from thecells. Stouthamer and Bettenhaussen (161)stressed the underestimation of maintenanceenergy and its influence on molar growthyields. They think that coupling between en-ergy production and growth rate is muchtighter than has been supposed hitherto. A cou-pled growth is of maximal efficiency when ATPis produced only when needed, and in theamount required, for biosynthetic mechanisms.Regulation is thus required, such as happens,e.g., in glycolysis in E. coli, where the energycharge of the adenylate system is increasedwhenever ATP is used by biosynthetic mecha-nisms. Phosphofructokinase is activated byADP or AMP and inhibited by phosphoenolpyr-uvate; pyruvate kinase is activated by AMPand F1,6P. We postulate here that the uncou-pled growth of Zymomonas occurs becausethese or similar control mechanisms either donot exist or function very poorly. The data ofForrest (62) may suggest some energy couplingin Zymomonas between 24 and 330C, althoughthe eventual nature and mechanism is un-known. Within this temperature range, therate of glucose consumption and the rate ofspecific cell growth both increased with thesame energy of activation, 11.1 kcal/mol. Abovethis temperature (from 33 to 390C), the rate ofglucose consumption continues to increase, butthe specific growth rate dropped. The couplingbetween anabolism and catabolism is thus notvery effective above 330C.The scenario for the energy flow in Zymo-

monas may thus be summarized as follows.Glucose catabolism occurs at a constant rate ofone ATP to one glucose, and the waste materialcomprises ethanol, CO2, and lactate. The com-position of the medium and the state of biosyn-thesis for the construction of the daughter cellseem to be of little importance for the mainATP production. If the medium and conditionsare suitable for growth and division of the cell,the biosynthetic pathways will use the ATPproduced; if the rate of growth decreases orstops, the energy source will continue to beutilized until it is exhausted. Lazdunski andBelaich (97) suggested from some preliminaryexperiments that the excess ofenergy in ATP isremoved by an adenosine triphosphatase.The apparent uncoupled growth of Zymo-

monas NCIB 8938 on sucrose is partially ac-counted for by the production of levan.

Aerobic growth and metabolism. Zymo-monas is not a strictly anaerobic organism.Zymomonas strains NCIB 8938 can grow in thepresence of oxygen (44). Belaich and Senez (17)prepared aerobic cultures of the same strain ona reciprocal shaker. The growth yield and divi-

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sion time were not different from an anaerobi-cally grown culture.Lindner (104) and Kluyver and Hoppenbrou-

wers (89) observed that glucose fermentation insemianaerobic conditions gave about one-thirdthe ethanol yield of strict anaerobiosis, a find-ing attributed to a very powerful respiratorysystem. This phenomenon was examined quan-titatively by Schreder et al. (144-147). Theirresults are summarized in Table 13. In thepresence of excess oxygen, Zymomonas did notbreak down glucose (in contrast to results ofother authors). When limited amounts of 02

were supplied, somewhat less ethanol wasformed, and there was a small increase in acet-aldehyde. Changes in other products were verysmall and nearly unimportant for the economyof the cell. The respiratory activity of Zymo-monas is present in anaerobically grown cells.The oxidative capacities of the cells seem to beaffected considerably by the medium on whichthey have been grown (17). Both aerobicallyand anaerobically grown cells oxidize glucose(44). The latter cells have a Q(02) of 60 to 80 andtake up 0.5 to 0.8 mol of 02 per mol of glucoseadded and the former ones have a Q(02) of 100to 140 and use 0.7 to 1.5 mol of 02 per mol of glu-cose. The aerobic dissimilation of glucose corre-

sponds to the equation: 1 glucose + 1 02 -_ 1

ethanol + 1.7 C02 + 1 acetate + 0.2 lactate (17).The mechanism seems to be limited to the oxi-dation of ethanol to acetic acid. Even anaero-

bically grown Z. anaerobia is able to oxidizeethanol without a lag (41). The transfer of elec-trons by the respiratory chain is apparently notcoupled with oxidative phosphorylation. Ac-cording to Gongalves de Lima et al. (70), strainsofZymomonas mobilis var. recifensis appear tobe more aerobic than other Zymomonas strains.However, several strains of the subspeciesmobilis in our collection were as aerotolerant.According to Magalhaes Neto et al. (108), strainAg 11 oxidizes the following sugars [Q(02) val-ues are given in parentheses]:fructose (38), glu-cose (35), sucrose (27), xylose (14), galactose (5),

mannose (3), maltose (3), and endogenous (1).Lactose and a few organic acids were not oxi-dized.The species name of Z. anaerobia suggests

that the organism is either strictly anaerobicor at least more anaerobic than Z. mobilis.McGill and Dawes (112) observed that this or-

ganism is facultatively aerobic, contrary to ear-lier reports (150, 151), which classified the orga-

