Biomass 17 (1988) 13-19

l)-Xylose Fermentation by Fusarium oxysporum Strains

E. G. Wene & A. A. A n t o n o p o u l o s *

Energy and Environmental Systems Division, Argonne National Laboratory, Argonne, Illinois 60439, USA

(Received 2 May 1988; revised version received 27 June 1988; accepted 29 June 1988)

ABS TRA CT

Fusarium species isolated from natural substrates were screened for the ability to ferment o-xylose. Almost all of the isolates produced some ethanol; the productivity on 1% o-xylose medium was from 0"4 to 4"4 g liter - ~ ethanol Using an inoculum volume of 10%, E oxysporum ANL 22-760 semi-aerobically fermented 2% D-xylose to 8"2 g liter- 1 ethanol in 72 h, which is a theoretical yield of 85%. Completely recycling the fungal biomass from 2% o-xylose fermentations to another 2% o-xylose fermen- tation reduced the fermentation time to 48 h with equal yields. This study shows that control of aeration and inoculum size are the most important factors for ethanol productivity in o-xylose fermentations.

Key words: Fusarium oxysporum, D-xylose, ethanol, fermentation.

INTRODUCTION

Interest in ethanol production from renewable resources and waste organics has accelerated, particularly since the oil short-fall in 1973. The recently expanded use of ethanol for blending into automotive gasoline is a major cause of this interest. A major source of ethanol is the fermen- tation of carbohydrates, and future feedstocks for these fermentations could be derived from plant biomass. Plants contain large amounts of cellulose and hemicellulose, which when thermochemically or bio- logically treated, yield o-glucose, o-xylose, and other sugars which can be fermented to ethanol. Although yeasts are efficient fermenters of D- glucose, those which convert o-xylose have fermentations rates which

13 *To whom correspondence should be addressed.

Biomass 0144-4565/88/S03.50 -- © 1988 Elsevier Science Publishers Ltd, England. Printed in Great Britain

14 E. G. Wene, A. A. Antonopoulos

are slow compared with D-glucose fermentation) Since xylose is produced in quantity from plant hemicelluloses, organisms which can ferment o-xylose to ethanol rapidly and in good yield need to be identi- fied.

Fusarium strains have been reported to be efficient fermenters of D- xylose. 2 The purpose of this study was to identify which Fusarium strains ferment D-xylose and to determine the conditions needed to produce high yields of ethanol.

MATERIALS AND METHODS

Isolation of organisms

Fusarium strains were isolated from soils, leaf litter, and plant debris using an isolation medium modified from that used by Mai-tin 3 and Papavizas. 4 The medium contained (in g liter-~): dextrose, 10.0; peptone, 5.0; pentachloronitrobenzene, 0.5; neomycin, 0.01; tetracyc- line, 0.02; rose bengal, 0.05; KH2PO4, 0.50; KNO3, 3.0; MgSO 4 . 7H20, 0.50; KCI, 0.50; FeSO4.7H20 , 0"10; (NH4)2504, 1.4; and agar, 15.0. Typical Fusarium colonies were transferred to potato-dextrose-agar (PDA) plates for identification and to PDA slants for maintenance and storage.

Fusarium species identification was based on the morphology of the macroconidia, number of cells per macroconidium, presence and arrangement of chlamydospores, morphology and arrangement of microconidia, and on the pigmentation in water agar medium.

Screening for xylose fermentation

Fusarium isolates were tested for their ability to ferment D-xylose to ethanol in 50-ml flasks with 25 ml of 1% o-xylose medium or in 20 x 150 mm test tubes with 10 ml of 1% D-xylose medium. The tubes or flasks were incubated in a shaking water bath at 30°C for 2-5 days.

Fermentation conditions

The composition of the D-xylose fermentation medium (in g liter- 1) was o-xylose (Sigma Chemical Co., St. Louis, Missouri, USA) 10-100; yeast extract (Difco, Detroit, Michigan, USA) 1-0; NHaNO 3 or (NH,)2SO4, 2.0; KH2PO4, 2.0; CaC12 . 5H20, 0.5; MgSO4.TH20, 0.2. Sugars were autoclaved separately and combined with other ingredients after steri-

D-xylose fermentation by Fusarium oxysporum strains 15

lization. The pH of the fermenting medium was adjusted to 5.2 with HC1 after sterilization and was uncontrolled during the fermentation. All fermentation was at 30°C.

