World Journal of Microbiology and Biotechnology 6, 281-284

Alcohol production from pineapple waste

L. Ban-Koffi and Y.W. H a n

Saccharomyces cerevislae and Zymomonas mobilis were grown on pineapple waste and

Introduction

their alcohol production characteristics compared. The pineapple waste consisted ol 19% cellulose, 22% heml-cellulose, 5 % Iignin and 53% cell soluble matters but concentration of soluble sugars, which included 5.2% sucrose, 3.1% glucose and 3.4% fructose, was relatively low and pretreatment ot the substrate was needed. Pretreatment of pineapple waste wilh cellulase and hemi-cellulese and then fermantation with S. cerevlslae or Z. mobllls produced about 8 % ethanol from pineapple waste In 48h.

Because of current interest in the economic conversion of renewable resources into alcohol, residues of a number of crops were evaluated as substrates for alcohol production ( H a n & Cho 1973; Bu'Lock 1979; Rogers et al. 1979; Weisz & Marshall 1980; Hertzmark et aL Jones et aL 1981). Pineapple waste was one. About 100,000 tons of pineapple are produced annually in the Ivory Coast and about 40 to 80% is discarded as waste, being composed of peel, cores and pomace (Muttamara & Nirmala 1982; Tewari et aL 1987). These materials, having high biochemical oxygen demand (BOD) and chemical oxygen demand (COD) values, cause a serious pollution problem if not disposed of properly (Burbank & Kumagai 1965). Pineapple waste, consisting mainly of cellulose and starch, was suggested as a substrate for production of valuable fermentation and nonfermentation products (Tewari et aL 1987). In the past, pineapple waste from canneries has been utilized as the substrate for bromelin, vinegar, wine, food/feed yeast and organic acids (Dev & Ingle 1982). This paper evaluates the potential of pineapple waste as a substrate for ethanol fermentation.

For French summary, see next page.

L. Ban-Koffi is with the Ivorian Center of Technological Research, Ministry of Scientific Research, Abidjan, Ivory Coast, and Y.W. Han is with the USDA, Southern Regional Research Center, New Orleans, LA, 70179, USA. Y.W. Han is the Corresponding Author.

Materials and Methods Substrate

Pineapples were obtained from local markets in New Orleans, USA. For analytical purposes, the pineapple was divided into three parts: crown (the top part), pulp (the inner part) and skin. For fermentation, the entire pineapple was ground with a Waring blender and sterilized at 121~ for 20 min. Pre-treatmenl

One g each of cellulase (5.1 U/mg solid, Sigma Chemical Co.) and hemi-cellulase (0.053 U/mg solid, Sigma Chemical Co.) were added to 1 kg of ground pineapple and the mixture was kept at room temperature (25 to 30~ for 24 h.

9 1990 Rapid Communications of Oxford Ltd.

Microorganisms and Fermentation Saccharomyces cerevisiae (NRRL Y-2034), grown on potato dextrose broth, and Zymomonas mobilis (NRRL B-14022), grown on a basal medium, were used as

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L. Ban-Ko/fl and Y.W. Han On a fail croitre Saccharomyces cerevisiae et Zymomonas mobilis sur des dbchets d'ananas, et on a compar(~ les caractdristiques de leur production d'alcool. Le d(~chet d'ananas consistait en 19% de cellulose, 22% d'h6micellulose, 5% de lignine et 53% de mati(~res cellulaires solubles. Mais la concentration en sucres solubles qui comprenait 5.2% de sucrose, 3.1% de glucose et 3.4% de fructose, 6tail relativement faible. Le pr(~traitement du substrat s'av6rait donc n6cessaire. Le pr(~traltement ces d(~chets d'ananas avec la cellulase et I'hemicellulase, suivi de la fermentation par S. cerevisiae ou Z. mobilis ont produit environ 8 % d'ethanol/~ partir de rbsidus d'ananas en 48 h.

inocula. The basal medium consisted of sucrose, 150 g; peptone, 2 g; yeast extract, 1.5 g; K2HPO4, 2 g; (NHa)2SO 4. 2 g; MgSO4.H20 , 0.1 g per litre of water. About 1 kg of ground pineapple waste in 2 1 Fernback flasks was inoculated with 100 ml of actively growing culture and kept at room temperature (25 to 30~ for up to 72 h. The high-solid fermentation mash contained about 25% (w/v) dry matter and the low-solid fermentation mash contained about 10% (w/v) dry matter. The low-solid fermentation mash was prepared by pressing the ground pineapple waste through three layers of cheesecloth and collecting the liquid.