nism as "microaeroduric." Z. anaerobia NCIB8227 grows when aerated vigorously on a gyra-

tory shaker. The yield of these cells and theirrate of growth are only 50o of anaerobicallygrown cells (38). It is likewise our own experi-ence that, as far as anaerobic response is con-

cerned, named mobilis and anaerobia strainsgrow in the same fashion. To stress the anaero-

bic features with a species name is not justified.The presence and possible role of the tricar-

boxylic acid cycle in Zymomonas NCIB 8227has been examined by Dawes et al. (43). Fourenzymes of the tricarboxylic acid cycle havebeen detected: citrate synthase, aconitase, iso-citrate dehydrogenase, and malate dehydrogen-ase. Aeration of intact cells does not inducehigher activities of these enzymes. The specificactivities of the Zymomonas enzymes are simi-lar to those reported for anaerobically grown E.coli, with the exception of malate dehydrogen-ase. The following enzymes were not detected:succinate-, a-ketoglutarate-, and glutamate de-hydrogenases; succinyl-CoA-, 8-amino-laevuli-nate-, and malate synthetases; fumarate hydra-tase, succinate thiokinase, and isocitrate lyase.Small amounts of all the intermediates of thecycle were detected. Whether they are formedby the cycle enzymes, present in too low activi-ties to be detected, or by some other mechanismis not known.The fragments of the tricarboxylic acid cycle

inZymomonas may be correlated with the pres-ence of the Entner-Doudoroff pathway. Bothmechanisms are typical of aerobic organisms,and their presence in preponderantly anaerobicorganisms is surprising. They suggest to us

TABLz 13. Effect of oxygen on the fermentation ofZymomonaS a

mol of product per mol of glucose fermented with added 02 (mol):Compounds formed

0 0.4 0.6 1.5

CO2 1.94 1.95 1.96 1.91 0Ethanol 1.99 1.92 1.91 1.70 0Acetaldehyde Trace 0.024 0.021 0.17 0Acetoin Trace 0.01 0.01 0.04 0Acetic acid 0.01 0.06 0.06 0.03 0Lactic acid 0.002 0.002 0.002 0.004 0Glycerol 0.004-0.03 0.008 0.009 0.019 0

Data from Schreder et al. (147).

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thatZymomonas is evolutionarily derived fromaerobic ancestors, by decrease or loss of severaltricarboxylic acid cycle enzymes and electrontransport enzymes. It is significant that strictlyaerobic, polarly flagellated Gluconobacterstrains likewise possess the Entner-Doudoroffmechanism and fragments of a tricarboxylicacid cycle. We suggest this evidence may con-stitute two more indications that Zymomonasand the acetic acid bacteria are remote rela-tives.Cytochromes. Z. mobilis NCIB 8938 displays

cytochrome bands at the temperature of liquidnitrogen (17). Aerobically grown cells have thetype c cytochrome 550 (,3 band 520 and 528) andthe type a2 cytochrome 620. Anaerobicallygrown cells have in addition the type b cyto-chrome 560. Low concentrations of cyanide(10-4 M) inhibit the respiration of this straincompletely. Z. mobilis probably respiresthrough a complete respiratory chain, in whichcytochrome c and a cytochrome oxidase ofthe a2type are constitutive (17).

Aerobically and anaerobically grown cells ofZ. anaerobia both contain a type b cytochromeand traces of c cytochrome (112). These differ-ences between both strains ofZymomonas areto be considered as minor and individual,rather than as criteria for species differentia-tion.We are not aware of a systematic compara-

tive investigation of cytochromes in Zymo-monas. A comparative study in many strains ofAcetobacter and Gluconobacter showed that acytochrome pattern has no taxonomic signifi-cance (118).The pigment P-503. A pigment absorbing at

503 nm has been detected in different spectraof yeast and several bacteria (74, 99). This pig-ment is not a hemoprotein; it displays the sameproperties as tetrahydroporphyrin (95, 124).Kropinski et al. (93) detected this pigment inZymomonas ATCC 10988; judging from theirdata, there is not very much present. The even-tual role of this pigment in the metabolism ofZymomonas is not clear. In E. coli this pigmentmay have some function in electron transport,because it is in kinetic equilibrium with flavo-protein (124).

