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Application of very high gravity technology to the cofermentation of sweet stem sorghum juice and sorghum grain
Industrial Crops and Products 11 (2000) 11 – 17 www.elsevier.com/locate/indcrop
Application of very high gravity technology to the cofermentation of sweet stem sorghum juice and sorghum grain J.M. Bvochora, J.S. Read, R. Zvauya * Department of Biochemistry, Uni6ersity of Zimbabwe, P.O. Box MP167, Mount Pleasant, Harare, Zimbabwe Received 27 January 1999; received in revised form 1 June 1999; accepted 1 June 1999
Abstract Ethanol production from mixtures of sweet stem sorghum juice and sorghum grain was investigated under normal and very high gravity (VHG) fermentation conditions. Fermentation was carried out using Saccharomyces cere6isiae yeast strain N96 at 30°C. For VHG fermentation, sucrose was added to the sweet sorghum juice to obtain a concentration of 34 g per 100 ml of dissolved solids. Fermentation was carried out for 96 h using malted and unmalted milled sorghum grain from sorghum cultivars DC-75 and SV-2. Under VHG conditions, maximum ethanol levels were about 16.8% (v/v) and 11% (v/v) for media containing malted and unmalted milled sorghum grain, respectively. Although fermentation did not occur to completion, levels of ethanol obtained under VHG conditions were three times higher than the levels obtained under normal fermentation conditions. Under VHG conditions, about 8 g/100 ml of dissolved solids remained in the fermentation media after ethanol production had ceased while under normal fermentation conditions, about 4 g/100 ml of dissolved solids remained unused in the fermentation media. There was an initial decline in free amino nitrogen (FAN) levels up to 34 h followed by an increase up to 96 h under VHG fermentation conditions. Levels of assayable proanthocyanidins (PAs) from sorghum cultivar DC-75 were reduced during fermentation. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Sweet sorghum; Very high gravity; Fermentation; Proanthocyanidin
1. Introduction The production of ethanol during fermentation is limited by the tolerance of yeast to high alcohol levels. In traditional brewing, worts of 11 – 12% dissolved solids are fermented to produce beers of * Corresponding author. Tel.: +263-4-303211; fax: + 263-4333678. E-mail address: [email protected] (R. Zvauya)
4–5% (v/v) ethanol (Casey et al., 1984). An opportunity exists for process improvements in the conversion of biomass to fuel alcohol which would result in more favourable production economics. Very high gravity (VHG) fermentation is one such process improvement aimed at increasing both the rate of fermentation and ethanol concentration. The technology involves preparation and fermentation of mashes containing 300 or more grams of dissolved solids per litre
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(Thomas et al., 1993). Under appropriate environmental and nutritional conditions, Saccharomyces cere6isiae can produce and tolerate high concentrations of ethanol (Thomas et al., 1995). VHG technology exploits the observation that growth of Saccharomyces yeast is promoted and prolonged when adequate but very low levels of oxygen are present and when assimilable nitrogen levels are not limiting (Casey and Ingledew, 1986). Use of VHG technology allows considerable saving of water, reduces distillation costs and allows more alcohol to be made with given plant capacity and labour costs (Thomas et al., 1995). The technology reduces capital costs, lowers energy cost per litre of alcohol and reduces the risk of bacterial contamination. Another advantage is an increase in opportunities for harvest of high protein spent yeast. Previous work on VHG fermentation of wheat mashes, sugarcane juice and molasses showed that the technology allows for production of up to 23% (v/v) alcohol in batch fermentations (Thomas et al., 1993, 1994, 1995; Jones et al., 1994). Although more fuel alcohol is produced from sugarcane sources than from any other material (Jones et al., 1994), it would be interesting to apply VHG technology to various other substrates and determine alcohol production. Field crops offer potential sources of fuels with sweet stem sorghum offering promise as a large scale energy crop based on its genetic diversity, climatic adaptation, biomass and sugar production (de Mancilha et al., 1984; Bulawayo et al., 1996). Due to their drought tolerance, grain sorghums are grown in semi-arid regions of Zimbabwe. A major drawback of grain sorghum as a food source is the high levels of polyphenols (tannins) associated with certain varieties of sorghum grain. High levels of polyphenolic compounds, mainly proanthocyanidins, have been shown to demonstrate several unfavourable characteristics including reduction in nutritional quality (Strumeyer and Malin, 1975), brewing quality and the rate of fermentation for fuel ethanol (Mullins and Nesmith, 1987). It is therefore necessary to identify ways of utilising sorghum which will improve household incomes of small scale farmers in semi-
arid regions thus improving food security. There are no reports of the application of VHG technology to produce higher concentrations of fermentation ethanol from sorghum. The aims of this study were to determine fuel ethanol production using a mixture of sweet stem sorghum juice and milled sorghum grain as a substrate for VHG fermentation and to determine any changes in levels of proanthocyanidins of the high tannin sorghum cultivar during fermentation.
