Vol.

80,1974] 241

ISOLATION OF ZYMOMONAS MOBILIS FROM PRIMED BEER*

By M. Richards and D. A. Corbey
{Bass Production Ltd., High Street, Burton, and Hope & Anclwr Brewery,
Clay Wheels Lane. Sheffield)

Received 11/A September, 1973

Isolation from beer of a sucrose-fermenting species of Zymomonas confirmed

as Z. mobilis, is recorded. Motility and H2S production were variable and
sensitive to precise environmental conditions. The beer-spoilage potential
of this species was established as limited. A strain of Z. anaerobia was isolated
which acquired sucrose-fermenting ability after growth in fructose. It is
doubtful, therefore, whether the creation of two distinct species within this
genus is valid.
Key words: bacteria, beer, taxonomy. isolate 2 appeared to ferment sucrose in addi
tion to glucose and fructose. Moreover, with
Introduction isolate 2, there appeared to be a metabolic
The physiology of Zymomonas mobilis and preference for sucrose, as onset of growth and
Z. anaerobia is very similar: the major fermentation (evolution of CO2) was more
difference between the two is the ability advanced in the presence of this sugar than
only of the former species to ferment suc in either the glucose or fructose tubes. When
rose.10 Additional characters differing be cultured on agar, colonies were large (1 mm
tween the species, viz., amino acid and to 2 mm diameter) on sucrose medium but
vitamin requirements,3-* have been inter pin-head on glucose and fructose media.
preted in other microbial genera, e.g. Saccharo- Utilization of the intact sucrose molecule
myces, as strain-specific rather than species- appeared to be a property of the Gram-
specific.2 Profound changes in vitamin re negative bacteria rather than a reflection of
quirements have recently been described for coincidental yeast contamination, as the
a strain of Z. anaerobia}2 thus supporting the test medium contained actidione, and yeast
opinion that such characters should be used cells were not observed microscopically. The
for strain designation only. Particularly possibility of contaminant yeast inverting
significant technologically is the demonstra sucrose and the bacterium then utilizing a
tion that both species of Zymomonas are sugar monomer was finally discounted by
tolerant to the highly limiting alcoholic- re-streaking and selecting from a single
acidic-anaerobic environment of fermented bacterial colony. Such a "clonal" isolate
beverages. In the light of these physiological still fermented sucrose and was shown to be
similarities and the demonstrated beer- free of yeast contamination by plating
spoilage potential of Z. anaerobia, it is sur (0-1 ml) on a nutrient medium containing
prising, therefore, that Z. mobilis has never aureomycin. Such preference for sucrose is
been isolated from beer. The present com unusual, as another isolate of Z. mobilis
munication documents such an isolation. revealed lower molar growth yields with
sucrose than with glucose and fructose.8
Results and Discussion Presence of a sucrose-fermenting zymo-
Occasional positive results (growth of monad was sought in brewery plant but both
Gram-negative rods, CO2 and H2S evolution dispatching and receiving breweries were free
from glucose medium) were recorded when of such an organism. It was assumed,
stout from road tanker was routinely screened therefore, that the source of infection was the
for the presence of Zymomonas. On testing road tanker.
against a wider range of sugars not all Confirmation of genus identity.—The genus
isolates were confirmed as Zymomonas; e.g. Zymomonas is characterized by a near-
the lactose-fermentation of isolate 3 (Table I) quantitative conversion of glucose to ethyl
excluded it from this genus. Isolate 1 was alcohol and COa. The sucrose-fermenting
confirmed as a typical Z. anaerobia, but isolate 2 produced 1-7 mole ethanol per mole
* Since submitting the present paper, similar results have been independently published: Dadds, M. J. S..
Martin,
artin, P. A. & Carr, J. G., Journal of Applied Bacteriology. 1973, 86. 631.
242 RICHARDS AND CORBEYt ZYMOMONAS MOBILIS
TABLE I
Fermentation Characteristics of Presumptive Zymomonads Isolated from Primed Beer
Isolate* Glucose Fructose Sucrose
1
2 + /+/+
3 + 1+1-
• Growth/CO,/H8S are shown as + = positive, — = negative for the different isolates.
of glucose. Carbon dioxide evolution was
confirmed quantitatively.13 A full carbon-
balance, therefore, was not established. How
ever, the molar yield of ethanol corresponds
to yields previously published8'10 for Z.
anaerobia. This high yield of ethanol was
taken as confirmation of genus.
Characterization of Z. tnobilis isolate
Cell tnorplwlogy.—From a liquid 48-hour
culture (isolation medium) cells were plump,
1 to 1-5 /*m in diameter to 4-6 /nm in length,
ends rounded. Cells occurred singly, in
pairs and occasionally as triplets: no chain
formation was evident. Neither tendency
towards cell elongation nor aggregation of
individual cells into rosette pattern was