Amino Acid Metabolism

Amino acid requirements. The first nutri-tional studies were carried out with strainNCIB 8938 by Belaich and Senez (17). A mix-ture of 20 amino acids, or even NH4Cl, couldpartially replace yeast extract and supportedgrowth in the presence of glucose and Ca-panto-thenate in the synthetic and minimal media,

respectively. The molar growth yields in thelatter media are only about half the value fromthe complex medium; the generation time andthe cellular carbon from glucose both increase.Ethanol formation remains approximately thesame (Table 14). The growth of ZymomonasNCIB 8227 is stimulated by individual aminoacids (Table 15), by NH4Cl, and by groups ofamino acids, but the complete set of aminoacids is necessary to obtain a growth yield com-parable to growth on peptone (42) (Table 16).From all the amino acids tested individually asa sole source of nitrogen for growth of Zymo-monas NCIB 8227, either arginine, tryptophan,glutamic acid, or cystine produced the bestgrowth (23, 42) (Table 15). Maximum growthwas supported by either glutamic acid, lysine,hydroxyproline, or cysteine with a Zymomonasisolate tested by Richards and Corbey (137).To establish the effect of amino acids on the

growth of 38 Zymomonas strains, Van Pee etal. (174) used the liquid synthetic medium ofBelaich and Senez (17), but added a mixture of10 vitamins (23), guanine, adenine, hypoxan-thine, cytosine, and uracil. This medium cov-ered the nutritional requirements of all Zymo-monas strains. Five transfers were made, andthe growth was measured. The omission of eachofthe 20 amino acids separately from the aminoacid mixture did not depress growth of Zymo-monas completely; i.e., none of the 20 acids isessential to Zymomonas when other aminoacids are present. The growth of 10 strains outof 38 showed no marked reduction upon with-drawal of any of the amino acids from the syn-thetic medium. In individual Z. mobilis subsp.mobilis strains, up to four amino acids reducedgrowth, whereas in the most exacting strain, Z.mobilis subsp. pomaceae ATCC 29192, thewithdrawal of any one of six amino acids causeda reduction ofgrowth (Fig. 9). From Fig. 10 it isseen that valine and leucine reduced growth in18 and 8 strains, respectively. The claim ofBexon and Dawes (23), that Z. anaerobia NCIB8227 is more exacting for amino acids than Z.mobilis, is contradicted by our results.The above information concerns the effect of

amino acids on growth and growth yield. Datafrom Belaich and Senez (17) show that abouthalf of the cellular carbon is derived from glu-cose. Clearly, the other half can come from oneor more amino acids. Some of them, thus, notonly serve as a nitrogen source, but also asa carbon source. Therefore, we determined theamino acid uptake by Zymomonas ATCC10988 growing in the synthetic medium ofBexon and Dawes (23) (J. De Ley, J. Swingsand J. Van Beeumen, unpublished data).Three amino acids only, aspartic acid, serine,

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TABLE 14. Growth characteristics ofZymomonas NCIB 8938 in complex, synthetic and minimal media"Molar growth Cellular car-

yield (YG) (g Generation Ethanol bon derivedMedium Nitrogen source [dry cells] per time (h) formed (mo e from glucosemol of glucose mol of glucose)fermented)

Complex Peptone + yeast extract 7.95 1.90 1.57 48Synthetic 20 Amino acids 4.98 2.62 1.52 62Minimal NH4Cl 4.09 3.0 1.50 100a Data from reference 17.

TABLz 15. Efficiency of individual amino acids forgrowth ofZymomonas NCIB 8227a

Amino acid (1.35%)

GlyIle, Leu, CysSerThrHypHisValTyrMetPheAlaCys-CysGluTrp, Arg

Net growth (jtg [drywt]Imi)

2325262832374351536373768387

a Data from reference 40.b Added to a medium containing 2% glucose, inor-

ganic salts, biotin, and lipoic acid (each, 20 pg/ml).

TABLE 16. Different nitrogen sources for growth ofZymomonas NCIB 8227a

NetSupplement (each amino acid at 0.067%)b growth(J~g [dry

wt]/ml)Ammonium chloride 57Groups of amino acidsArg + Cys + Cys-Cys + Met 51Ile + Leu + Val 48Phe + Trp + Tyr 60Asp + Glu + His + Thr 53Ala + Gly + Hyp + Ser 58

All amino acids 121All amino acids + NH4Cl 139Synthetic medium (17) 146Peptone 172

a Data from reference 42.b Added to a basal medium containing 2% glu-

cose, inorganic salts, biotin, and lipoic acid (20.&g/ml each).

and cysteine, were taken up in appreciableamounts. Six other amino acids were not takenup at all from the medium (Tyr, Pro, Phe, Glu,Ala, Trp). From the remaining amino acids,small amounts were consumed, but less than

Ln 11z

IL-Wo \I

I

II

0 3 6

NUMSER Of AMINO ACIDS CAUSINGREDUCED GROWTH

FIG. 9. Number of amino acids that reducedgrowth in individual Zymomonas strains.

