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Appl Microbiol Biotechnol (1984) 19:252-255 Applied

o. Microbiology
Biotechnology
© Springer-Verlag 1984

Formation o f levan and sorbitol from sucrose by Zymomonas mobilis


Liisa Viikari
VTI', Biotechnical Laboratory, Tietotie 2, SF-02150 Espoo 15, Finland

Summary. Ethanol yields produced by Zymomonas In the present work, the formation of levan and
strains from sucrose are significantly lower than from other byproducts was studied.
glucose or fructose. The low yield is a consequence of
the formation of both levan and sorbitol as by-prod-
ucts. Most of the levan is in a non-precipitable form, Materials and methods
indicating low molecular weight. Formation of sor-
bitoi was observed with both the Zymomonas strains Strains. The organisms used were Zymomonas mobilis
studied. The measured amounts of levan and sorbitol VTT-E-78082, originally isolated as a beer contaminant and
Zymomonas mobilis CP 4.
were 8% and 11% of the original sucrose content,
respectively. Media. The strains were maintained on media containing 10 g - I- l
Bacto peptone, l(I g , 1-I yeast extract and 20 g • 1-~ glucose. The
cultures were renewed every 3 weeks.
The fermentation medium contained l g . I -j KH2POa,
l g - I -I (NH4)2SO4, l g . I -I M g S O 4 - 7 H 2 0 , 2 . 5 g . I -I yeast
Introduction extract and 150g, I-~ sucrose. Th.e inocula were grown on the
same medium with the exception of the sucrose concentratkm,
Zymomonas bacteria are known as efficient ethanol which was 50 g . 1-l.
producers on glucose-based media (Rogers 1982). Ethanolproduction. The fermentations were carried out in a New
The yields from fructose are also high, 94% of Brunswick Magnaferm fermenter, overall volume of 7 1, containing
theoretical (Lee et al. 1981), but the yields from 4.5 1 of medium. The inocula (0.5 I) were cultivated for 1 day at
sucrose may be as low as 70% of theoretical. The 28°C in conical flasks without agitation. The pH of the fermen-
tation was maintained at 5.1,) by addition of 5% NH4OH. The
yield decreases with increasing sucrose concentration
temperature was 30° C and the stirring speed 200 rpm. The original
(Lyness and Doelle 1981). dissolved oxygen was not flushed by nitrogen. After some carbon
The reduced ethanol yield has been attributed to dioxide evolution the fermentation was considered to be anae-
levan formation (Dawes et al. 1966). In a batch robic.
fermentation on 25% sucrose medium the amount of Analyses. T h e samples were centrifuged at 7,000 rpm for 5 min at
levan was only 2% of the initial sucrose, whereas in a 0 ° C and the supernatants were boiled immediately for 5 min.
continuous culture the levan accounted for 10% of Ethanol was analysed by determining the specific gravity of
the original 15% sucrose. No levan was detected in the distilled samples.
Sugars were determined by high-performance liquid chroma-
continuous culture when the fructose concentration tography (Optilab 931 HSRt) using an Aminex 87-C c o l u m n
was limiting (Lee et al. 1981). No other major (Bio-Rad), temperature 85° C, with water as eluent (0.8 ml/h) and
by-products have been reported. a refractometer (Optilab Multiref 9(12 C) as detector.
The mechanism of sucrose hydrolysis and levan Levan was precipitated by 75% ethanol. CaCI 2 was added to
facilitate the precipitation and the pH was adjusted to 10 with 0. t
formation is not clear. Two distinct enzymes may be
M K O H (Avigad 1965). The precipitate was centrifuged at 4,500
involved in the sucrose hydrolysis: levansucrase (EC rpm for 10 min and washed with 75% ethanol. The precipitate was
2.4.1.10) and invertase (EC 3.2.1.26). According to resuspended and hydrolysed in 0.1 M HCI for 1 h at 11,)(}° C. The
Blackbeard and Doelle (1983) the sucrose hydrolysis content of levan was analysed as fructose by the DNS method or by
is independent of glucose utilization. The rate of HPLC.
Total levan was analysed without precipitation after hydrolysis
ethanol formation is limited by the catabolism of for I h at }00°C in (I.l M HCI.
glucose and fructose rather than by the hydrolysis of Sorbitol was analysed by IlPLC an d bY a sorbitol dehydro-
sucrose. genase method in which the reduced N A D H is removed by a
L. Viikari: Formation of tevan and sorbitol from sucrose by Zymomo*ms mobitis 253

subsequent reaction with iodonitrotetrazolium chloride (Boehrm- fructose. In addition, the fructose concentration
ger. M a n n h e i m ) . increased because of hydrolysis of levan. The total
Acetic acid was analysed by gas chromatography and lactic
amount of levan was corrected for fructose originat-
acids as N A D H after addition of L- and D-lactate dehydrogenase
(Boehringer, Mannheim). ing from the hydrolysed sucrose. The amount of
For the determination of cell concentrations, centrifuged cells levan, assuming a complete hydrolysis, was equiva-
were resuspended in water, the turbidity was measured and the cell lent to almost 10% of the original sucrose. The
concentrations were calculated using a calibration curve. increase in the glucose concentration indicated that