Ana[ytical Procedures Cellulose, hemi-cellulose, lignin, cell soluble matter (CSM), ash and in vitro rumen digestibility were determined by the method of Goering & Van Soest (1970). Crude protein was determined as total N, determined by the micro-Kjeldahl method, x 6.25, according to Perrin (1953). Sugars and alcohol were determined by high-performance liquid chromatography (HPLC) (Waters sugar analyser, Waters Assoc. Inc. Milford, MA, USA) with a refractive index detector and Amines HPX-87 column (Bio-Rad Corp., Richmond, CA, USA). Water was used as the mobile phase. Total fermentable sugar was the sum of sucrose, glucose and fructose. Cell growth was determined by plate counts.

Results and D i s c u s s i o n Table 1 shows the chemical and nutritional composition of the various parts of pineapple. The crown contained less cell soluble matter and more cellulose compared with other parts. The composition of the pulp was similar to that of the skin. The amounts of fermentable sugars in pineapple varied depending on the maturity of the fruit. A fresh pineapple contained 5.2% sucrose, 3.1% glucose and 3.4% fructose, which amounts to 11.7% (w/w) of total fermentable sugar. The protein content was about 4% for all parts of the fruits. The pulp was most digestible (72%); the crown was the least digestible (60%). Digestibility was inversely proportional to the cellulose content of the sample. The pH of the fresh pineapple juice was about 3.4. Relatively low levels of fermentable sugars (11.7% total sugar) and high amounts of fibre necessitated a pre-treatment of pineapple waste to increase the sugar content prior to fermentation. Initial sugar concentrations of more than 20% have been suggested as being the necessary minimum for economic alcohol fermentation of grains (Anon. 1980). Cellulase and hemi-cellulase were used to saccharify the fibrous matter, which increased the total sugar content to 14.4% in the substrate, a 23% increase from the initial level (Table 2). Combinations with other pre-

Table 1. Chemical and nutritional r

of pineapple waste ( % dry matter).* Component

Cellulose Hemi-cellulose Lignin

Ash CSM Total sugar

Protein Digestibilityl-

Whole

Skin

Crown

Pulp

19.4 22.4 4.7 0.7 53.4

14.0 20.2 1.5 0.6 64.8

29.6 23.2 4.5 0.4 42.5

14.3 22.1 2.3 0.2 61.4

11.7 4.4

-4.1

-4.2

-4.6

63.2

64.3

60.1

72.1

* Average of triplicate samples. t In vitro rumen digestibility.

282

A lcoholfrom pineapple waste treatments, such as heat, acid and alkali, might release more sugar. Tewari et al. (1987) reported that heat and acid saccharified pineapple waste up to 80%; whereas only 57% saccharification was achieved by cellulase treatment, The enzyme treatment also hydrolysed sucrose and increased the glucose and fructose levels. Figure 1 depicts the characteristics of pineapple fermented by S. cerevisiae and Z. mobilis. Almost all of the fermentable sugar initially present (about 14.7% of the fermentation mash) was converted to alcohol and cell mass. S. cerevisiae produced about 8% alcohol in 24 h, while Z. mobilis produced 7.6% alcohol in 48 h: these alcohol concentrations stayed the same throughout the 72 h fermentation period. The fermentation characteristics of the both organisms were similar. In both cases, about 98% of the total fermentable sugar was consumed, and the alcohol yield was a little more than the theoretical maximum, probably because of the presence of other fermentable sugars, besides sucrose, glucose and fructose, in the fermentation mash. The pH of the fermentation mash stayed the same throughout fermentation for both organisms. Addition of extra nitrogen or phosphorus was not needed for growth of the organisms. The alcohol production rate, expressed as volumetric production efficiency (VPE), was higher (4.7-fold for S. cerevisiae and 2.7-fold for Z. mobilis) for high-solid fermentation than for low-solid fermentation (Table 3). Since the VPE compares alcohol production per unit time with the unit fermenter volume, it determines the size of fermenter needed and has a large impact on fermentation cost. Solids remaining after fermentation could be used as animal feed because fermentation increases their nutritional value (protein, amino acids, vitamins, etc.) Total utilization of the fermentation product increases the economic feasibility of alcohol production from pineapple waste.

Table 2. Sugar release from pineapple waste by enzyme treatments*. Sugar

Sucrose Glucose Fructose Total sugar

Untreated

Enzyme treated*

(%)

(%)

5.2 3.1 3.4

1.1 6.6 6.7

11.7

14.4

*A mixture of cellulase (5100 units) and hemi-cellulase (53 units) was added to 1 kg of ground pineapple, and the treated material was kept to room temperature for 24 h.