2. Materials and methods Stalks of sweet stem sorghum were obtained from fields in Chiredzi, Zimbabwe. The crop was harvested, defoliated and the stalks cut at the nodes. Juice from the stalks was extracted using laboratory scale blenders and kept frozen at − 20°C until required for use. A high tannin, bird resistant sorghum variety (DC-75) and a low tannin line (SV-2) were obtained from Research and Specialist Services (R&SS), Harare, Zimbabwe. The yeast strain used in this study was a Saccharomyces cere6isiae (N96) strain.
2.1. Fermentation media The juice used for the cofermentations was supplemented to give the following concentrations (g/l); urea, 1.6; (NH4)2HPO4, 1.2. For VHG fermentations, the juice was freeze-dried to obtain VHG levels of sucrose (34 g/100 ml). For the cofermentations, sucrose (250 g/l) was added to the juice to obtain VHG levels. Milled sorghum grain, both malted and unmalted, (87.5 g), was autoclaved at 121°C for 15 min and added to 500 ml of autoclaved juice. The final pH of all the media used was adjusted to 4.5 using 0.5 M H2SO4 before autoclaving.
2.2. Fermentation Saccharomyces cere6isiae (N96) was maintained on malt extract agar and stored at 4°C. Precultures were prepared by inoculating a loopful ofyeast cells into 10 ml of supplemented juice in 25-ml universal bottles. The bottles were incu-
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bated at 30°C and 150 rpm for 48 h in a shaking incubator. Fermentations were carried out in 1-L Erlenmeyer flasks with a working volume of 500 ml juice, and 87.5 g milled grain for the cofermentations. The flasks were inoculated with 10 ml precultures and incubated at 30°C and 150 rpm for 96 h. Samples were collected at 10 – 12-h intervals. A subsample was freeze-dried for tannin analysis while another subsample was left for microbial analysis. The rest of the sample was centrifuged at 3000 rpm for 10 min and the supernatant kept at −20°C until required for analysis.
2.3. Analysis For microbial analysis, samples were serially diluted using peptone water and spread in duplicates on Rose Bengal Chloramphenical agar plates for yeast colony counts. The plates were incubated at 30°C for 48 h. Dissolved solids were estimated by measuring the refraction at room temperature of centrifuged sample using a sugar refractometer. Dissolved solids were recorded as grams of dissolved solids (expressed as sucrose) per 100 ml. Proanthocyanidin levels were determined on 70% acetone extracts of the freeze-dried samples using the butanol-HCl method (Reed et al., 1987). Ethanol concentrations were measured
Fig. 1. Changes in dissolved solids during normal cofermentation of sweet stem sorghum juice and sorghum grain meal. Media components: — —, malted DC-75 meal + juice; — —, DC-75 meal+juice; — —, malted SV-2 meal + juice; — — , SV-2 meal +juice.
Fig. 2. Changes in dissolved solids during very high gravity cofermentation of sweet stem sorghum juice and sorghum grain. Media components: — — , malted DC-75 meal + juice; — — , DC-75 meal +juice; — — , malted SV-2 meal + juice; — — , SV-2 meal +juice.
by gas chromatography using a PEG 20M column. The column temperature and gas flow rate were 55°C and 10 ml/min respectively. Free amino nitrogen (FAN) was measured colorimetrically using the ninhydrin method (European Brewery Convention, 1987) with glycine as the standard.
