World

Journal

of Microbiology

and Biotechnology

7, 490493

High-yield Jerusalem

C. Barthomeuf,

Fermentation conditions were optimized for the production of ethanol from Jerusalem artichoke with a strain of Sacc/taromyces cerevlsiae able to use high-concentration juice and undiluted pulp. Yields (95 to 125 g ethanol/i = 85 to 98% of the theoretical value) exceeded those obtained with strain of Kbyveromyces used classically. The authors are with the Laboratoire de Pharmacognosie et Biotechnoiogie, UFR Pharmacie, 28 Place Henri-Dunant, 63001 Clermont-Ferrand CtSdex, France. TBI: 73.60.80.00; Fax: 73.27.79.07. H. Pourrat is the corresponding author.

Jerusalem

490

Communications

World

Journal

of Oxford

of Microbiology

Ltd.

F. Regerat

from

and H. Pourrat

Current increases in oil prices have re-kindled interest in ethanol as an alternative fuel. Its production by biotechnological means is now classical but, for the costs to be kept low, biomass materials must be cheap and fermentative processes optimized. Among possible carbohydrate substrates for ethanol production, Jerusalem artichokes have numerous advantages. The sugar content of the tubers represents 80% of the dry matter and is made up exclusively of inulin, a fructose 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 of tubers/ha (Sachs et al. 1981; Duvnjac et al. 1982; Williams 1982). The plant grows well on poor-quality soils, requires minimal fertilizer additions, is able to resist many common pests and diseases, and grows in cold climates. It is a hardy perennial crop able to grow on land unsuitable for other purposes. 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

@ 1991 Rapid

ethanol production artichoke tubers

and Methods A rticbokes

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

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 by extraction or diffusion from potential of the strain but, for itself. Accordingly, subsequent

Jerusalem artichoke was studied on juice prepared tubers. These preliminary studies were to test the industrial use, the feedstock must be the tuber tests were carried out on pulp.

Fermentation oj Juice Tests in liquid media were carried out on juice containing increasing concentrations 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 Total sugars (0)

Juice

1. Ethanol

productlon

Fermentation time (h)

by S. cerevislaeY-481 Flnal ethanol concentration [g/l (% v/v)]

from

Conversion W)

Jerusalem

artichoke

Specific ethanol productlvlty (ofW (llnal fermentation time)

We

and pulp.

Specific

substrate uptake (g/kg wet fresh tubers)

fermentations

a4

42 (5.2)

99.1

0.583

70

72 75

60 (7.6) 110 (13.8) 124 (15.5)

98.4 98.3

0.833 1.527 1.663

101 a5 72

83

131 (16.4)

1.578

50

80

49 (6.1)

a7 89

85 (10.6) 91 (11.3)

80 87

0.612 0.977

89 93

85

1.022

91

72 72

120 220 250 280

97.1 91.1

Pulp fermentations 120 200 220

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 Tourliere (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.

TIME

Figure

1. Fermentation

(h)

of Jerusalem artichoke juice containing total sugars at 120 g/l (V), 220 g/l (m), 250 g/l (e) and 280 g/l (A).

492

World

Journa/

of Microbiology

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 it, eventually being released by ‘explosive disgorgement’. Vigorous mixing of the medium is thus necessary to ensure gas exchange. We used a mechanical system equipped with a bladed rotor of diameter 80% of that of the fermenter. To optimize yield, the operating parameters of the fermenter were systematically studied. Like

and Biotechnology,

Vol 7, 19%

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‘acdarom_ycescereuisiae Y-481 gives good results indicating that its use for industrial-scale production of ethanol deserves study.

References 24

72

TIME

96

(hl

Figure 2. Fermentation of Jerusalem artichoke pulp containing total sugars at 120 g/l (V), 200 g/l (0) and 220 g/l (W).

DE MINIAC, M. 1989 Contamination de fermentations levures du genre Brettanom_yces. Industries Alimentaires

alcooliques industrielles par les et .Agricoles (juillet-aolit) 559--

563.

Z., KOSARIC, N., KILZA, S. & HAYES, D. 1982 Production of alcohol from Jerusalem artichokes by yeast. Biotechnology and Bioengineering 24, 2297-2308. GIBBONS, W.R. & WESTBY, CA. 1984,-l bstracts of the *4 nnual Meeting of the -4 merican .SocieII DUVNJAC,

of MicrobioLogy

3, p. 189.

solid-phase fermentation of Jerusalem artichoke tubers. Journal oy Fermentation and Bioengineering 67, 25S-265. GLIIRAUD, J.P., DIX, T. & GAJ.ZY, I?. 1981 Selection of yeast strains for ethanol production from inulin. Folia Microbiologica 24, 147-150. MARGARJTIS, A. & BAJPAI, P. 1982 Ethanol production from Jerusalem artichoke tubers using Kluyyljeromyces marxianus and J‘accbarom_yces rosei. Biotechnology and Bioengineering 24, GIBBONS,

W.R. 1989 Batch and continuous

941-953.

POURRAT, H., BARTHOMEUF, C., REGERAT, F. & CARNAT, A.P. 1983 Production d’kthanol i partir de topinambours par des souches de Saccharomyces cherensiensis et Sacrharon/yces beticus. Industries ,4limentaires et Agricoles (mai) 181-190. ROSA, M.F., CORREIA, I. Sr\ & NOVAJS, J.M. 1987 Production of ethanol at high temperature in the fermentation of Jerusalem artichoke juice and a simple medium by K/~veromyces

marxianus.

Biotechnology

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9, 441-444.

SACEIS,R.M., Low, C.B., VASAVADA, A., SUJ,L~, M.J., WJJ.LIAI\IS, L.A. & ZIOBRO, G.C. 1981 Fuel alcohol from Jerusalem artichokes. Calfornia Agricu/ture 35, 4-G. TOURJ.JERE, R. 1985 L’kthanol de fermentation. Ses possibilitt?s, ses limitcs. Industries Alimentaires et Agricoles (juillet-abut), 749753. TREVEJ.YAN, W.E. & HARRISON, J.S. 1956 Studies on yeast metabolism. Biochemical Journal 63, 2S28.

WESTBY, C.A. & GIBBONS, W.R. 1982 Farm-scale production of fuel ethanol and wet grain from corn in a batch process. Biotechnology and Bioengineering 24, 1681-1699. WKLJA~~S, L.A. 1982 Proceedings of the Fifth International ~4lcohol Fuel Jjmposium 1, 55-61. WILLIAMS, L.A. & ZIOBRO, G. 1982 Processing and fermentation of Jerusalem artichokes for ethanol production. Biotechnology Letters, 4, 4550. ZIOBRO, G.C. & WII.LIAIMS, L.A. 1983 Pilot scale fermentation of Jerusalem artichoke tuber pulp mashes. Developments in Industrial hlicrobiology, 24, 22&229. (Receiljed

12 November

1990;

rezlisfd

World Journal

20 February

of Microbiology

1991;

accepted

and Biotechnology,

23 February

Vol 7, 1991

1991)

493