apparent in old cultures.
Motility.—From liquid sucrose medium,
cells were highly motile, but in fructose (20%
motile) and glucose (5% motile) cells became
progressively non-motile.
Mode of flagellation was not established.
Colony morphology.—Mature surface
colonies on sucrose plates (incubated anaero
bically for seven days at 30 CC) were slightly
oval in appearance, 1-2 mm in diameter at
the widest point, of moist, translucent
appearance with an entire margin. The
colony had a slightly convex elevation and
was not adherent to the medium surface.
Growth characteristics in liquid medium
(Beer-yeast extract plus glucose).—Dense
turbidity was produced within 18 hours at
30 °C accompanied by abundant CO2 and
H2S evolution. Older cultures sedimented
leaving the supernatant substantially clear.
This growth pattern was followed whether
the organism was incubated anaerobically or
aerobically as long as the culture tube was
filled with medium. When incubation was
aerobic growth commenced at the bottom of
the tube and progressed upwards as anaerobic
conditions extended towards the liquid/air
[J. Inst. Brew.
Galactose Maltose Lactose Mannose Ethanol
. . , ,
'. I.
interface. In shallow culture, growth occurred
only when the organism was incubated
anaerobically.
Under these conditions the growth pattern
of this Zymomonas mobilis isolate closely
followed that described for Z. anaerobia by
Shimwell.11
Carbohydratesfermented.—Glucose, fructose
and sucrose only (Table I).
Vitamin requirements.—Only pantothenate
was required absolutely, though growth was
further stimulated when biotin was also
supplied. A requirement for pantothenate
has previously been described for an authenti
cated strain of Z. mobilis3 but this can no
longer be considered diagnostic for the
species in the light of a derived strain of
Z. anaerobia also requiring only this vitamin.12
Amino acid requirements.—Maximum
growth was supported by any one of the
following amino acids: glutamic acid, lysine,
hydroxyproline, cysteine. Simple nitrogen
requirements characterize Z. mobilis3 and
distinguish it from Z. anaerobia.*
Identity.—The characteristics of bacterial
isolate 2 conformed to those described for
Z. mobilis.b
Virulence of Zymomonas mobilis as a beer
spoilage organism
Primed pale ale was infected with Z. mobilis
(beer isolate) at levels of 1, 10, 100 and 1000
cells per 250 ml of beer. After 2 weeks at
28 °C all levels of inoculation produced a
slight bacterial deposit but this was insuffi
cient to produce a visible haze when re-
suspended in the bulk of the beer. Absence
of beer spoilage even at high levels of infec
tion with Z. mobilis contrasts dramatically
with the dense beer turbidities produced
within 12 hours by Z. anaerobia.11 In view
of the extent of the physiological similarities
between the species the substantially different
beer spoilage properties appear surprising.
Vol. 80,1974] RICHARDS AND CORBEY: ZYMOMONAS MOBILIS
Variability of strain characteristics:
Taxonomic implications
Hydrogen sulphide evolution and motility
of Z. mobilis were both readily susceptible to
variation as a response to different environ
ments. The first of these characters was
particularly easily "lost." Commonly, HaS
evolution was pronounced upon initial isola
tion but absent after only one laboratory
sub-culture. Sulphury aromas result from
Zymomonas infection, so evaluation of this
character is important technologically. No
taxonomic significance is, however, attached
to this character.
Expression of motility was directly asso
ciated with the sugar available, this character
being fully expressed when cultured with
sucrose but substantially diminished with
glucose. Glucose repression of motility has
also been recognized with Esclterichia coli.1
The mode of flagellation is of significance in
the taxonomy of the genus Zymomonas, but
the importance of standardizing the environ
ment for the evaluation of bacterial motility
is already well recognized.
The most disconcerting variation in culture
characteristics, however, was observed with
isolate 1, an apparently typical strain of
Zymomonas anaerobia (Table I). Simple
subculture into fructose converted this
culture from a sucrose non-fermenter into a
sucrose fermenter. Serial subcultures
through glucose, however, failed to render the
culture sucrose-fermenting, and presumably
the phenomenon is explained by induction of
the enzyme /J-fructofuranosidase by fructose.
This enzyme is not inducible by glucose. The
possibility arises, therefore, of conversion of
Zymomonas anaerobia into Z. mobilis. In
the light of the preceding findings, the obser
vation that, of six clones established from
isolate 2 (Z. mobilis), one was a sucrose non-
fermenter assumes a greater significance. It
now appears possible that the sucrose non-
fermenter was a non-induced cell whilst the
sucrose fermenters arose from induced cells
of Z. anaerobia.
Despite additional differences in cyto-
chrome content,8'8 differences in amino acid