15% of the amount supplied (Table 17).There is evidence that, in many bacterial

genera and species, the amino acid require-ments are strain specific (31, 45, 113, 140). InZymomonas also many amino acids stimulategrowth, and the degree of stimulation variesfrom strain to strain. Amino acid requirementsare therefore of little taxonomic use in Zymo-monas.Formation of higher alcohols. Plant juices,

beers, and ciders, infected with Zymomonas,develop typical fruity aromas, which are veryimportant for the commercial value of theseproducts. The aromas of common alcoholic bev-erages are largely due to esters and fusel alco-hols. Therefore, Verachtert and collaborators(21, 22, 133, 175) determined the fusel alcoholsproduced by Zymomonas. These bacteria pro-duce about 0.6 mg of total fusel alcohols per g ofglucose fermented; this is about 40 times lessthan Saccharomyces in the same conditions.The most important components of the fuselalcohol fraction are propanol and isoamyl alco-hol in Zymomonas (Table 18), and isobutanoland isoamyl alcohol in S. cerevisiae. No consid-erable differences in fusel alcohol productionwere observed among nine Zymomonas strainsstudied. The nature and the very small amountof fusel alcohols formed by resting Zymomonascells in glucose solution suggest that they arederived mainly from amino acids and much less

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V0

0

15wL)0w

z

0

(n

zlO

U,

z

:;)

U-

0

wS

W

z

za5

,W

-zz

BI .- V

AMINO ACID OMITTED FROMTHE SYNTHETIC MEDIUM

FIG. 10. Number of Zymomonas strains showingreduced growth by omission of individual aminoacids from the synthetic medium. Thirty-eightstrains were tested.

from carbohydrate metabolism. Direct proofwas obtained by the addition of a variety ofcompounds. The addition of a-ketobutyric acid,threonine, propionaldehyde, and homoserine,in that order, stimulates the formation of pro-panol. Isobutyraldehyde and valine activateisobutanol production. Isoleucine increases theformation of i-amyl alcohol. Forssman (63)demonstrated that DL-methylethylacetalde-hyde is reduced to amyl alcohol by activelyfermenting Zymomonas. a-Ketoisocaproic acid,isovaleraldehyde, and leucine stimulate theformation of isoamyl alcohol. Oxaloacetate ap-pears to stimulate the production of severalfusel alcohols, on the one hand by decarboxyla-tion, on the other hand by transamination toaspartic acid. The general pathways existing inyeasts and in Zymomonas for the formation offusel alcohols are represented in Fig. 11.Amino acid decarboxylases. Of 38 Zymo-

monas strains tested, 31 contain L-arginine de-

carboxylase, 18 contain Llysine decarboxylase,and 4 contain L-ornithine decarboxylase (50).Five do not possess any of the above-mentionedthree amino acid decarboxylases. The testswere read after 10 to 15 days of incubation.

Growth Factor RequirementsCider sickness strains B and C required pan-

tothenic acid, riboflavin, biotin, and nicotinicacid (11).Zymomonas NCIB 8938 required 50 ng of

pantothenate per ml only for growth in syn-thetic or minimal medium (17). Van Pee et al.(174), however, found with two other subcul-tures, 410 and ATCC 10988, of the same orga-nism, that both pantothenate and biotin arerequired. In synthetic or minimal medium con-taining excess pantothenate, the molar growthyield and the growth rates of ZymomonasNCIB 8938 were only about half ofthe values inthe complex medium. Both remained constantover a series of successive transfers. Belaichand Senez (17) tried unsuccessfully to restorethe molar growth yield to the level of the com-plex medium by separate or simultaneous addi-tion of several compounds, such as ascorbicacid, menadione, nicotinic acid, etc.

Stephenson et al. (158) showed that the nutri-tional requirements of Zymomonas NCIB 8227did not remain constant. When first checked in1970 (23), this strain required lipoic acid orbiotin for growth. Upon reexamination of threesubcultures of the same strain a few yearslater, the vitamin requirement had changed topantothenate (Table 19) (158). The same au-thors reported that another Zymomonas strainshowed a requirement for p-aminobenzoic acid,folic acid, biotin, and cyanocobalamine uponisolation. Five months later, one of the subcul-tures showed a requirement for Ca pantothen-

TABLE 17. Uptake ofamino acids by ZymomonasATCC 10988 during growth in the medium ofBexon

and Dawes (23)a

Amino acids taken up from themedium for. Amino acids not

taken up from the50% of ini- 15% or less of ini- mediumtial concn tial concn

Asp Arg TyrSer Gly PheCys Ile Ala

Leu ProOrn and/or Lys GluHis TrpMetValThr

a Unpublished data from J. De Ley, J. Swings,and L. Van Beeumen.