3. Results and discussion All results are means of three independent experiments. Figs. 1 and 2 show the changes in dissolved solids during normal and VHG fermentations, respectively. Not all the dissolved solids in the media were utilisable by the yeast. Under VHG conditions, about 8 g/100 ml of dissolved solids remained in the fermentation media after ethanol production had ceased, while under normal conditions 4–5 g/100 ml of dissolved solids remained unused in the fermentation media. There have been reports that with increasing fermentation temperature, above 25°C, the amount of sugar that can be fermented decreases (Jones and Ingledew, 1994b). In this study, a temperature of 30°C was employed as in the fuel industry. ‘Stuck’ fermentation may have been due to thermal stress which has been reported with fermentation of wheat mashes (Jones and Ingledew,
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1994b). Fermentation was carried out in shake flasks, where conditions may not be optimal to ensure complete fermentation. Further research is necessary to determine the optimum processing parameters to attain complete utilisation of the dissolved solids. Tables 1 and 2 show microbial counts from the various cofermentations under VHG and normal fermentations, respectively. In all the fermentations, yeast cell multiplication occurred during the course of fermentation. Yeast cell multiplication and the time required to attain maximum cell numbers in fermentation varied with the substrates used. After 57 h of fermentation, loss of yeast viability was observed under normal fermentation conditions and in the VHG media containing unmalted sorghum meal. Ethanol production had ceased at this stage. Under normal fermentation conditions, the loss of yeast viability may be due to limitation of essential nutrients such as free amino nitrogen which was depleted at 24 h of fermentation (Fig. 6). Under VHG conditions, loss of yeast viability was observed in cases where the unmalted sorghum meal was used, probably due to the lack of hydrolytic enzyme activity and nutrients available in the malts. The maximum ethanol levels obtained under VHG fermentation conditions are about 16.8% (v/v) for the media containing malted sorghum grain meal and 11% (v/v) for the media containing unmalted sorghum grain meal from an initial dissolved solids content of 34% (w/v) (Fig. 3). Malted sorghum grain meal is a better substrate for fermentation than unmalted sorghum grain meal due to the higher nutrient content which develops as a result of the
malting process. Ethanol levels obtained under VHG fermentation conditions are an improvement over the 12.9% (v/v) ethanol produced from VHG molasses at 30°C though not as high as the 18.6% (v/v) achieved at 30°C from the fermentation of VHG wheat mash (Jones and Ingledew, 1994b). However, shake flasks were used in the present experiments while the levels quoted above were obtained using bioreactors. Ethanol levels obtained under VHG fermentation conditions are about three times those obtained under normal conditions (Figs. 3 and 4). The concentration of fermentable sugar in the media, the amount of assimilable nitrogen available and the pitching rate all play important roles in determining the rate of fermentation and the final ethanol yield. There was no cultivar difference observed for ethanol production under VHG fermentation conditions while under normal conditions, higher ethanol levels were recorded for DC-75 malt compared to SV-2 malt. Previous studies have shown that DC-75 produces a better malt quality than SV-2 (Bvochora et al., 1999). After an initial decline, there was an increase in the FAN levels for all the VHG fermentations (Fig. 5). The increase in FAN may be due to the release of nutrients by lysing yeast cells. Yeast cell lysis may be due to exposure to high concentrations of ethanol for long periods of time andosmotic stress exerted by the unfermented sugars (Jones and Ingledew, 1994a). Though the media was supplemented with urea and diammonium phosphate as nitrogen sources, fermentation did not occur to completion. Similar results have been reported for VHG fermentation of wheat at 30°C
Table 1 Microbial analysis of VHG fermentation samples Time (h)
0 9.5 24 33.5 48.5 57.5 96
Yeasts (log cfu/ml) DC-75+juice
DC-75 malt+juice
SV-2 malt+juice
SV-2+juice
8.13 9.01 9.79 10.54 10.78 9.78 9.18
8.20 8.81 9.53 9.69 9.79 10.00 10.30
8.22 8.79 9.53 10.17 10.11 10.18 10.48
7.95 8.76 9.43 10.20 10.28 9.70 7.70
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Table 2 Microbial analysis of ‘normal’ cofermentation samples Time (h)
0 10 24 34 57 73 80 96
Yeasts (log cfu/ml) DC-75+juice
DC-75 malt+juice
SV-2 malt+juice
SV-2+juice
6.72 7.58 8.74 8.81 10.20 9.19 7.95 7.16
6.71 7.63 8.85 9.11 10.40 9.54 8.34 8.36
6.85 7.45 8.55 9.20 10.00 9.46 8.62 7.85
6.82 7.44 8.71 9.14 10.53 8.95 8.08 8.34
(Jones and Ingledew, 1994a). Under normal conditions, an initial decrease in FAN levels was observed up to 24 h then depletion of FAN occurred for the rest of the fermentation (Fig. 6). FAN levels may have limited the fermentation to completion of media under normal fermentation conditions. However, yeast cell lysis may not have occurred under normal fermentation conditions as the increase in FAN levels observed under VHG conditions was not observed. Fig. 7 shows the proanthocyanidin levels during fermentation of various media under VHG conditions. Initial levels of proanthocyanidins were highest in media containing unmalted DC-75 grain meal. Media containing malted DC-75 grain meal had lower initial proanthocyanidin levels showing
that malting reduces the levels of assayable proanthocyanidins in sorghum grain meal. For the medium containing malted SV-2 grain meal, levels of assayable proanthocyanidins were negligible. The results obtained indicate that fermentation reduced the levels of assayable proanthocyanidins in sorghum grain meal. Under normal conditions (Fig. 8), a general decrease in proanthocyanidins (PAs) was observed.