Conclusion Pineapple waste had a relatively low content of sugars for alcohol fermentation. Therefore, pre-treatment to increase the sugar level was needed. Fermentation

a

b

12-

10

"?.0 m

"6.0 vI

=

O e"

,I .

-5.0

"4.0 0 "3.0



7z Time (hr)

Time (hr)

Figure 1, Cell growth, alcohol production, pH and residual sugars in pineapple waste fermented by S, cerevisiae (a) and Z. mobilis (b). A--Viable cell count; O--alcohol; rT--pH; ~--total sugar.

283

L. Ban-Koff and Y. IV. Han Table 3. Comparison of alcohol yield by high- and low-solid fermentation. Organism

Total sugar (%, w/v)

Alcohol (%, w/v)

VPE* (g/I/h)

14.7 10.2

8.1 3.5

3.37 0.73

14.7 10.2

7.6 3.0

1.66 0.62

S. cerevisiae

High-solid1" Low-solid~ Z. mobilis

High-solid'l" Low-solid~t

* Volumetric production efficiency. 1 High-solid fermentation mash contained about 25% (w/v) dry matter. :1:Low-solid fermentation mash contained about 10% (w/v) dry matter.

with a high-substrate concentration also increased the alcohol yield. Once an efficient pre-treatment method is developed pineapple waste could be used for the economic production of alcohol. Using residues as animal feed further increases the economic feasibility.

Acknowledgement This investigation was supported by the USDA, OICD, Cochran Middle Income Country Program and USDA, Southern Regional Research Center.

References ANON, 1980 Fuel from Farms: A Guide to Small-scale Ethanol Production, Solar Energy Research Institute Report No. SERI/SP-451-519 UC-61. Golden, CO: SERI. BU'LOCK, J.D. 1979 Industrial alcohol: In Microbial Technology: Current State, Future Prospects. Symposium of the Societyfor GeneralMicrobiology, eds. Bull, A.T., Ellwood, D.C. & Ratledge, C. Vol. 29, pp. 309-325. Cambridge: Cambridge University Press. BURBANK, N.C. & KAMAGAI, J.S. 1965 Study of pineapple cannery waste. Proceedings of the 20th-lndustrial Waste Conference, Engineering Extension Series 118, Vol. 20, pp. 365-397. Purdue University. DEV, D.K. & INGLE, U.M. 1982 Utilization of pineapple by-products and wastes--review. Indian Food Packer 36, 15-22. GOERING, H.K. & VAN SOEST, P.J. 1970 Forage Fiber Analysis. Agriculture Handbook No. 379, p. 20. United States Department of Agriculture, Washington, DC: Agricultural Research Service. HAN, Y.W. & CHO, Y.K. 1983 Effect of gamma-ray irradiation on alcohol production from corn. Biotechnologyand Bioengineering25, 2631-2640. HERTZMARK, D., RAY, D. & RICHARDSON, S. 1981 Economic Impact of Ethanol Fuel from Crops, Solar Energy Research Institute Report No. SERI/TR-734-1320. Golden, CO : SERI. JONES, R.P., PAMMENT,N. & GREENFIELD,R.F. 1981 Alcohol fermentation by yeasts the effect of environmental and other variables. ProcessBiochemistry 16, 42-49. MUTTAMARA,S. & NIRMALA,D.J. 1982 Management of industrial wastewater in developing nations. Proceedingsof International Symposium (Alexandria, Egypt, March 1981). eds Stuckey, D. & Hamza, A. pp. 445-452. Oxford: Pergamon Press. PERRIN, C.G. 1953 Rapid modified procedure for the determination of Kjeldahl nitrogen. Analytical Chemistry 25, 968--971. ROGERS, P.L., LEE, K.J. & TRIBE, D.E. 1979 Kinetics of alcohol production by Zymomonas mobil# at high sugar concentrations. BiotechnologyLetters 1, 165-170. TEWARI, H.K., MARWAHA, S.S., RUPAL, K. & KENNEDY, J.F. 1987 Bio-utilization of pineapple waste for ethanol generation. In Wood and Cellulosics: Industrial utilization, biotechnology, structure and properties, eds. Kennedy, J.F., Phillips, G.O. & Williams, P.A. pp. 251-259. Chichester: Ellis Horwood. WEISZ, P.B. & MARSHALL, J. 1980 Fuel from Biomass. A Critical Analysis of Technology and Economics. New York: Marcel Dekker.

(Received 22 January 1990; revised and accepted 26 March 1990)

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Alcohol production from pineapple waste.

Saccharomyces cerevisiae andZymomonas mobilis were grown on pineapple waste and their alcohol production characteristics compared. The pineapple waste...
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