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BIOLOGY OF ZYMOMONAS

TABLz 18. Formation of fusel alcohols by Zymomonas and Saccharomycesa

Alcohol formed (yg/ml)

Organisms State of cells I l al Avg total,Propanol i-Butanol n-Butanol hoAmyl oohao l higher alco-hol cohol ~hols

Zymomonas Growing culture 4.1-21.4 Traces Traces Traces 6.6-17.6 25.96Resting cells in 5% Traces Traces 0 0 0.81 0.81

glucose solution,pH 6.4

Saccharomyces Growing culture 13.71 779.2 0 60.22 95.65 948.78cerevisiae

Resting cells in 5% 4.79 25.93 0 11.59 33.98 76.29glucose solution,pH 6.4

a According to Bevers (21) and Bevers and Verachtert (22). The Zymomonas values are the mean fromnine strains tested. All cells were grown on the medium of Gibbs and DeMoss (67).

propionaldehyde - n-propanolCH3-CH2-CH2-OH

homoserine - threonine - a-ketobutyricacid

a-methylbutyral->--amyl alcoholdehyde CH3-CH2-CH-CH2-OH

aspartic isoleucine CH3acid

a-acetolactate > a-ketoisovaleric > isobutyric alde- > isobutanolacid

valine {

a-ketoisocaproicacid

hydeCHK

I,,'CH-CH2-OHCH3

isoamyl aldehyde-> isoamyl alcoholCH3\

C CH-CH2-CH2-OHCH3

FIG. 11. Formation offusel alcohol compounds.

ate only. Strains ofZ. anaerobia, examined byDadds (32), had an absolute requirement forpantothenate. A new isolate from beer requiredpantothenate, but was also stimulated by biotin(137).Ca pantothenate and biotin are growth fac-

tors for the 38 strains tested by Van Pee et al.(174) (Fig. 12). In 32 strains, they are even theonly growth factors required. There are a fewstrains with some additional requirements,

such as 70.9 and 70.10 (vitamin B12), VP2 (p-aminobenzoic acid), 7.4 (riboflavin), and thepomaceae strain ATCC 29192 (thiamine andriboflavin). VP3 is the most exacting strain,since it requires cyanocobalamine, lipoic acid,folic acid, and riboflavin, in addition to panto-thenate and biotin.

In conclusion it may be said that (i) most orall strains require pantothenate and biotin, andoccasionally some other growth factors, (ii) re-

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TABLE 19. Changes in growth factor requirements ofZymomonas NCIB 8227

Requirement for: Lag pe-Report and origin of Reference Growth characteristics *i*c Ca pan- riod (in

strain no. Biotin Lpoc tothen- definedacid ate medium)acd ate mei)

Bexon and Dawes 23 + or + - -NCIB

Stephenson et al. 158 - - + +Culture H (maintained No wall growth; no

at Hull) clumping; slow sedi-mentation

Culture AB Wall growth; clumping; - - +(maintained at Allied fast sedimentationBreweries)

Culture T (newly Wall growth; clumping; - - +obtained from NCIB) fast sedimentation

Van Pee et al. 174NCIB Clumping; slow sedimen- + - +

tationQueensland No clumping; slow sedi- + - +

mentation

sults with the same strain differ from one au-thor to another, (iii) upon aging, some strainsappear to require pantothenate only, and (iv)differences in growth factor requirements haveno taxonomic meaning in Zymomonas.

H2S Formation and Sulfur Metabolism"Sulfury" aromas are of great technological

importance in beer and cider. They are due toH2S and other unknown compounds (6).The formation of H2S by Zymomonas has

been described by Shimwell (150), Millis (115),Gongalves de Lima et al. (70), Carr and Pass-more (29), Dadds et al. (36), and Richards andCorbey (137). The latter authors claim that H2Sproduction is easily lost upon subculturing inthe laboratory. The production of H2S may be astable feature in some strains, e.g., in Zymo-monas NCIB 8227 and NCIB 8938, which havebeen subcultured for years and still producedH2S when tested by Millis (115), Dadds et al.(36), and ourselves.Forty-two Zymomonas strains of our collec-

tion do not form H2S (50). According to Ander-son and Howard (5), the main source of H2S inbeer and wort is sulfate (Table 20). On increas-ing sulfate concentrations from 0 to 500 mg/liter, the amount of H2S produced increases.Pantothenate has a reverse effect: when its con-centration increases from 0 to 40 pg/liter, H2Sformed decreases; higher growth factor concen-trations have no additional effect. Zinc ions alsostimulate H2S production (Fig. 13).Traces of dimethylsulfide and dimethyldisul-

fide are produced by Zymomonas NCIB 8938,B70, and NCIB 8227 (36).

Cellular sulfur is mainly of organic origin (5)(Table 21). Methionine, thiamine, sulfite, sul-fate, and cysteine can be used as sources ofsulfur in a synthetic medium (35).