Fig. 3. Changes in ethanol levels during very high gravity cofermentation of sweet stem sorghum juice and sorghum grain. Media components: — —, malted DC-75 meal + juice; — — , DC-75 meal + juice; — —, malted SV-2 meal+ juice; — — , SV-2 meal +juice.
Fig. 4. Changes in ethanol levels during normal cofermentation of sweet stem sorghum juice and sorghum grain. Media components: — — , malted DC-75 meal +juice; — — , DC-75 meal +juice; — —, malted SV-2 meal +juice; — — , SV-2 meal +juice.
4. Conclusion The results of this research show that sweet stem sorghum juice and sorghum grain meal can be
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Fig. 5. Changes in free amino nitrogen levels during very high gravity cofermentation of sweet stem sorghum juice and sorghum grain. Media components: — —, malted DC-75 meal+ juice; —— , DC-75 meal+ juice; — —, malted SV-2 meal +juice; — —, SV-2 meal + juice.
Fig. 7. Changes in water soluble proanthocyanidins during very high gravity cofermentation of sweet stem sorghum juice and sorghum grain. Media components: — — , malted DC-75 meal+ juice; —— , DC-75 meal + juice; —— , malted SV-2 meal + juice; — — , SV-2 meal +juice.
employed to facilitate efficient batch VHG fermentation for fuel ethanol production. VHG fermentation technology, therefore, has potential as a process improvement to increase the profitability of fuel alcohol production from sorghum which would otherwise have limited uses. Levels of proanthocyanidins in sorghum are reduced during fermentation hence high PA cultivars may be used in VHG technology.
Fig. 8. Changes in water soluble proanthocyanidins during normal cofermentation of sweet stem sorghum juice and sorghum grain. Media components: — —, malted DC-75 meal + juice; — — , DC-75 meal + juice.
Acknowledgements Fig. 6. Changes in free amino nitrogen levels during normal cofermentation of sweet stem sorghum juice and sorghum grain. Media components: — —, malted DC-75 meal + juice; — —, DC-75 meal+ juice; — —, malted SV-2 meal + juice; — — , SV-2 meal + juice.
The work was supported by grants from the McKnight Foundation Collaborative Research Program, USA and the University of Zimbabwe Research Board. We thank Dr I. Ncube for help with G.C. analysis.
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References
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optimization of temperature for efficient very high gravity fermentations. Appl. Environ. Microbiol. 60, 1048 – 1051. Jones, A.M., Thomas, K.C., Ingledew, W.M., 1994. Ethanolic fermentation of blackstrap molasses and sugarcane juice using very high gravity technology. J. Agric. Food Chem. 42, 1242 – 1246. Mullins, J.T., NeSmith, C.C., 1987. Acceleration of the rate of ethanol fermentation by addition of nitrogen in high tannin grain sorghum. Biotechnol. Bioeng. 30, 1073 – 1076. Reed, J.D., Tedla, A., Kebede, Y., 1987. Phenolics, fibre and fibre digestibility in the crop residue from bird-resistant and non-bird resistant sorghum varieties. J. Sci. Food Agric. 39, 113 – 121. Strumeyer, D.H., Malin, M.J., 1975. Condensed tannins in grain sorghum: isolation, fractionation and characterisation. J. Agric. Food Chem. 23, 909 – 914. Thomas, K.C., Hynes, S.H., Jones, A.M., Ingledew, W.M., 1993. Production of fuel alcohol from wheat by very high gravity technology. Effect of sugar concentration and fermentation temperature. Appl. Biochem. Biotechnol. 43, 211 – 226. Thomas, K.C., Hynes, S.H., Ingledew, W.M., 1994. Effects of particulate material and osmoprotectants on very high gravity ethanolic fermentation by Saccharomyces cere6isiae. Appl. Environ. Microbiol. 60, 1519 – 1524. Thomas, K.C., Hynes, S.H., Ingledew, W.M., 1995. Practical and theoretical considerations in the production of high concentration of alcohol by fermentation. Process Biochem., 321 – 331.