and vitamin requirements,3-4 and different
modes of flagellation,5 sucrose fermentation
remains the main taxonomic character
separating these two species. The conver
sion of a Z, anaerobia culture into Z. mobilis
by simple modification to culture conditions
seriously undermines the validity of this
243
distinction. The remaining differences seem
insufficient to support the recognition of two
species. It is proposed, therefore, that the
genus Zymomonas be represented by one
species only, viz. Z. mobilis with the second
species relegated to variety level as Z. mobilis
var. anaerobia. This conforms to the existing
recognition of the variety potnaceae10 and
would create a satisfactory situation whereby
differing spoilage potential would be recogn
ized only as different varieties.
Experimental
Isolation procedure.—The deposit centri-
fuged from 25 ml of beer was inoculated into
25 ml of the following medium contained in
1-oz MacCartney bottles: beer (pH 4-0;
300 ppm sulphate) supplemented with glucose
(4%, w/v) and yeast extract (0-3%, w/v). An
inverted Durham tube was placed in the
McCartney bottle for the detection of CO2.
Sterilization was by autoclaving at 121 °C
for 15 min, after which a pre-sterilized lead
acetate paper strip was placed under the
screw-cap for the detection of HaS.
Incubation was aerobic at 30 °C for up to
4 days.
Confirmation procedure.—Fructose, sucrose,
lactose, maltose, galactose, mannose or ethanol
(all at 4%, w/v) were substituted individually,
in the above medium, for glucose. A loop
inoculum was transferred from the initial
isolation tube to confirmatory media. All
confirmatory tubes were incubated anaerobic-
ally employing the GasPak system (Becton
Dickinson U.K. Ltd., Wembley, England).
CO2 and H2S production were detected as
described in the isolation procedure.
Isolation of discrete colonies.—Nutrient
plates were prepared from "isolation medium"
plus agar (2%, w/v). Cultures were "streaked"
on the surface of the plate to produce discrete
colonies. Two further "streaks" were per
formed sequentially, using a single colony
from the preceding selection on each occasion.
All plates were incubated anaerobically at
30 °C.
Evaltialion of amino acid and vitamin
requirements.—The defined medium of
Belaich & Senez3 was employed. This
comprised tris-maleate buffer, 0-2m, pH 6-8,
1 litre; KH2PO4, 10mg; NH4C1, lg; MgSO4
7H2O, 238 mg; CaCla, 1-1 mg; FeSO4 7HaO,
50 mg; ZnSO4 7H2O, 7-2 mg; MnSO4H2O,
4-2 mg; CuSO4 5HaO, 1-4mg; CoSO4, 7H2O,
1-4 mg; KC1, 50 mg and NaCl, 50 mg.
244 RICHARDS AND CORBEY: ZYMOMONAS MOBILIS
Glutamic acid, lysine, hydroxyproline and
cysteine were added at a concentration of
60 mg per litre. Calcium D-pantothenate
and biotin were added to concentrations of
20 ^cg/ml.
Basal medium (minus amino acids and
vitamins) was dispensed into 1-oz McCartney
bottles and autoclaved at 121 °C for 15 min.
Stock amino acid and vitamin solutions were
sterilized by membrane nitration (Millipore;
0-2 /ttm pore size) and added ascptically to
sterile basal medium. The final volume of
medium per McCartney bottle was 25 ml.
Zymomonas cultures were washed once and
suspended in physiological saline, before
loop-inocula were transferred to denned
media. All such tubes were incubated
anaerobically at 30 °C.
Confirmation of C02 production.—Gases
evolved during growth were bubbled into a
saturated solution of calcium hydroxide.
Demonstration of motility.—This was
evaluated by the hanging-drop method.
Evaluation of beer-spoilage potential of
Zymomonas mobilis {beer isolate).—A total
count (haemocytometer) was performed upon
a 24-hour culture (in "initial isolation
medium") of this isolate. One ml of diluted
culture was then inoculated into 250 ml of
bottled pale ale (2-6 vol CO2) to achieve
infection rates of 1, 10, 100 and 1000 cells
per bottle. Bottles were then closed with
crowns previously sterilized by immersion in
methylated spirit (75% v/v). Inoculated
beers were stored at 28 CC for 14 days, then
opened and examined for signs of spoilage.
Quantitative estimation of sugars in "isola
tion medium".—Individual sugars were separ
ated by paper chromatography (Whatman
No. 3 paper; developing solvent, butanol
60: acetic acid 15: water 25), followed by
elution and individual estimation by the
an throne procedure.7
Quantitative estimation
of elhanol.—This
was performed on a Varian 1200 gas-liquid
[J. Inst. Brew.
chromatograph fitted with flame ionization
detector. A Carbowax 1540 column on
diatomite C-AW-DMCS (80-100 mesh) was
used at a temperature of 40°-100 °C pro
grammed at 2 °C per min. n-Butanol was
used as internal standard.
Culture supernatants for ethanol estima
tion were equilibrated at 25 °C for 20 min
prior to injection of head-space vapour into
the gas-liquid chromatograph.
Chemicals.—Wherever available, analytical
grade reagents were used.
Acknowledgement.—The authors wish to
thank the Directors of Bass Production Ltd.,
for permission to publish this paper.
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