Various FeaturesPresence of catalase. The presence of this

enzyme in Zymomonas has been demonstratedby Kluyver and Hoppenbrouwers (89), Millis(115), Carr and Passmore (29), and Dadds et al.(36). It is present in all 45 Zymomonas culturesof our collection. Belaich and Senez (17) ob-served considerable variations in catalase ac-tivity ofZymomonas NCIB 8938, depending onthe composition of the medium. The specificactivities of catalase in the minimal and in thesynthetic medium were 12 and 5 times higher,respectively, than in the complex medium. Thecatalase activity remained unchanged afterstrong aeration for 4 h prior to the assay.Presence of gelatinase. No gelatinase activ-

ity was found in 45 strains of our collection,thus extending the results of Shimwell (151)and Millis (115).Presence of urease. This reaction was posi-

tive but very slow in 24% of the strains of ourcollection; at least 3 days are needed to show apositive reaction (50).Oxidase reaction. Dadds et al. (36) reported

a negative oxidase reaction in ZymomonasNCIB 8938 and B70. Our 45 strains were like-wise oxidase negative.

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Dz -4°Emr- <wC

_O---->

_ m

dOnZ>ZC) -4M0 0 zmC.>5~~~~~~~~

no0

-4I:

z

U

NCIB 8227409ATCC 10988410ATCC 29192

FIG. 12. Growth factor requirements (darksquares) ofZymomonas strains.

Hydrolysis of Tweens. Tween 60 and Tween80 were not attacked (50).

Nitrate reduction. This test was negative in45 of our Zymomonas cultures, a result thatagrees with these of Shimwell (151) and of Mil-lis (115).Formation of indole. Indole was found to be

absent by Shimwell (150), Millis (115), and inour 45 Zymomonas strains.

CONCLUSIONS AND SUMMARYZymomonas cells are gram-negative rods; a

minority of the strains are motile, with 1 to 4polar flagella. These organisms need glucose,fructose, or (for some strains) sucrose in themedium of growth. They are very unusual mi-

croorganisms since they ferment these sugarsanaerobically by way of the Entner-Doudoroffmechanism, followed by pyruvate decarboxyla-tion. The oxidation-reduction balance betweenG6P dehydrogenase and triosephosphate dehy-drogenase on one hand and ethanol dehydro-genase on the other hand is mediated throughNAD+. Sucrose is converted to some small ex-tent into levan and consumed mainly by a hy-drolytic split. Sucrose metabolism seems to bean inducible feature. Sugar fermentation is ac-companied by formation of a small amount of

TABLE 20. Sulfur sources used by ZymomonasNCIB 8227 and Saccharomyces carlsbergensisa

Cellular S (%) from:H2S

Prepn from and Me-me- S04 thio-thio- ninenine

Zymnomonas in:Wort 71 56Beer 62 35 _b

Saccharomyces carls-bergensisin wort 83 21 18

Data from Anderson and Howard (5).No methionine was detected in beer.

r-0nv

3:

EcmI

0w

0U-

UI)x

Il

4

3

2

1 - PANTOTHENATESo0- -

0 200 400S 7- (mg/I1)

0 1000 200 0OPantothenate (4g / l)

I I

0 500 1000Zn 4g9/I)

FIG. 13. Effect of Zn2+, pantothenate, and So42-on H2S production (data from Anderson and Howard[5D).

I

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lactic acid, with traces of acetaldehyde and ace-toin. The molar growth yield indicates thatZymomonas is only about 50% efficient in con-verting its carbon and energy sources. Thegrowth is partially uncoupled. About 2% of theglucose substrate is the source of about half ofthe cellular carbon. Several amino acids alsoserve as carbon sources. Some strains growonly anaerobically; others display various de-grees of microaerophily. Apparently, the maineffect of oxygen is the oxidation of part of theethanol to acetic acid. A few enzymes of theKrebs cycle and some cytochromes are alsopresent. Most strains are alcohol tolerant (10%)and grow in up to 40% glucose. None of the 20amino acids is essential to Zymomonas whenother amino acids are present. Several aminoacids, though, improve growth. This degree ofgrowth stimulation varies from strain to strain.Zymomonas produces a small amount ofhigheralcohols, mainly propanol and isoamyl alcohol,from aspartic acid, homoserine, threonine, andleucine. Most or all strains require pantothen-ate and biotin; upon aging, some strains ap-pear to require pantothenate only. All strainsare oxidase, nitrate, and indole negative. Thewide pH range for growth, from 3.5 to 7.5, andthe acid tolerance are quite typical.We have reviewed methods for the detection

and isolation of Zymomonas, and provided aminimal description for its identification.Zymomonas is one of the very important