R e s u l t s and d i s c u s s i o n

Ethanol production 120

Ethanol production by the strain VTT-E-78082 on 100


15% sucrose is presented in Fig. 1. The sucrose was
hydrolysed rapidly, with consequent accumulation of 80 40 "~
fructose and glucose. During the first 6 h of fermen-
tation the concentrations of fructose and glucose 60 aoig
remained constant because the hydrolysis of sucrose o)
7__
was balanced by the production of ethanol and levan.
After 26 h the concentration of sucrose had decreased
} /..--- • ,r 20 o
,n

O3 ,'a o
to 1 g . 1-1 and the monomeric sugars were con- 2 10

sumed. T h e ethanol yield was 75% of theoretical. I


~ " . z ---g_.,
This yield is similar to those obtained with other 10 20 30 40 50
strains (Lyness and Docile 1981; Rogers et al. Fermentation time (h)

1982). Fig. 1. Fermentation of sucrose to ethanol by Z. mobilis strain


VTT-E-78082. Sucrose /x A; glucose [] E]; fructose
• • ; ethanol 0 0 ; cells • V; precipitated
levan • Q
Levan formation
Levan formation was observed immediately after
inoculation as cloudiness and quantified by precipi- Table 1. Distribution of carbon during sucrose fermentation to
cells, ethanol, sugars, and precipitated levan (all expressed as
tation (Fig. 1). As expected the amount of precipi- glucose), Z. mobilis strain VTI'-E-78082. Theoretical yield factors
table levan reached its maximum at the end of sucrose used were I).51 for ethanol and 11.5 for cells
hydrolysis. After this the concentration of precipi . . . . . .
table levan decreased slightly. Fermen- Cells Ethanol Sugars Levan Total
ration ( g , l -t) (g-l-t) ( g - I j) ( g . l -I) ( g . l -I)
The carbon balance during the fermentation, time
expressed as glucose, is presented in Table 1. After (h)
10 h fermentation the sum of the products decreased,
indicating that other products in addition to ethanol 0 0.1 5.0 161 0.8 167
1.5 0.1 8.(1 t57 0.8 166
and precipitated levan had been formed. It was 3 0.2 8.0 157 1.(1 167
expected that part of the levan could be in a 6 0.5 ltl.tl 156 1.(1 167
non-precipitable form. To verify this the samples m 1.3 24.0 128 1.7 155
were hydrolysed with mild acid and the sugars were 22 3.5 120 4.2 2,7 130
analysed (Table 2), All the sucrose was hydrolyscd, 34 3.5 t26 1.8 2.4 134
48 3.5 121 1.6 2.5 129
causing an increase in the amount of glucose and

Table 2. Sugar concentrations before and after hydrolysis of the samples and the calculated total a m o u n t of levan (as fructose)

Fermentation Before hydrolysis After hydrolysis Total


time Lcvan
(h) Sucrose Glucose Fructose Sucrose Glucose Fructose ( g . I - t)
( g ' l -I ) ( g - I l) ( g ' l -I) ( g ' l 1) ( g - I I) (g.l-l)

22 2.9 1.2 (I.0 (I.0 4. t 15.9 14.4


26 1.0 1.0 0.0 0.0 3.5 13.1 12.6
34 0.8 0.8 0.0 11.0 2.8 12.6 12.2
48 1).8 11.6 ..... 0.0 0.0 3. I 12.6 12.2
254 L. Viikari: Formation of levan and sorbitot from sucrose by Zymornonas mobilis

the hydrolysed fructose polymers may also have


contained glucose. The precipitable levan contained
only fructose. The demonstration of the non-precipi-
table levan in the early phase of the fermentation was
~2:~::~-polymer s hampered by the high original sugar concentra-
sucrose tion.
glucose

