Short Communication: Ethanol fermentation by a cellulolytic fungusAspergillus terreus

S. Pushalkar and K.K. Rao*

The cellulolytic fungus Aspergillus terreus showed an additional property of fermenting glucose to ethanol. In

addition to glucose, A. terreus also fermented other hexoses, pentoses and disaccharides to ethanol. Of the soluble

carbon sources tested, glucose yielded maximum (2.46% (w/v)) ethanol.

Key words: Alcohol dehydrogenase; Aspergillus terreus; ethanol fermentation.

The utilization of hemicellulose and cellulose components

of plant tissue for the production of liquid fuel would be a

very economically attractive process (Deshpande et al.

1986). The major sugars obtained after hydrolysis are

glucose, xylose and cellobiose. Many microorganisms

which produce extracellular cellulase when grown in

medium containing cellulose or cellulase inducers cannot

ferment sugar to ethanol. Conversely, most of the micro-

organisms capable of fermenting sugars to ethanol lack

the hydrolytic enzymes needed to break down cellulose

(Gong et al. 1981). Only a few ®lamentous fungi Ð Monilia

sp., Fusarium oxysporum VTT-D-80134, Fusarium oxyspo-

rum F-3 and Neurospora crassa Ð have been reported to

ferment sugars to ethanol (Gong et al. 1981; Suihko 1983;

Deshpande et al. 1986; Christakopoulos et al. 1989).

The cellulolytic fungus Aspergillus terreus produces

extracellular endo-b-1,4-glucanase, exo-b-1,4-glucanase

with high levels of b-glucosidase and has the ability to

ferment glucose to ethanol (Pushalkar & Rao 1995). The

present work was undertaken to study the ability of this

fungus to ferment different soluble carbon sources.

Materials and Methods

Fungal StrainA. terreus Thom isolated locally from soil and identi®ed at theInternational Mycological Institute, Surrey, UK (IMI 355964) wasused for the present investigation.

Media and Growth ConditionsEthanol production was carried out in two stages. The inoculumwas grown aerobically (pregrown aerated culture), and then

transferred to nonaerated fermentation ¯asks. The fungal cul-ture was grown in 250-ml Erlenmeyer ¯asks, each containing100 ml of sterile liquid medium (Pushalkar & Rao 1995) sup-plemented with glucose 1.0% (w/v) and inoculated with 1% (v/v) of the conidial suspension (108 conidia/ml, approx.). The¯asks were incubated at 28 � 2 °C on a rotary shaker (180 rev/min) for 24 h and contents were transferred to the above-men-tioned medium containing 5.0% (w/v) of the carbon substratesand kept for nonaerated fermentation. At intervals samples wereremoved and the ethanol concentration in the culture ®ltrate wasestimated by gas chromatography with a Chromosorb 101 (80/100 mesh) column. No ethanol was detected in substrate-freespent media.

Growth was measured in dry weight. The mycelia from each¯ask were harvested separately, ®ltered, washed and dried at80 °C in an oven to constant weight.

Analytical MethodsResidual sugars were estimated by the methods of Miller (1959)and Handel (1968). Protein was determined by the Lowrymethod with bovine serum albumin as a standard. The theo-retical yield of ethanol from hexoses, pentoses and thedisaccharide, cellobiose, was calculated as described by Christ-akopoulos et al. (1989).

Results and Discussion

The fermentation of hexoses, pentoses and disaccharides

at 5.0% (w/v) concentration by A. terreus was investi-

gated for a period of 7 days. With DD-glucose as soluble

substrate, the ethanol production was maximum in 5

days, exhibiting a yield of 2.46% (w/v) ethanol (96.5%

theoretical yield). Other hexoses, DD-fructose and DD-man-

nose were fermented to give high ethanol yields of 2.16%

(w/v) (85% conversion) in 5 days and 1.98% (w/v) (78%

conversion) in 6 days, respectively. With DD-maltose as

carbon source, 1.5% (w/v) ethanol was produced yield-

ing 59% theoretical yield in 6 days. Low levels of ethanol

World Journal of Microbiology & Biotechnology, Vol 14, 1998 289

World Journal of Microbiology & Biotechnology 14, 289±291

ã 1998 Rapid Science Publishers

S. Pushalkar and K.K. Rao are with the Department of Microbiology and Bio-technology Centre, Faculty of Science, M.S. University of Baroda, Baroda390002, India; fax no. (91) (265) 481080. *Corresponding author.