ethanol-producing microorganisms in theworld. It has been isolated from ferment-ing agave sap in Mexico, from fermenting palmsaps in Zaire, Nigeria, and Indonesia, fromfermenting sugarcane juice in Northeast Brazil.Undoubtedly, they are important contributorsto the fermentation ofplant saps in many tropi-cal areas of America, Africa, and Asia. Thesebeverages have a great variety of names ac-cording to the localities where they are pro-duced. Considerable amounts are sometimesconsumed yearly by the local populations. Inmany cases, these plant wines are produced ona small scale by skilled artisans, but there aresome reports of large-scale technological appli-cations, and of therapeutic use. Zymomonas,undoubtedly, has a great social and economicimportance.These bacteria also occur in ciders, perries,

and beers. So far they have been detected inthis habitat in the United Kingdom only. Theyproduce the cider sickness disease, accompa-nied by much gas formation, change in aromaand flavor, reduction in sweetness, and an un-pleasant taste. As a beer contaminant, theycause unpleasant aromas and taste. Recently,Zymomonas has also been isolated from ripen-

ing honey and bees. Zymomonas strains areextremely well adapted to all these ecologicalniches. Glucose, fructose, or sucrose, aminoacids, and growth factors are needed by thebacteria and are provided by the plant juices.We reviewed critically several proposals

made in the literature on the nomenclature andthe classification of this genus. A numericalanalysis shows that the phenotypical similaritybetween all the strains is very high; they allform one cluster above 88% SSM. The DNA ge-nome size of all our strains is about 1.5 x 109,expressed as molecular weight. All strains ex-cept three from the subspecies pomaceae are ge-netically very similar. The degree of genome-DNA relatedness is at least 76% D in the maingroup. The cider sickness organism ATCC29192 has less than 32% genetic similarity withall other zymomonads. The protein electropher-ograms of all strains except the cider sicknessorganisms are very similar. There seem to be atleast two serological groups, the importance ofwhich is still unclear. Our extensive reexami-nation of many strains ofZymomonas with theabove-mentioned modern methods allowed amore realistic classification. Nearly all existingstrains ofZymomonas, except three, are so sim-ilar, in spite of their greatly diverse origins,that they deserve to be united in one taxon,Zymomonas mobilis subsp. mobilis (Kluyverand van Niel) De Ley and Swings 1976, with thelectotype strain ATCC 10988 = NCIB 8938, andthe phenotypic centrotype strain Z6 = ATCC29192 = NCIB 11999. We proposed a secondsubspecies, Z. mobilis subsp. pomaceae (Millis)De Ley and Swings 1976, the cider sicknessorganism, with the lectotype strain ATCC29192 = NCIB 11200.

Since all the Zymomonas strains, except thecider sickness strains, are genetically and phe-notypically almost identical, we suggest thatZymomonas is either of recent evolutionary ori-gin, and has not yet had time for genetic diver-sity, or else it is a genetically very stable genus,in spite of its worldwide distribution.We also examined the relationship between

Zymomonas and other genera. Zymomonas re-sembles most closely the acetic acid bacteria,and is closer to Gluconobacter than to Acetobac-ter, because of the polar flagella, the occurrenceof the Entner-Doudoroff mechanism, the in-complete Krebs cycle, and the ready consump-tion of glucose. Several other points of similar-ity were indicated. We suggest that these threegenera are of a common phylogenetic origin.This implies that the ancestors ofZymomonaswere aerobic organisms. Indeed, nearly all or-ganisms with the Entner-Doudoroff mechanismare aerobic. We assume that the aerobic ances-

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tors gave rise to the largely anaerobic Zymo-monas by loss ofsome Krebs cycle enzymes andperhaps also some electron transport enzymes.

ACKNOWLEDGMENTSOne of us (J.D.L.) is indebted to the Fonds voor

Kollektief Fundamenteel Onderzoek and to the Na-tionaal Fonds voor Wetenschappelijk Onderzoek(Belgium) for several research, equipment, and per-sonnel grants over the last two decades. J.S. isindebted to the Instituut tot Aanmoediging van hetWetenschappelijk Onderzoek in Nijverheid enLandbouw for a scholarship. We are indebted toseveral colleagues, mentioned in the text, for per-mission to quote their unpublished data.

LITERATURE CITED1. Adriaens, E. L. 1951. Contribution a l'6tude

des vins de palme au Kwango. Inst. R. Co-lon. Belge Bull. Seances 22:334-351.

2. Adriaens, E. L. 1951. Recherches sur l'ali-mentation des populations au Kwango. Bull.Agr. Congo Belge 42:473-552.

3. Adriaens, E. L. 1952. Les vins de palmier. Bull.Agr. Congo Belge 43:556-558.

4. Ahmad, M., A. R. Chaudhury, and K. U. Ah-mad. 1954. Studies on toddy yeast. Mycologia46:708-720.

5. Anderson, R. J., and G. A. Howard. 1974. Theproduction of hydrogen sulphide by yeastand by Zymononas anaerobia. J. Inst. Brew.London 80:245-251.