Bulawayo, B., Bvochora, J.M., Muzondo, M.I., Zvauya, R., 1996. Ethanol production by fermentation of sweet stem sorghum juice using various yeast strains. World J. Microbiol. Biotechnol. 12, 357–360. Bvochora, J.M., Reed, J.D., Read, J.S., Zvauya, R., 1999. Effects of fermentation processes on proanthocyanidin levels in sorghum during preparation of mahewu, a non-alcoholic beverage. Process Biochem. (in press). Casey, G.P., Ingledew, W.M., 1986. Ethanol tolerance in yeasts. CRC Crit. Rev. Biotechnol. 13, 219–280. Casey, G., Magnus, C.A., Ingledew, W.M., 1984. High gravity brewing: effects of nutrition on yeast composition, fermentative ability, and alcohol production. Appl. Environ. Microbiol. 48, 639 – 646. de Mancilha, I.M., Pearson, A.M., Waller, J., Hogaboam, G.J., 1984. Increasing alcohol yield by selected yeast fermentation of sweet sorghum. I. Evaluation of yeast strains for ethanol production. Biotechnol. Bioeng. XXVI, 632– 634. European Brewery Convention, 1987. Free amino nitrogen. In: Analytica-EBC, 4th ed. Brauerei- und Getranke-Rundschau, Zurich, pp. E141–E142. Jones, A.M., Ingledew, W.M., 1994a. Fuel alcohol production: appraisal of nitrogenous yeast foods for very high gravity wheat mash fermentation. Process Biochem. 29, 483–488. Jones, A.M., Ingledew, W.M., 1994b. Fuel alcohol production:
.
Application of very high gravity technology to the cofermentation of sweet stem sorghum juice and sorghum grain J.M. Bvochora, J.S. Read, R. Zvauya * Department of Biochemistry, Uni6ersity of Zimbabwe, P.O. Box MP167, Mount Pleasant, Harare, Zimbabwe Received 27 January 1999; received in revised form 1 June 1999; accepted 1 June 1999
Abstract Ethanol production from mixtures of sweet stem sorghum juice and sorghum grain was investigated under normal and very high gravity (VHG) fermentation conditions. Fermentation was carried out using Saccharomyces cere6isiae yeast strain N96 at 30°C. For VHG fermentation, sucrose was added to the sweet sorghum juice to obtain a concentration of 34 g per 100 ml of dissolved solids. Fermentation was carried out for 96 h using malted and unmalted milled sorghum grain from sorghum cultivars DC-75 and SV-2. Under VHG conditions, maximum ethanol levels were about 16.8% (v/v) and 11% (v/v) for media containing malted and unmalted milled sorghum grain, respectively. Although fermentation did not occur to completion, levels of ethanol obtained under VHG conditions were three times higher than the levels obtained under normal fermentation conditions. Under VHG conditions, about 8 g/100 ml of dissolved solids remained in the fermentation media after ethanol production had ceased while under normal fermentation conditions, about 4 g/100 ml of dissolved solids remained unused in the fermentation media. There was an initial decline in free amino nitrogen (FAN) levels up to 34 h followed by an increase up to 96 h under VHG fermentation conditions. Levels of assayable proanthocyanidins (PAs) from sorghum cultivar DC-75 were reduced during fermentation. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Sweet sorghum; Very high gravity; Fermentation; Proanthocyanidin
1. Introduction The production of ethanol during fermentation is limited by the tolerance of yeast to high alcohol levels. In traditional brewing, worts of 11 – 12% dissolved solids are fermented to produce beers of * Corresponding author. Tel.: +263-4-303211; fax: + 263-4333678. E-mail address: [email protected] (R. Zvauya)
4–5% (v/v) ethanol (Casey et al., 1984). An opportunity exists for process improvements in the conversion of biomass to fuel alcohol which would result in more favourable production economics. Very high gravity (VHG) fermentation is one such process improvement aimed at increasing both the rate of fermentation and ethanol concentration. The technology involves preparation and fermentation of mashes containing 300 or more grams of dissolved solids per litre
0926-6690/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 6 - 6 6 9 0 ( 9 9 ) 0 0 0 2 9 - 1
12