(0.98% (w/v)) were obtained with DD-galactose, giving

38% conversion in 6 days. (Figure 1) The pentose sugar,

DD-xylose was poorly fermented by A. terreus, yielding

only 0.36% (w/v) (14% conversion) ethanol in 6 days.

An ethanol yield of 0.51% (w/v) (50% conversion) was

obtained with DD-arabinose in 5 days, indicating that DD-

arabinose was fermented more ef®ciently than DD-xylose.

The fermentation of disaccharides, DD-sucrose and DD-cel-

lobiose by A. terreus resulted in yields of 2.13% (w/v)

ethanol in 6 days and 2.37% (w/v) ethanol (88% con-

version) in 5 days, respectively (Figure 2).

The growth of the organism, ethanol production and

sugar utilization is related to the type of substrate used.

Table 1 shows the growth of A. terreus on different sugars

and their consumption during fermentation on the day

seven. Compared with other sugars, growth was best on

maltose but ethanol yield was higher with glucose. Xy-

lose appeared to be poor substrate for growth and eth-

anol yield, and approximately 60% remained unutilized.

Fructose, sucrose and cellobiose proved to be better

substrates, with glucose being utilized maximally.

The above results show that A. terreus is able to

ferment hexoses, pentoses and disaccharides such as

cellobiose which are the common constituents of ligno-

cellulosic and hemicellulosic hydrolysates (depending on

the type of agricultural residues and the hydrolysis

method used). The ethanol values and the theoretical

yields produced by A. terreus with glucose and cellobiose

were comparable to or higher than that reported by other

fungal species, except for xylose which was very poorly

fermented (Gong et al. 1981; Suihko 1983; Deshpande et

al. 1986; Christakopoulos et al. 1989).

To conclude, A. terreus may be included among the

few ®lamentous fungi, (Monilia sp., F. oxysporum VTT-

D-80134, F. oxysporum and N. crassa) which are known to

ferment sugars to ethanol. Because A. terreus produces

hydrolytic enzymes for cellulose degradation, further

studies on the ability of this fungus to ferment cellulosic

materials may prove its usefulness in the conversion of

lignocellulose to ethanol.

Acknowledgement

The authors are grateful to the Gujarat Energy Devel-

opment Agency (GEDA), Government of Gujarat, for a

research grant to Prof. K.K. Rao.

References

Christakopoulos, P., Macris, B.J. & Kekos, D. 1989 Direct fer-mentation of cellulose to ethanol by Fusarium oxysporum.Enzyme and Microbial Technology 11, 236±239.

Deshpande, V., Keskar, S., Mishra, C. & Rao, M. 1986 Directconversion of cellulose/hemicellulose to ethanol by Neuro-spora crassa. Enzyme and Microbial Technology 8, 149±152.

Gong, C.S., Maun, C.S. & Tsao, G.T. 1981 Direct fermentation ofcellulose to ethanol by a cellulolytic ®lamentous fungus,Monilia sp. Biotechnology Letters 3, 72±82.

Handel, E.V. 1968 Direct microdetermination of sucrose. Ana-lytical Biochemistry 22, 280±283.

Figure 1. Fermentation of hexoses by Aspergillus terreus: (,)

glucose, (n) fructose, (h) mannose, (e) maltose and ( |́±) galactose.

Figure 2. Fermentation of pentoses and disaccharides by Aspergillus

terreus: (±m±) xylose, (±n±) arabinose, (±r±) cellobiose, (±.±) and

sucrose.

Table 1. Growth of Aspergillus terreus on different sugars and

their relative consumption during fermentation.

Substrate Mycelial dry wt Residual sugar

(5.0% (w/v)) (g %) (% (w/v))

Arabinose 0.68 2.47

Cellobiose 0.71 ±

Fructose 0.70 2.09

Galactose 0.75 2.56

Glucose 0.73 0.19

Maltose 0.88 1.06

Mannose 0.73 2.15

Sucrose 0.79 1.18

Xylose 0.62 2.97

±, Not estimated.

290 World Journal of Microbiology & Biotechnology, Vol 14, 1998

S. Pushalkar and K.K. RaoS. Pushalkar and K.K. Rao

Miller, G.L. 1959 Use of dinitrosalicylic acid reagent for deter-mination of reducing sugars. Analytical Chemistry 31, 426±428.

Pushalkar, S. & Rao, K.K. 1995 Production of b-glucosidase byAspergillus terreus. Current Microbiology 30, 255±258.

Suihko, M.-L. 1983 The fermentation of different carbon sourcesby Fusarium oxysporum. Biotechnology Letters 5, 721±724.

(Received in revised form 9 July 1997; accepted 11 July 1997)

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