World Journal of Microbiology and Biotechnology 7, 490493

High-yield ethanol production from

Jerusalem artichoke tubers

C. Barthomeuf, F. Regerat and H. Pourrat

Fermentation conditions were optimized for Current increases in oil prices have re-kindled interest in ethanol as an alternative
the production of ethanol from Jerusalem fuel. Its production by biotechnological means is now classical but, for the costs
artichoke with a strain of Sacc/taromyces
to be kept low, biomass materials must be cheap and fermentative processes
cerevlsiae able to use high-concentration
juice and undiluted pulp. Yields (95 to 125 g optimized. Among possible carbohydrate substrates for ethanol production,
ethanol/i = 85 to 98% of the theoretical Jerusalem artichokes have numerous advantages. The sugar content of the tubers
value) exceeded those obtained with strain represents 80% of the dry matter and is made up exclusively of inulin, a fructose
of Kbyveromyces used classically. polymer containing a terminal glucose. It is a raw material available in large
quantities at fairly low prices. The crop yield is on average 27 to 51 metric tonnes
The authors are with the Laboratoire de
Pharmacognosie et Biotechnoiogie, UFR of tubers/ha (Sachs et al. 1981; Duvnjac et al. 1982; Williams 1982). The plant
Pharmacie, 28 Place Henri-Dunant, 63001 grows well on poor-quality soils, requires minimal fertilizer additions, is able to
Clermont-Ferrand CtSdex, France. TBI: resist many common pests and diseases, and grows in cold climates. It is a hardy
73.60.80.00; Fax: 73.27.79.07. H. Pourrat is perennial crop able to grow on land unsuitable for other purposes.
the corresponding author.
The proper choice of micro-organism for bioconversion of Jerusalem artichoke
sugars to ethanol is vital. Important yeast characteristics include high ethanol
productivity, rapid growth and fermentation rates and relatively high ethanol
tolerance. Use of a micro-organism with inulinase activity, thus able directly to
metabolize inulin, is desirable because it would eliminate preliminary acidic or
enzymatic hydrolysis and thereby reduce processing costs. Two main types of
micro-organism are used; bacteria of the genus Zymomonas and, more often, yeasts
of the genus Kltiyveromyces, especially K. marxianus and K. fragilir. Traditional
brewing yeasts (Saccharomyces cerevisiue), used by distillers for their high ethanol
productivity, are for the most part unable directly to ferment inulin (Guiraud et
al. 1981; Ziobro & Williams 1983). This necessitated prior hyrolysis of the inulin
to simple sugars, either by acido-thermic or enzymatic processes. However,
previous work has shown that a strain from our laboratory collection, Saccbaromyces
cerevisiue Y-481, was able to ferment inulin directly (Pourrat et al. 1983). By
selection, we obtained this particularly productive strain, S. cerezjisiue Y-481. This
was tested in media containing high concentrations of inulin.

Materials and Methods

Jerusalem A rticbokes
Tubers of Jerusalem artichoke (Helianthw ttrberosq var. Mammoth French White)
@ 1991 Rapid Communications of Oxford Ltd. were washed, allowed to air dry and sealed in paper sacks for storage at 4 to 5°C.

490 World Journal of Microbiology and Biotechnology, Vol 7, 1991

 Ethanol from Jerusalem artichokes

To prepare pulp, the tubers were sliced with a blade homogenizer and deep-frozen.
Juices (280 g/l total sugars) were prepared for inulin extraction by two successive
steepings of fresh pulp with water for 1 h at 70°C each followed by vacuum
filtration and storage of the extract at -20°C. Diluted juices were obtained by
dilution of the extract with de-ionized water.

Micro-organism and Growth

The strain of Saccharomyces cerevisiae Y-481 from sherry (‘Zerez’) from our
laboratory collection was used throughout. 40 mg of the lyophilized yeast was
incubated on dilute Jerusalem artichoke juice (100 g/l total sugars) containing
mineral salts [KH,PO, (1 g/l), (NH&SO, (1 g/l), MgS04 (0.3 g/l), NaCl (0.4 g/l),
FeCl, (0.004 g/l), MnSO, (0.007 g/l)] p reviously sterilized at 110°C for 30 min. It
was incubated with periodic agitation at 30°C for 24 h and then inoculated at
either 4% (v/v) for juice fermentation or at 10% (v/v) for pulp fermentation. For
all trials the inoculum contained 0.2 to 1.0 x IO’ cells/ml.