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(Thomas et al., 1993). Under appropriate environmental and nutritional conditions, Saccharomyces cere6isiae can produce and tolerate high concentrations of ethanol (Thomas et al., 1995). VHG technology exploits the observation that growth of Saccharomyces yeast is promoted and prolonged when adequate but very low levels of oxygen are present and when assimilable nitrogen levels are not limiting (Casey and Ingledew, 1986). Use of VHG technology allows considerable saving of water, reduces distillation costs and allows more alcohol to be made with given plant capacity and labour costs (Thomas et al., 1995). The technology reduces capital costs, lowers energy cost per litre of alcohol and reduces the risk of bacterial contamination. Another advantage is an increase in opportunities for harvest of high protein spent yeast. Previous work on VHG fermentation of wheat mashes, sugarcane juice and molasses showed that the technology allows for production of up to 23% (v/v) alcohol in batch fermentations (Thomas et al., 1993, 1994, 1995; Jones et al., 1994). Although more fuel alcohol is produced from sugarcane sources than from any other material (Jones et al., 1994), it would be interesting to apply VHG technology to various other substrates and determine alcohol production. Field crops offer potential sources of fuels with sweet stem sorghum offering promise as a large scale energy crop based on its genetic diversity, climatic adaptation, biomass and sugar production (de Mancilha et al., 1984; Bulawayo et al., 1996). Due to their drought tolerance, grain sorghums are grown in semi-arid regions of Zimbabwe. A major drawback of grain sorghum as a food source is the high levels of polyphenols (tannins) associated with certain varieties of sorghum grain. High levels of polyphenolic compounds, mainly proanthocyanidins, have been shown to demonstrate several unfavourable characteristics including reduction in nutritional quality (Strumeyer and Malin, 1975), brewing quality and the rate of fermentation for fuel ethanol (Mullins and Nesmith, 1987). It is therefore necessary to identify ways of utilising sorghum which will improve household incomes of small scale farmers in semi-
arid regions thus improving food security. There are no reports of the application of VHG technology to produce higher concentrations of fermentation ethanol from sorghum. The aims of this study were to determine fuel ethanol production using a mixture of sweet stem sorghum juice and milled sorghum grain as a substrate for VHG fermentation and to determine any changes in levels of proanthocyanidins of the high tannin sorghum cultivar during fermentation.
2. Materials and methods Stalks of sweet stem sorghum were obtained from fields in Chiredzi, Zimbabwe. The crop was harvested, defoliated and the stalks cut at the nodes. Juice from the stalks was extracted using laboratory scale blenders and kept frozen at − 20°C until required for use. A high tannin, bird resistant sorghum variety (DC-75) and a low tannin line (SV-2) were obtained from Research and Specialist Services (R&SS), Harare, Zimbabwe. The yeast strain used in this study was a Saccharomyces cere6isiae (N96) strain.
2.1. Fermentation media The juice used for the cofermentations was supplemented to give the following concentrations (g/l); urea, 1.6; (NH4)2HPO4, 1.2. For VHG fermentations, the juice was freeze-dried to obtain VHG levels of sucrose (34 g/100 ml). For the cofermentations, sucrose (250 g/l) was added to the juice to obtain VHG levels. Milled sorghum grain, both malted and unmalted, (87.5 g), was autoclaved at 121°C for 15 min and added to 500 ml of autoclaved juice. The final pH of all the media used was adjusted to 4.5 using 0.5 M H2SO4 before autoclaving.
2.2. Fermentation Saccharomyces cere6isiae (N96) was maintained on malt extract agar and stored at 4°C. Precultures were prepared by inoculating a loopful ofyeast cells into 10 ml of supplemented juice in 25-ml universal bottles. The bottles were incu-
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13
bated at 30°C and 150 rpm for 48 h in a shaking incubator. Fermentations were carried out in 1-L Erlenmeyer flasks with a working volume of 500 ml juice, and 87.5 g milled grain for the cofermentations. The flasks were inoculated with 10 ml precultures and incubated at 30°C and 150 rpm for 96 h. Samples were collected at 10 – 12-h intervals. A subsample was freeze-dried for tannin analysis while another subsample was left for microbial analysis. The rest of the sample was centrifuged at 3000 rpm for 10 min and the supernatant kept at −20°C until required for analysis.
