Short communication

Microbial ecology of solid cellulosic materials during aerobicdigestion and anaerobic fermentation

Charlene P. D'Souza, R.H. Balasubramanya

Central Institute for Research on Cotton Technology, Adenwala Road, Matunga, Mumbai 400 019, India

Revised 23 October 1998; accepted 28 October 1998

Abstract

A process for anaerobic digestion of cotton willow dust, pressmud and baggase after a preliminary alkaline aerobic digestion had

been developed. The microbial ecologies during the aerobic digestion and anaerobic fermentation of these substrates were inves-

tigated, with respect to the isolation, identi®cation and characterization of aerobic microorganisms. The study produced infor-

mation on the survival of aerobic and facultative bacteria, actinomycetes and fungi, under anaerobic conditions. Ó 1999 Elsevier

Science Ltd. All rights reserved.

1. Introduction

Information on biogas production from solid cellu-losic materials is scanty. The solid cellulosic materialsunder consideration in this investigation were cottonwillow dust (Balasubramanya et al., 1981, 1986,1988, 1994), bagasse (Shaikh, 1990; Balasubramanyaet al., 1994) and pressmud (Lakshmanan et al., 1990;Balasubramanya et al., 1994). Willow dust, a textile millprocessing residue which is available in abundancehad been tested for the production of biogas by aerobicfollowed by anaerobic solid-state fermentations(Balasubramanya et al., 1981, 1986, 1988, 1994). Thistechnology had been extended to process bagasseand pressmud (Balasubramanya et al., 1994), theby-products of the sugar industry, for biogas pro-duction.

The microbiology of anaerobic digesters with respectto facultative and obligate anaerobes has been exten-sively investigated. The microbial ecology of aerobicdigestion and the fate of the microorganisms undersubsequent anaerobic fermentation of cellulosic mate-rials under high solid loadings has not been investigatedin detail. The present investigation dealt with a study onmicrobial ecology during two-stage digestion of twoindustrially important, cellulose-rich, materials onwhich technologies had been standardized earlier for theproduction of biogas.

2. Methods

2.1. Materials

Willow dust was obtained from a local textile millprocessing cotton, and bagasse and pressmud from thesugarcane crushing industry.

2.2. Analysis of materials

The materials were analysed for cellulose, pentosansand ether extractives (Halliwell, 1965), nitrogen (Perrin,1953), lignin (Sarkar et al., 1944) and ash (Narayananand Dabadghao, 1972). Moisture was determined byheating the samples at 105°C to constant weight ac-cording to the A.O.C.S. (1988a) method.

2.3. Aerobic digestion and anaerobic fermentation ofcellulosic materials

Willow dust (100 g), was mixed with 150 ml of watercontaining 1 g NaOH and incubated for 72 h for aerobicdigestion in an open container. In the case of pressmudand bagasse, 50 g of each were mixed together with 150ml of water containing 2 g NaOH, and kept for aerobicdigestion. Samples (about 25 g) were removed after 24,48 and 72 h for the enumeration of bacteria, fungi andactinomycetes.

Bioresource Technology 69 (1999) 285±287

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PII: S 0 9 6 0 - 8 5 2 4 ( 9 8 ) 0 0 1 8 6 - 2

In order to carry out anaerobic fermentation studies,eight sets of aerobically digested material (72 h) weremixed with 10% inoculum from a mixed microbialconsortium maintained separately (Balasubramanyaet al., 1994) and transferred to 500 ml capacity side-arm¯asks. The tops of the ¯asks were stoppered with rubberbungs and side arms were connected to air traps with¯exible rubber hoses. At weekly intervals, one ¯ask ineach case was removed and used for microbialenumeration. The concentration of alkali and timeof aerobic digestion had been standardized earlier(Balasubramanya et al., 1981).

2.4. Microbial enumeration and identi®cation

The above samples were transferred to 90 ml sterilesaline in 250 ml Erlenmeyer ¯asks and serially diluted.Appropriate dilutions (0.1 ml) were placed on solidi®edmedia and spread. Nutrient agar for bacteria, K�usters'agar for actinomycetes, and Rosebengal-Streptomycinagar for fungi were used. The plates were incubated atroom temperature under aerobic conditions for 3±5 daysand the viable counts were expressed as number per unitweight of the original material on a moisture free basis.

Predominant colonies were transferred on to agarslopes of nutrient agar for bacteria and actinomycetesand potato dextrose agar (PDA) for fungi, and trans-ferred to new slopes at monthly intervals. The isolateswere identi®ed to genus level by various morphologicaland biochemical tests (Balows et al., 1992; Thom andRaper, 1945; Gilman, 1957; Buchanan and Gibbons,1974).