Fermentation
Juice fermentations were made in a Biolafitte 2 litre fermenter. The trials were
carried out at 30°C on 1.3 1 of Jerusalem artichoke juice containing mineral salts
(same composition/concentration as per inocula preparation). The pH was then
adjusted with 18 M HaSO,. For pulp fermentations, 1 kg artichoke pulp was placed
in 2 1 stainless steel containers for fermentation. As trials with or without addition
of mineral salts gave similar results, fermentations with pulp were made without
mineral salts. The pH of the pulp was adjusted to 3.2 to 3.3, and it was then
sterilized at 110°C for 30 min and incubated.

-4 nabtical Methods
Samples were measured for ethanol, total carbohydrates and yeast population.
Ethanol was measured by gas chromatography as described previously (Pourrat
et al. 1983), using a 1.2 m PORAPAK type QSO to 100 mesh column. Total sugars
were assayed by the anthrone method (Trevelyan & Harrison 1956). The yeast
cell population in juice samples was estimated by counting using a Malassez cell,
but in fermenting pulp samples was determined by the plate count method (Westby
& Gibbons 1982). Fermentation was also monitored by thin layer chromatography
on silica gel using chloroform/methanol/water (61: 32: 7, by vol.) as the elution
solvent. Sugars were revealed by spraying anisaldehyde solution.

Results and Discussion

The production of ethanol from Jerusalem artichoke was studied on juice prepared
by extraction or diffusion from tubers. These preliminary studies were to test the
potential of the strain but, for industrial use, the feedstock must be the tuber
itself. Accordingly, subsequent tests were carried out on pulp.

Fermentation oj Juice
Tests in liquid media were carried out on juice containing increasing concentra-
tions of inulin (84 to 280 g/l) after determining optimal operating conditions.
The most important parameters are aeration and stirring. Previous assays have
shown that aeration must be discontinuous. A systematic study was carried out
with aeration rates of 0.17, 0.3, 0.34, 0.5 and 0.85 vol/vol/min for 15 min to 2 h
at 15 min increments one, two or three times a day. The higher ethanol yields
and specific rate of production were obtained with aeration at flow rate of 0.34
vol/vol/min for 50 min every 8 h the first day, then once a day every 12 h, with
a stirring rate of about 160 rev/min. Any departure from this value either way
caused a large drop in yield.

World Journal of Microbiology and Biotechnology, Vol 7, 1991 491

C. Barthomeuf

Table 1. Ethanol productlon by S. cerevislaeY-481 from Jerusalem artichoke We and pulp.

Total Fermentation Flnal ethanol Conversion Specific ethanol Specific substrate

sugars time concentration W) productlvlty uptake
(0) (h) [g/l (% v/v)] (ofW (g/kg wet fresh
(llnal tubers)
fermentation
time)

Juice fermentations
a4 72 42 (5.2) 99.1 0.583 70
120 72 60 (7.6) 98.4 0.833 101
220 72 110 (13.8) 98.3 1.527 a5
250 75 124 (15.5) 97.1 1.663 72
280 83 131 (16.4) 91.1 1.578 50

Pulp fermentations
120 80 49 (6.1) 80 0.612 89
200 a7 85 (10.6) 87 0.977 93
220 89 91 (11.3) 85 1.022 91