ethanol
Sorbitol formation

sorbito, The detection of the non-precipitable levan did not


explain the carbon balance completely; about 10% of
the original carbon was still missing. The HPLC
analysis of the samples revealed a new by-product
(Fig. 2), which was identified as sorbitol. The for-
Fig. 2. HPLC analysis of the end-products of the sucrose
fermentation (48 h), Z. mobilis strain VTT-E-78082
mation of sorbitol seemed to be connected with
ethanol production, reaching its maximum, 17 g • 1-1,
simultaneously with the highest ethanol concentra-
tion (Fig. 3). Theoretically, sorbitol and ethanol are
alternative products leading to oxidation of the
20 2.0 reduced coenzymes. Whether sorbitol formation is
connected with the regeneration of NADH or
o
NADPH (reduction of glucose or fructose) will be
15 1.5
clarified in further experiments.
"7
L_ To test whether sorbitol production is a strain-de-
g g pendent phenomenon, a comparative fermentation
"~10 1.0 was carried out with the Zymomonas strain CP 4.
o
This strain also produced sorbitol (Fig. 4). The
O9
ethanol concentration was slightly higher and the
5 0.5 sorbitol concentration slightly lower than with the
strain VTT-E-78082.
t
10 20 30 40 50
Fermentation time (hi
Acid production
Fig. 3. Formation of sorbitol and acids during sucrose fermentation Small amounts of acetic and lactic acids were
by Z. mobilis strain VTI'-E-78082. Sorbitol © .0; acetic acid
• • ; lactic acids (D- and t.-) ~ ........ A
produced (Fig. 3). Both isomers of lactic acid (D- and
L-) were formed in approximately equal amounts.

Distribution of substrate
~ O
60 The distribution of the substrate to different products
i was calculated Using theoretical yield factors (Ta-
en 50 ble 3). In addition to ethanol, sorbitol accounted for

40
Table 3. Distribution of substrate to different products. Theore-
--- 30 tical yield factors from glucose or fructose used were 0.5 for cells,
o 0.51 for ethanol, 1.0 for sorbitol, levan, and lactic acids and 0.7 for
acetic acid
2(3
Product % of substrate
10
Cells 2
Ethanol 75
10 20 3Jo 40
Sorbitol 11
Fermentation time (hi
Levans 8
Fig. 4. Production of ethanol and sorbitol by Z. mobilis strain CP 4 Acids 1
on 15~ sucrose medium. Ethanol (3- -O; sorbitol Total 97
L. Viikari: Formation of levan and sorbitol from sucrusc by Zymomorms mobihs 255

an a p p r e c i a b l e part of the original substrate. T h e Jakeman and Miss Birgitta Sipponen is gratefully acknowl-
total a m o u n t of levan was also high, but lower than edged.
the a m o u n t of sorbitol. It is therefore a p p a r e n t that
Zyrnornonas strains with r e d u c e d levan and sorbitol
p r o d u c t i o n are necessary for increased e t h a n o l yields References
from s u c r o s e .
Avigad G (1965) Levan. In: Whistler RL, BeMiller JN, Wolfrom
ML (eds) Methods in carbohydrate chemistry, vol V, Aca-
demic Press, New York London, pp 161-165
Conclusions Blackbeard JR, Doelle HW (t983) The effect of glucose on the
sucrose hydrolysing activity of Zyrnomonas mobilis, Eur J Appl
T h e e t h a n o l yield from the f e r m e n t a t i o n of sucrose by Microbiol Biotechnol 17:261-263
Dawes EA, Ribbons DW, Rees DA (1966) Sucrose utilization by
Z y m o m o n a s strains is low b e c a u s e of f o r m a t i o n of Zymomonas mobilis: Formation of a levan. Biochem J
levan a n d sorbitol. T h e f o r m e r is c o n n e c t e d with the 98:804-812
hydrolysis of sucrose and the l a t t e r with e t h a n o l Lee KJ, Skotnicki ML, Tribe DE, Rogers PL (1981) The kinetics
f e r m e n t a t i o n . Most of the levan p r o d u c e d is in a of ethanol production by Zymomonas mobilis on fructose and
sucrose media. Biotechnol Lett 3:207-212
n o n - p r e c i p i t a b l e form. The glucose p r e s e n t in the
Lyness E, Doelte HW (1981) Fermentation pattern of sucrose to
h y d r o l y s a t e s o f the p o l y m e r s indicates the possible ethanol conversion by Zymomonas mobilis. Biotechnol Bioeng
existence of h e t e r o p o l y m e r s . Sorbitol is p r o b a b l y 23 : 1449-1460
f o r m e d b e c a u s e of i m b a l a n c e in the N A D H - or Rogers PL, Lee KJ, Skotnicki ML, Tribe DE (1982) Ethanol
N A D P H levels in the early phase of the f e r m e n t a - production by Zymomonas mobilis. Adv Biochem Eng
23 : 37-84
tion. T h i s h y p o t h e s i s , however, needs f u r t h e r clari-
fication.

Acknowledgements. The HPLC analyses were carried out by Mrs.


Raija Gisler, M.Sc. The technical assistance of Miss Sarah ReceivedOctober 31, 1983

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