2.5. Production of organic acids during aerobic digestionand anaerobic fermentation

The aerobically digested and anaerobically fermentedmaterials were sampled at regular intervals and thesamples extracted into heptane and concentrated by¯ash evaporation on a hot water bath. The extracts were

analysed for organic acids by gas±liquid chromatogra-phy (GLC), using a Shimadzu GC 14A model gaschromatograph equipped with a computerized datastation with ¯ame ionization detector (FID), by theA.O.C.S. (1988b) method.

3. Results and discussion

Willow dust and pressmud contain 25% cellulose and15% hemicellulose. Bagasse contains around 55% cel-lulose and 28% hemicellulose. The percentage of ash inpressmud is around 25%, 10% in willow dust and 2.5%in bagasse. The nitrogen percentage is around 1.5% inwillow dust and pressmud and 0.4% in bagasse. Thesematerials have been found to be suitable for biogasproduction in batch digestion under high solid loadings(Balasubramanya et al., 1994).

The populations of bacteria, fungi and actinomycetesare given in Table 1. The populations increased duringaerobic digestion and remained throughout the anaer-obic fermentation excepting fungi, where there was adecline by about ten times. Though the exact role playedby the aerobic or the facultative organisms in anaerobicenvironment is not very well understood, the highnumbers indicate that they might be participating in theproduction of organic acids and also hydrolysis of car-bohydrates, proteins and fats.

The dominant aerobic and/or facultative ¯ora com-prised both gram-positive and gram-negative bacteria.Various species of the endospore-forming Bacillus werepredominant among the gram-positive bacteria in bothwillow dust and bagasse + pressmud samples. In sam-ples containing bagasse and pressmud, gram-positive togram-variable Sporolactobacillus and Cellulomonas werepresent. From willow dust the gram-negative ¯oracomprising Beijerinckia sp. and non-¯uorescent Pseu-domonas sp. were recovered, whereas isolates frombagasse and pressmud belonged to Beijerinckia sp. and¯uorescent-pigment-producing Pseudomonas sp.

Table 1

Microbial population during aerobic digestion and anaerobic fermentation

Incubation time Willow dust Bagasse + Pressmud

Bacteria Actinomycetes Fungi Bacteria Actinomycetes Fungi

Control (untreated) 14 ´ 107 12 ´ 106 6 ´ 103 a 18 ´ 107 a 17 ´ 106 a 4 ´ 103

b 89 ´ 108 b 19 ´ 107 a 13 ´ 107

Aerobic digestion 72 h 27 ´ 1010 20 ´ 108 68 ´ 104 67 ´ 109 89 ´ 108 33 ´ 105

Anaerobic fermentation (weeks)

1 36 ´ 108 40 ´ 108 10 ´ 104 99 ´ 109 13 ´ 109 11 ´ 105

4 59 ´ 108 51 ´ 108 3 ´ 104 11 ´ 1010 24 ´ 108 44 ´ 103

8 84 ´ 108 25 ´ 108 5 ´ 104 68 ´ 109 7 ´ 108 8 ´ 105

a Bagasse.b Pressmud.

All viable counts expressed per g of sample on moisture free basis.

Averages of three replications.

286 C.P. D'Souza, R.H. Balasubramanya / Bioresource Technology 69 (1999) 285±287

Cellulomonas (a cellulose degrading bacterium) hadbeen reported to be present in samples of bagasse (Ba-lows et al., 1992) and species of cellulolytic Bacillus,Pseudomonas, Cellulomonas, Nocardia and Streptomyceshave been isolated from a Gobar gas digester (Goreet al., 1985). The exact role of the fungi and actinomy-cetes (apart from initiating aerobic hydrolysis) has notyet been established even through their presence has beenobserved at various stages of anaerobic digestion (Goreet al., 1985; Price and Cheremisino�, 1981; DST, 1981).

The results on the production of volatile fatty acidsduring aerobic digestion and anaerobic fermentation aregiven in Table 2. The production of small amounts oforganic acids, namely acetic, propionic, iso-butyric andn-valeric acids, during aerobic digestion could be due tothe formation of anaerobiosis in the system when alkali-pretreated materials were kept in deep open vessels. Theconcentrations of some organic acids were higher duringanaerobic fermentation with the formation of n-butyric,iso-caproic and n-caproic acids. These acids were notdetected in the aerobic digestion. The continuous pro-duction of methane exhausted the acids without accu-mulation and souring of the process. The pH remainedalkaline (7.5) throughout the fermentation period.