6. Anderson, R. J., and G. A. Howard. 1974. Theorigin and occurrence of volatile sulphurcompounds in British ales and lagers. J.Inst. Brew. London 80:357-370.

7. Arai, T., S. Kuroda, and Y. Koyama. 1963.Infrared absorption spectra of whole cells ofNocardia and related organisms. J. Gen.Appl. Microbiol. 9:119-136.

8. Auclair, B. 1955. Le framboise des cidres. Ind.Agr. Aliment. 72:185-186.

9. Ault, R. G. 1965. Spoilage bacteria in brew-ing-A review. J. Inst. Brew. London71:376-391.

10. Bain, N., and J. M. Shewan. 1968. Identifica-tion ofAeromonas, Vibrio and related orga-nisms, p. 79-84. In B. M. Gibbs and D. A.Shapton (ed.), Identification methods for mi-crobiologists. The Society for Applied Bacte-riology. Technical series, no. 2, part B. Aca-demic Press Inc., New York.

11. Barker, B. T. P. 1948. Some recent studies onthe nature and incidence of cider sickness.Annu. Rep. Agric. Hort. Res. Stn. Long Ash-ton Bristol, p. 174-181.

12. Barker, B. T. P., and V. F. Hillier. 1912. Cidersickness. J. Agric. Sci. 5:67-85.

13. Bassirr, 0. 1968. Some Nigerian wines. W.Afr. J. Biol. Appl. Chem. 10:42-45.

14. Bauchop, T., and S. R. Elsden. 1960. Thegrowth ofmicroorganisms in relation to theirenergy supply. J. Gen. Microbiol. 23:457-469.

15. Belaich, J. P. 1963. Thermogenese et croiss-ance de Pseudomonas lindneri en glucoselimitant. C. R. Soc. Biol. 157:316-322.

16. Belaich, J. P., A. Belaich, and P. Simonpietri.1972. Uncoupling in bacterial growth: effectof pantothenate starvation on growth ofZymomonas mobilis. J. Gen. Microbiol.70:179-185.

17. Belaich, J. P., and J. C. Senez. 1965. Influenceof aeration and pantothenate on growthyields of Zymomonas mobilis. J. Bacteriol.89:1195-1200.

18. Belaich, J. P., J. C. Senez, and M. Murgier.1968. Microcalorimetric study of glucose per-meation-in microbial cells (Zymomonas mob-ilis). J. Bacteriol. 95:1750-1757.

19. Belaich, J. P., P. Simonpietri, and A. Belalich.1969. Uncoupling ofbacterial growth by pan-tothenate starvation. J. Gen. Microbiol.58:vii.

20. Bergeret, B. 1957. Note pr6liminaire a l'6tudedu vin de palme au Cameroun. Med. Trop.17:901-904.

21. Bevers, J. 1970. Studie van de biosynthese vande hogere alkoholen bij het geslacht Zymo-monas. Eindwerk Fak. Landbouwweten-schappen. Katholieke Universiteit, Leuven.

22. Bevers, J., and H. Verachtert. 1976. Synthesisof higher alcohols in the genus Zymomonas.J. Inst. Brew. London 82:35-40.

23. Bexon, J., and E. A. Dawes. 1970. The nutri-tion of Z. anaerobia. J. Gen. Microbiol.60:421-425.

24. Bidan, P. 1959. La maladie du framboise dansles cidres. Ind. Aliment. Agric. 76:31-33.

25. Bois, D. 1937. In Les plantes alimentaires. P.Lechevalier, Paris.

26. Booth, G. H., J. D. A. Miller, H. M. Paisley,and A. M. Saleh. 1966. Infrared spectra ofsulphate-reducing bacteria. J. Gen. Micro-biol. 44:83-87.

27. Capelle, F. 1641. Rapport de F. Capelle auComte J.-M. de Nassau. Rivalites Luso-n6er-landaises au Sohio, Congo 1600-1675 (1966).Bull. Inst. Hist. Belge de Rome 37:221.

28. Carr, J. G. 1974. Genus Zymomonas Kluyverand van Niel 1936, 399, p. 352-353. In R. E.Buchanan and N. E. Gibbons (ed.), Bergey'smanual of determinative bacteriology, 8thed. The Williams & Wilkins Co., Baltimore.

29. Carr, J. G., and S. M. Passmore. 1971. Discov-ery of the "cider sickness" bacterium Zymo-monas anaerobia in apple pulp. J. Inst.Brew. London 77:462-466.

30. Corner, E. J. H. 1966. The natural history ofpalms. Weidenfeld and Nicolson, London.

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The Biology ofZymomonas · BIOLOGY OFZYMOMONAS 3 and dry farm-made cider. The manufacturers encountered great difficulties, as sweet ciders easily develop a secondary fermentation,