HEMICELLULASE SUPPLEMENTATION OF REGULAR AND HYBRID RYE GRAIN MASHES FOR ETHANOL FERMENTATION

Zbigniew Czarnecki, Jacek Nowak
Department of Fermentation and Biosynthesis, Institute of Food Technology of Plant Origin, The August Cieszkowski Agricultural University of Poznan, Poland

ABSTRACT

Rye hybrids were proved to be a very good raw-material for ethanol production, as good as a regular variety. The most profound difference found in chemical composition of hybrids and a regular variety was higher level of pentosanes, reaching from 7.6 to 9.2% of dry matter. Applying hemicellulase enzyme in the mashing process as the additional enzyme is particularly reasonable for rye hybrids. Taking into account higher productivity of hybrid varieties, the ethanol yield from hectare of land might be up to 40% higher in comparison with a regular rye grain.

Key words: ethanol, rye hybrids, hemicellulase.

INTRODUCTION

Rye hybrid varieties are started to be planted in Poland, but so far not in the amounts to be important in the ethanol industry. The first hybrid variety – “Marder” was registered in Poland in 1995. Nowadays 8 hybrid varieties are registered in Poland [11]. Intensive work on the assessment of genetic diversity existing in rye is going, which will fruit in some more available rye hybrids [5, 9, 10, 12].

Rye hybrids are characterized by 15-30% higher yield of grain. Some authors suggest also that the hybrids might get better resistance to plant diseases. In the ethanol industry, normally, rye is pressure cooked at the temperature 140-150°C before mashing, but HTST extrusion also might be applied [4, 7]. The influence of saccharification with added hemicellulases on non-starch carbohydrates of regular rye as well as on some quality factors of mashes and stillages were examined. Laboratory experiments showed the significant influence of the type of pretreatment and hemicellulases enrichment on the dynamic of saccharification and efficiency of ethanol fermentation. It was concluded that the increase of ethanol yield as a result of hemicellulase supplementation is not connected with pentosans fermentation but with the increased availability of hexose (starch) bound with pentose chain for amylolytic enzymes [3]. Because of the high yield of rye hybrids, this grain might be a potential source of raw materials in the distillery industry. In this research four hybrid varieties named H 1 to H 4 were tested to compare with regular variety R for their usefulness as raw material for ethanol production.

MATERIALS AND METHODS

Four hybrid varieties coded as H 1, H 2, H 3 and H 4 as well as the regular variety R were obtained from The August Cieszkowski Agricultural University of Poznań Experimental Station in Swadzim.

Pressure cooking (121°C, 1h) was used for gelatinising the ground rye material during the laboratory experiments.

Commercial a-amylase GAMALPHA HT 120L and amyloglucosidase GAMMYLO 300L (GAMMA CHEMIE) were applied for saccharification. In some experiments additionally CELLULASE AC (SOLVAY) with activity of xylanases, cellulases and b-glucanases was used. For liquefing GAMALPHA HT 120L at 75°C during 20 min and GAMMYLO 300L for saccharification at 55-60°C (80 min) were used. CELLULASE AC was used together with saccharyfing enzymes.

Polish commercial distillery yeast D-2 was used for laboratory and large scale experiments. Industry scale experiments were carried out in the distillery plant of the Swadzim Experimental Station of the August Cieszkowski Agricultural University of Poznań.

The samples were inoculated with D-2 yeast and incubated at 30°C for 72 h. The experiments in the distillery plant were done 5 following days for experimental and control batches and the results were estimated everyday.

Carbohydrates were estimated after enzymatic hydrolysis as reducing sugars by DNS-method [6] and calculated as starch.

Total pentosans and soluble in water pentosans by the method of Czarnecki et al. [2] as well as relative viscosity according to Antoniou et al. [1] were tested in the mashes. The relative viscosity of mashes and stillages were estimated using capillary type viscometer.

Dynamics of saccharifing process was controlled after 20 and 100 min.

Ethanol (by distillation method) as well as extract (by areometric method), reducing sugars, pentosanes and relative viscosity were estimated after fermentation.

RESULTS AND DISCUSSION

The most significant difference found in chemical composition of hybrids and a regular variety, from the point of view of ethanol fermentation by yeast, was higher level of pentosanes, reaching from 7.5 to 9.2% of dry matter, 1-1.5% more than popular variety R (Table 1). There were no significant differences in starch content between tested varieties. All the tested varieties were characterized by similar but slightly higher starch level in comparison to the most popular varieties on the market (Motto, Dańkowskie Złote).

There were also no differences in the saccharification kinetics and fermentation efficiency between hybrids and a regular variety found but slightly lower for H 1. Mashes got from hybrids attributed significantly higher viscosity than those from regular (R) (Table 2). From hybrid varieties the lowest viscosity of mashes was found for H 4. Generally rye mashes are characterized by high viscosity which is undesirable in the process.