2.3. Analysis For microbial analysis, samples were serially diluted using peptone water and spread in duplicates on Rose Bengal Chloramphenical agar plates for yeast colony counts. The plates were incubated at 30°C for 48 h. Dissolved solids were estimated by measuring the refraction at room temperature of centrifuged sample using a sugar refractometer. Dissolved solids were recorded as grams of dissolved solids (expressed as sucrose) per 100 ml. Proanthocyanidin levels were determined on 70% acetone extracts of the freeze-dried samples using the butanol-HCl method (Reed et al., 1987). Ethanol concentrations were measured
Fig. 1. Changes in dissolved solids during normal cofermentation of sweet stem sorghum juice and sorghum grain meal. Media components: — —, malted DC-75 meal + juice; — —, DC-75 meal+juice; — —, malted SV-2 meal + juice; — — , SV-2 meal +juice.
Fig. 2. Changes in dissolved solids during very high gravity cofermentation of sweet stem sorghum juice and sorghum grain. Media components: — — , malted DC-75 meal + juice; — — , DC-75 meal +juice; — — , malted SV-2 meal + juice; — — , SV-2 meal +juice.
by gas chromatography using a PEG 20M column. The column temperature and gas flow rate were 55°C and 10 ml/min respectively. Free amino nitrogen (FAN) was measured colorimetrically using the ninhydrin method (European Brewery Convention, 1987) with glycine as the standard.
3. Results and discussion All results are means of three independent experiments. Figs. 1 and 2 show the changes in dissolved solids during normal and VHG fermentations, respectively. Not all the dissolved solids in the media were utilisable by the yeast. Under VHG conditions, about 8 g/100 ml of dissolved solids remained in the fermentation media after ethanol production had ceased, while under normal conditions 4–5 g/100 ml of dissolved solids remained unused in the fermentation media. There have been reports that with increasing fermentation temperature, above 25°C, the amount of sugar that can be fermented decreases (Jones and Ingledew, 1994b). In this study, a temperature of 30°C was employed as in the fuel industry. ‘Stuck’ fermentation may have been due to thermal stress which has been reported with fermentation of wheat mashes (Jones and Ingledew,
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1994b). Fermentation was carried out in shake flasks, where conditions may not be optimal to ensure complete fermentation. Further research is necessary to determine the optimum processing parameters to attain complete utilisation of the dissolved solids. Tables 1 and 2 show microbial counts from the various cofermentations under VHG and normal fermentations, respectively. In all the fermentations, yeast cell multiplication occurred during the course of fermentation. Yeast cell multiplication and the time required to attain maximum cell numbers in fermentation varied with the substrates used. After 57 h of fermentation, loss of yeast viability was observed under normal fermentation conditions and in the VHG media containing unmalted sorghum meal. Ethanol production had ceased at this stage. Under normal fermentation conditions, the loss of yeast viability may be due to limitation of essential nutrients such as free amino nitrogen which was depleted at 24 h of fermentation (Fig. 6). Under VHG conditions, loss of yeast viability was observed in cases where the unmalted sorghum meal was used, probably due to the lack of hydrolytic enzyme activity and nutrients available in the malts. The maximum ethanol levels obtained under VHG fermentation conditions are about 16.8% (v/v) for the media containing malted sorghum grain meal and 11% (v/v) for the media containing unmalted sorghum grain meal from an initial dissolved solids content of 34% (w/v) (Fig. 3). Malted sorghum grain meal is a better substrate for fermentation than unmalted sorghum grain meal due to the higher nutrient content which develops as a result of the