References

A.O.C.S. o�cial method Aa 3±38, 1988a. O�cial and Tentative

methods for American oil chemists society. 3rd ed., including

revision upto 1988. American Oil Chemists, Illinois.

A.O.C.S. o�cial method Ce-1-62, 1988b. Fatty acid composition by

G.L.C.

Balasubramanya, R.H., Khandeparkar, V.G., Betrabet, S.M., Sund-

aram, V., 1981. Production of biogas from willow dust by a batch

fermentation process. J. Text. Assn. 42, 145±149.

Balasubramanya, R.H., Khandeparkar, V.G., Sundaram, V., 1986.

Production of biogas and biomanure from textile mill processing

residue, willow dust, by anaerobic fermentation. Agric. Wastes

16, 295±302.

Balasubramanya, R.H., Khandeparkar, V.G., Sundaram, V., 1988.

Large scale digestion of willow dust in batch digesters. Biol.

Wastes 25, 25±32.

Balasubramanya, R.H., Gangar, H.U., Khandeparkar, V.G., 1994.

Biogas from cellulosic wastes by solid state fermentation. In:

Proceedings of the MICON-International-94 and 35th Annual

Conference of Association of Microbiologists of India, 9±12

November, 1994. In: Sankaran, R., Manja, K.S. (Eds.), Microbes

for Better Living, pp. 235±240.

Balows, A., Turper H.G., Dworkin, M., Harder, W., Schleifer, K.H.,

1992. The Prokaryotes, 2nd ed., A Handbook on the biology of

bacteria, Ecophysiology Isolation, Identi®cation, Applications,

Vol. I±IV, Springer, New York, pp. 1028, 2140, 3132, 4126.

Biogas Technology and Utilisation ± A status Report, 1981. Depart-

ment of Science and Technology Government of India, New Delhi.

Buchanan, R.E., Gibbons, N.E., 1974. BergeyÕs Manual of Determi-

native Bacteriology 8th ed. The Williams and Wilkins Company,

Baltimore, p. 1268.

Gilman, J.C., 1957. A Manual of Soil Fungi, 2nd ed. Oxford and IBH,

Calcutta, p. 450.

Gore, J.A., Ranade, D.R., Godbole, S.H., 1985. Celluloytic organisms

from fermenting slurry of a gobar gas plant. Indian J. of

Microbiol. 25, 105±106.

Halliwell, G., 1965. Cellulose. In: Bergmeyer, H.O. (Ed.), Methods of

Enzymatic analysis, pp. 64±71.

Lakshmanan, A.R., Kuppuswamy, G., Jeyabal, A., 1990. Biogas

generation from sugarcane ®lter cake. Laboratory scale and pilot

plant studies. Urja 27, 25±28.

Narayanan, T.R., Dabadghao, P.M., 1972. Forage Crops of India.

ICAR Publication, New Delhi, p. 373.

Perrin, C.H., 1953. Rapid modi®ed procedure for determination of

Kjeldahl Nitrogen. Anal. Chem. 25, 968±971.

Price, E.C., Cheremisino�, P.N., 1981. Biogas Production and Utili-

zation. Ann Arbor Science Publishers, Michigan, p. 146.

Sarkar, P.B., Bandopadhyay, S.G., Nodder, C.R., 1944. The rela-

tionship between the chemical ®bre characters and spinning

quality of jute. Int. Cent. Jute Comm. Technological Res. memoir

No. 6.

Shaikh, A.J., 1990. Blending of cotton stalk pulp with bagasse pulp for

paper making. Biol. Wastes 31, 37±43.

Thom, C., Raper, K.B., 1945. A Manual of the Aspergilli. Baillere

Tindall and Cox. London, p. 373.

Table 2

Concentrations of volatile fatty acids during aerobic digestion and anaerobic fermentation of willow dust and bagasse + pressmud

Substrate Volatile fatty acids (ppm)

Acetic Propionic Iso-butyric n-butyric n-valeric Iso-caproic n-caproic

Willow dust

72 h a 10.7 4.1 10.6 ± 10.5 ± ±

4 weeks b 5.1 2.7 6.9 0.4 0.5 1.9 7.3

Bagasse + Pressmud

72 h a 14.0 5.5 14.1 ± 4.6 ± ±

4 weeks b 3.9 2.5 6.3 0.3 0.3 1.5 6.9

a Aerobic.b Anaerobic.

C.P. D'Souza, R.H. Balasubramanya / Bioresource Technology 69 (1999) 285±287 287