Taking into account the high amount of pentosanes in the hybrid varieties, hemicellulase enzyme (CELLULASE AC) was used additionally together with amylo-glucosidase. The yield of ethanol counted for 100 kg of starch was enhanced from 2.2-3.6 L for hybrid varieties and 1.5 L for a regular rye variety (Tables 2 and 3). Addition of hemicellulase resulted in mashes viscosity decrease. Sweet rye mashes without hemicellulase were characterized by relative viscosity range 23.2-32.8 while the viscosity of mashes prepared with hemicellulase decreased to 12.8-15.7 (Tables 2 and 3). Viscosity of mashes is an important factor in small distilleries as most of them have no possibilities to mix fermentation medium during the process [8].

During laboratory experiments rye hybrids were proved to be a very good raw-material for ethanol production, as good as a regular variety. Taking into account higher productivity of hybrid varieties, the ethanol yield from hectare of land might be up to 40% higher in comparison with control grain (Table 4). Applying of hemicellulase enzyme in the mashing process as the additional enzyme is particularly reasonable for rye. The decrease of viscosity of sweet mashes and fermented mashes is more important for rye than for other starch material as rye mashes are more viscous which cause problems during fermentation and distillation [3].

The laboratory results were partly confirmed in a rural distillery plant scale. There were no statistically important differences between varieties in extract of sweet mash. Some differences in apparent extract in the fermented mash were observed. The efficiency of fermentation and distillation process was very similar, but because of higher grain yield from the unit of land got for hybrid varieties, the ethanol yield counted for 1 ha of land was estimated for hybrid variety H 1 as 128% and for H 2 as 144% of that obtained from the regular variety R. This result indicates the economic evidence of hybrid varieties for bioethanol production.

Results presented in this research and our previous papers show that hybrid varieties of rye are excellent raw-material for ethanol fermentation. Especially beneficious is their grain yield and connected with it high ethanol yield from hectare of land.

Very effective for ethanol yield improvement and viscosity decrease was the supplementation of mashing process with hemicellulolytic enzymes. Hemicellulolytic enzymes action allowed more effective hexose (starch) availability for saccharification and fermentation process.

REFERENCES

1. Antoniou T. C., Marquardt R. R., Misir R., 1983. The utilisation of rye by growing chicks as influenced by autoclave treatment, water extraction, and water soaking. Poultry Sci., 62, 91-102.

2. Czarnecki Z., Czarnecka M., Żuromska M., 1993. Oznaczanie sumy pentozanów i ich form rozpuszczalnych w składnikach paszowych [Total and soluble pentosans determination in animal feed components]. Biul. Inf. Przem. Pasz., 1, 22-35 [in Polish].

3. Czarnecki Z., Nowak J., 2001. Effects of rye pretreatment and enrichment with hemicellulolytic enzymes on ethanol fermentation efficiency. Electronic Journal of Polish Agricultural Universities.(www.ejpau.media.pl.) 4, 2, Food Sci. Technol., 1-7.

4. Czarnecki Z., Nowak J., 1997. Ethanol fermentation of HTST extruded rye grain by bacteria and yeasts. Acta Biotechnol., 17, 1, 63-71.

5. Dojczew D., Sobczyk M., Gożdzicki K., Haber T., 2004. The influence of pre-harvest sprouting grains on the breadmaking wheat, rye and tricale flour. Acta Sci. Pol., Technol. Aliment., 3 (2), 127-136.

6. Miller G. L., 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugars. Anal. Chem., 31, 3, 426-428.

7. Nowak J., Czarnecki Z., Kamiński E., 2000. Bacterial and yeast by-products formation in ethanol fermentation of glucose medium and rye mashes. Pol. J. Food Nutr. Sci., 9, 4, 49-51.

8. Piasecki M., Kujawiński J., Kumider J., Gruchała L., 1995. Stan aktualny i perspektywy rozwoju przemysłu spirytusowego [Current situation and progress perpectives of bioethanol industry]. Science and practic paralels symphosium. POLAGRA 95, v. 2, edited by PTTŻ - Oddział Wielkopolski, Poznań, 111-148 [in Polish].

9. Rafalski A., Madej L., Wiœniewska I., Gawe M., 2002. The genetic diversity of components of rye hybrids. Cell. Mol. Biol. Lett., 7, 471-475.

10. Roux S. R., Miedaner T., Geiger H. H., Knopf E., Wilde P., Wortmann H., 2003. Combining ability vs. population performance of genetic resources in rye plant breed. Seed Sci., 48, 2, 45-48.

11. Rozbicki J., 2002. Produkcja i rynek zbóż [Production and market of cereals]. Wieœ Jutra, Warszawa, 26-31 [in Polish].

12. Szambelan K., Nowak J., Chrapkowska K. J., 2004. Comparison of bacterial and yeast ethanol fermentation yield from jerusalem artichoke (Helaintus tuberosus L.) tubers pulp and juices. Acta Sci. Pol., Technol. Aliment., 3 (1), 45-53.

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