The optimal pH for ethanol production was 4.5 but since values between 3.2
and 4.5 give quite similar results, the starting pH was set at 3.4 to 3.5 without
regulation. Under these conditions, cell growth rate was maximal. Maximum cell
density (2.08 x 10’ cells/ml) was reached in 17 to 19 h. According to Gibbons
(1989), bacterial contamination was avoided and acidity stayed low enough to
reduce the risk of secondary contamination by other yeasts (De Miniac, 1989).
The inoculum for the fermentation had to be grown on artichoke juice; increasing
the size of inoculum from 4% (v/v) to 12% (v/v) did not significantly change the
specific ethanol productivity rate.
Under the optimal conditions thus established, all the sugars were consumed,
and the conversion was close to 98% of the theoretical yield for concentrations
less than, or equal to, 250 g/l (Table 1). This is markedly better than the yields
of 90% obtained with Kluyveromyces (Margaritis & Bajpai 1982; Rosa et al. 1987)
with practically identical specific ethanol productivity rates. According to Tour-
liere (1985), for a strain to be useful industrially, it has to produce ethanol to at
least 8.5 to 10% (v/v) and with a yield of at least 62 1 ethanol per 100 kg sugar.
S. cerevisiae Y-481 meets both criteria: with a juice containing 220 to 250 g total
sugars/l, it produces an ethanol concentration of 13.8 to 16% (v/v) with a yield
of 58 to 62 1 ethanol per 100 kg sugar. However, as the sugar concentration
increased, the specific substrate uptake, expressed as 1 of ethanol per tonne of
fresh raw material, decreased.

Fermentation of Pulp
Three types of trial were performed: with undiluted pulp, and pulp diluted with 10
and 30% (v/v) in water. According to Ziobro and Williams (Williams & Ziobro
1982; Williams 1982; Ziobro & Williams 1983), when the dilution of the pulp is
below 50%, it is not advisable to use a conventional system of stirring. The
high viscosity of the medium inhibits the normal circulating patterns in the tanks,
which then reduces heat dissipation and prevents adequate temperature control.
The lack of pulp homogeneity also causes a floating cap of solids to form in the
fermenter. Carbon dioxide compresses this cap and becomes trapped underneath
TIME (h)
it, eventually being released by ‘explosive disgorgement’. Vigorous mixing of the
Figure 1. Fermentation of Jerusalem arti-
medium is thus necessary to ensure gas exchange. We used a mechanical system
choke juice containing total sugars at 120 g/l
(V), 220 g/l (m), 250 g/l (e) and 280 g/l equipped with a bladed rotor of diameter 80% of that of the fermenter. To optimize
(A). yield, the operating parameters of the fermenter were systematically studied. Like

492 World Journa/ of Microbiology and Biotechnology, Vol 7, 19%


24
Figure 2. Fermentation of Jerusalem
choke pulp containing total sugars at 120 g/l
(V), 200 g/l (0) and 220 g/l (W).
Ethanol from Jerusalem artichokes
Gibbons (1989), we found prior thermal treatment useful to facilitate diffusibility
of polyfructosans and, therefore, their assimilation by the yeast. The medium,
adjusted to pH 3.2 to 3.5, was sterilized for 30 min at 110°C. The best results
were obtained with undiluted or slightly diluted pulp with a stirring rate of 300
rev/min and an aeration rate of 0.34 vol/vol/min applied every 8 h during the first
24 h. The other parameters were the same as for the juice.
The fermentation profile of the Jerusalem artichoke juice and pulp are given
in Figures 1 and 2. The strain was not inhibited when ethanol was less than 95
to 110 g/l.
With pulp, the maximum cell density (3.2 to 3.0 x IO’ cells/g of fresh pulp)
was obtained in 25 to 27 h (instead of 17 to 19 h with the juice), resulting in a
lengthening of the duration of the fermentation (87 to 89 h instead of 72 to 75 h)
and hence a slowing of the conversion. However, the conversion was improved
(91 to 93 g instead of 72 to 85 g ethanol/kg fresh tubers), with 95 to 97% of the
sugars present being consumed with a 85 to 87% conversion into ethanol. The
ethanol yield was thus particularly high (85 to 91%).
These results are an improvement upon those of Gibbons (41 to 53%) (Gibbons
1989), Ziobro and Williams (50 to 75%) (Ziobro & Williams 1983) and Williams
and Ziobro (72 to 83%) (Williams & Ziobro 1982) with K. fTagi/is. They
correspond to an ethanol yield of 110 to 112 I/t, i.e. at the maximum feasible level
(100 to 110 l/t) according to the revised estimate of Gibbons and Westby (1984).
In summary, the fermentation of the inulin of Jerusalem artichokes by Xa‘acdar-
om_ycescereuisiae Y-481 gives good results indicating that its use for industrial-scale
production of ethanol deserves study.
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World Journal of Microbiology and Biotechnology, Vol 7, 1991 493