Fermentation was carried out anaerobically, semi-aerobically, and with controlled aeration. Anaerobic fermentations were in 250-ml sidearm flasks with tubing connected to a water trap. The headspace was purged with nitrogen gas for 10 rain following inoculation. Agitation was provided by a Teflon stirring bar or a shaking water bath at 80 rpm. Semi-aerobic fermentations used 50-100 ml medium in a 250-ml Erlemneyer in a shaking water bath at 100 rpm. Consecutive controlled aeration fermentations were done in a 500-ml Bioflo C-30 (New Brunswick Scientific, Edison, New Jersey, USA) fermentor, with filtered air at a flow rate of 0.1 v/v min-~ bubbled under the bottom impeller (125 rpm). After 48 h of fermentation, the culture medium was asepti- cally removed and centrifuged at 1000 g. The pellet was returned to the fermentor and fresh 2% D-xylose medium was added. This procedure was repeated four times.

Inoculum for fermentations

Fusarium inoculum for shake flask fermentations was grown for 3 days in 250-ml shake flasks, in 100 ml of fermentation medium with 1% i> xylose. For some experiments the culture broth was centrifuged at 1000 g and the pellet was used for inoculum.

Analytical procedures

Ethanol and acetic acid were determined with a Varian 3700 (Varian Instruments Division, Palo Alto, California, USA), or a Gow-Mac 750-P (Gow-Mac, Bridgewater, New Jersey, USA)gas chromatograph. Ethanol quantities were determined with a glass colunm (6 mm × 160 cm) packed with Porapak Q, a flame ionization detector at 200°C, column tempera- ture 170°C, injection port temperature 180°C, and helium carrier gas flow at 45 ml min-~. Isopropanol was used as an internal standard. Acetic acid determinations were as above, except for a column packed with 100/120 mesh Chromosorb WAW with 15% SP-1220 and 1% H3PO4 (Supelco Co., Bellefonte, Pennsylvania, USA). The temperature of the column started at 100°C for 2 min then rose at 10°C per rain to 140°C. The injection port was held at 150°C. Samples were acidified to pH 2-0 with H2SO 4 prior to injection.

Samples for cell dry weight determinations were prepared by filtering 20 ml of fermentation broth through a preweighed Metricel 0-45-ktm

16 E. G. Wene, A. A. Antonopoulos

filter (Gelman Sciences, Inc., Ann Arbor, Michigan, USA) and drying at 80°C overnight.

RESULTS

The most commonly isolated Fusarium species (in descending order) were F. oxysporum, F. solani, F. tricinctum, and F. rnoniliforme. Over 2000 Fusarium isolates were tested for ethanol production on 1% D- xylose medium. Almost all of the isolates produced some ethanol; the productivity ranging from 0.4 to 4.4 g liter- 1 of ethanol.

The best ethanol-producing isolates from initial screenings were grown on 100 ml of 1% D-xylose medium in 250-ml flasks in a shaking water bath. The inoculum was 10 ml of 3- to 5-day-old culture. Three of the isolates produced from 4.1 to 4.4 g liter-1 ethanol in 72 h. These screenings were essentially semi-aerobic fermentations because the headspace above the media contained oxygen. Comparisons of ethanol and cell mass production by the Fusarium oxysporum ANL 22-760 isolate under semi-aerobic and anaerobic conditions using 10% v/v inoculum showed more ethanol was produced under semi-aerobic cond- itions in 72 h, and ethanol was consumed under semi-aerobic conditions but not under anaerobic conditions (Fig. 1). Not shown in Fig. 1 is the production of acetic acid, up to 0.5 g liter-l under semi-aerobic cond- itions, but none was found under anaerobic conditions.

Comparisons of ethanol production using inoculum volumes of 10% v/v and the pellet from 100 ml of centrifuged F. oxysporum culture

Fig. I.

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Anaerobic and semi-aerobic batch fermentation of 1% D-xylose by Fusarium oxysporum ANL 22-760 strain.

o-xylose ferrnentation by Fusarium oxysporum strains 17

showed that increasing the inoculum size reduced fermentation times (Figs 2 and 3). Fermentations of 2% D-xylose medium with 10% v/v inoculum were completed in 72 h, compared to 48 h for the pellet inoculum. Seven days were required for fermentation of 5% D-xylose medium with 10% inoculum, compared to 5 days for pelletized inoculum. There was little difference in fermentation time for 10% D- xylose medium using larger inoculum volume.

Consecutive controlled-aeration fermentations with centrifuged and recycled inoculum produced consistent ethanol yields. Within 48 h of recycling the fungal biomass and adding fresh medium, ethanol concentrations reached 8.1, 8-0, and 8-1 g liter- 1 in consecutive fermen- tations (Fig. 4). Aeration rates higher than 0" 1 v/v min- 1 reduced ethanol accumulation and increased cell mass production.

Fig. 2.

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Ethanol production from D-xylose fermentation by Fusarium oxysporum.

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Ethanol production from D-xyiose fermentation by Fusarium oxysporum ANL 22-760, using recycled inoculum.

18 E. G. Wene, A. A. Antonopoulos

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Fig. 4. Ethanol production from consecutive fermentation of 2% o-xylose by Fusarium oxysporum ANL 22-760. At the * point of fermentation, contents of the 500 ml-fermen- tor were removed, centrifuged, and the pellet was returned to the fermentor along with

2% o-xylose.

DISCUSSION

Selected Fusarium oxysporum strains were able to ferment D-xylose to ethanol with high yields; the maximum ethanol production was 4% (w/v). Only D-xylose was fermented in this study, although Suihko 5 has shown that F. oxysporum ferments all the main sugars in the hydrolysates of hemicellulose.

Fermentations using recycled and centrifuged Fusarium inoculum in shake flasks with controlled aeration reduced fermentation times and shortened the lag phase, particularly in 2% D-xylose fermentations. Aeration control was the most important factor affecting ethanol yield from fermentations by the Fusariurn isolates. Anaerobic fermentations had high ethanol yields but fermentation times were longer. High aera- tion reduced ethanol yields and increased the cell mass. Semi-aerobic conditions increased ethanol yields and shortened fermentation times.

Fusarium oxysporum ANL 22-760 has an ethanol yield similar to that from other Fusarium isolates, 5-7 but fermentation rates are higher for ANL 22-760 than other reported isolates. This study shows that control of aeration and inoculum size are important factors in ethanol fermen- tation rates, which suggests that continuous fermentation and/or use of immobilized cells would increase ethanol productivity.

A C K N O W L E D G M E N T

This work was supported by the Solar Energy Research Institute through an agreement with the US Department of Energy, under Contract DX-2-02-41.

o-xylose fermentation by Fusarium oxysporum strains 19

R E F E R E N C E S

1. Sliniger, R J., Bothast, R. J., van Cauwenberge, J. E. & Kurtzman, C. R, Conversion of D-xylose tO ethanol by the yeast Pachysolen tannophilus. Bio- technology and Bioengineering, Z 4 ( 1982) 371-84.

2. Antonopoulos, A. A., Fusarium species: Their potential for transforming biomass to ethanol. Argonne National Laboratory Report ANL/EES-TM- 38, 1979.

3. Martin, J. P., Use of acid, rose bengal and streptomycin in the plate method for the estimation of soil fungi. Soil Science, 69 (1950) 215-32.

4, Papavizas, G. C., New medium for the isolation of Thielaviopsis brassicola on dilution plates from soil and rhizosphere. Phytopathology, 54 (1964) 1475-81.

5. Suihko, M.-L., The fermentation of different carbon sources by Fusarium oxysporum. Biotechnology Letters, 5 ( 1983) 721-4.

6. Batter, T. R. & Wilke, C. R., A study of the fermentation of xylose to ethanol by Fusarium oxysporum. Lawrence Berkeley Laboratory Report No. 6365, 1977.

7. White, M. G. & Willaman, J. J., Biochemistry of plant diseases X, Ferment- ation of pentoses by Fusariurn lini. BiochernicalJournal, 22 (1928) 583-91.