malting process. Ethanol levels obtained under VHG fermentation conditions are an improvement over the 12.9% (v/v) ethanol produced from VHG molasses at 30°C though not as high as the 18.6% (v/v) achieved at 30°C from the fermentation of VHG wheat mash (Jones and Ingledew, 1994b). However, shake flasks were used in the present experiments while the levels quoted above were obtained using bioreactors. Ethanol levels obtained under VHG fermentation conditions are about three times those obtained under normal conditions (Figs. 3 and 4). The concentration of fermentable sugar in the media, the amount of assimilable nitrogen available and the pitching rate all play important roles in determining the rate of fermentation and the final ethanol yield. There was no cultivar difference observed for ethanol production under VHG fermentation conditions while under normal conditions, higher ethanol levels were recorded for DC-75 malt compared to SV-2 malt. Previous studies have shown that DC-75 produces a better malt quality than SV-2 (Bvochora et al., 1999). After an initial decline, there was an increase in the FAN levels for all the VHG fermentations (Fig. 5). The increase in FAN may be due to the release of nutrients by lysing yeast cells. Yeast cell lysis may be due to exposure to high concentrations of ethanol for long periods of time andosmotic stress exerted by the unfermented sugars (Jones and Ingledew, 1994a). Though the media was supplemented with urea and diammonium phosphate as nitrogen sources, fermentation did not occur to completion. Similar results have been reported for VHG fermentation of wheat at 30°C
Table 1 Microbial analysis of VHG fermentation samples Time (h)
0 9.5 24 33.5 48.5 57.5 96
Yeasts (log cfu/ml) DC-75+juice
DC-75 malt+juice
SV-2 malt+juice
SV-2+juice
8.13 9.01 9.79 10.54 10.78 9.78 9.18
8.20 8.81 9.53 9.69 9.79 10.00 10.30
8.22 8.79 9.53 10.17 10.11 10.18 10.48
7.95 8.76 9.43 10.20 10.28 9.70 7.70
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Table 2 Microbial analysis of ‘normal’ cofermentation samples Time (h)
0 10 24 34 57 73 80 96
Yeasts (log cfu/ml) DC-75+juice
DC-75 malt+juice
SV-2 malt+juice
SV-2+juice
6.72 7.58 8.74 8.81 10.20 9.19 7.95 7.16
6.71 7.63 8.85 9.11 10.40 9.54 8.34 8.36
6.85 7.45 8.55 9.20 10.00 9.46 8.62 7.85
6.82 7.44 8.71 9.14 10.53 8.95 8.08 8.34
(Jones and Ingledew, 1994a). Under normal conditions, an initial decrease in FAN levels was observed up to 24 h then depletion of FAN occurred for the rest of the fermentation (Fig. 6). FAN levels may have limited the fermentation to completion of media under normal fermentation conditions. However, yeast cell lysis may not have occurred under normal fermentation conditions as the increase in FAN levels observed under VHG conditions was not observed. Fig. 7 shows the proanthocyanidin levels during fermentation of various media under VHG conditions. Initial levels of proanthocyanidins were highest in media containing unmalted DC-75 grain meal. Media containing malted DC-75 grain meal had lower initial proanthocyanidin levels showing
that malting reduces the levels of assayable proanthocyanidins in sorghum grain meal. For the medium containing malted SV-2 grain meal, levels of assayable proanthocyanidins were negligible. The results obtained indicate that fermentation reduced the levels of assayable proanthocyanidins in sorghum grain meal. Under normal conditions (Fig. 8), a general decrease in proanthocyanidins (PAs) was observed.
Fig. 3. Changes in ethanol levels during very high gravity cofermentation of sweet stem sorghum juice and sorghum grain. Media components: — —, malted DC-75 meal + juice; — — , DC-75 meal + juice; — —, malted SV-2 meal+ juice; — — , SV-2 meal +juice.
Fig. 4. Changes in ethanol levels during normal cofermentation of sweet stem sorghum juice and sorghum grain. Media components: — — , malted DC-75 meal +juice; — — , DC-75 meal +juice; — —, malted SV-2 meal +juice; — — , SV-2 meal +juice.
4. Conclusion The results of this research show that sweet stem sorghum juice and sorghum grain meal can be
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Fig. 5. Changes in free amino nitrogen levels during very high gravity cofermentation of sweet stem sorghum juice and sorghum grain. Media components: — —, malted DC-75 meal+ juice; —— , DC-75 meal+ juice; — —, malted SV-2 meal +juice; — —, SV-2 meal + juice.
Fig. 7. Changes in water soluble proanthocyanidins during very high gravity cofermentation of sweet stem sorghum juice and sorghum grain. Media components: — — , malted DC-75 meal+ juice; —— , DC-75 meal + juice; —— , malted SV-2 meal + juice; — — , SV-2 meal +juice.
employed to facilitate efficient batch VHG fermentation for fuel ethanol production. VHG fermentation technology, therefore, has potential as a process improvement to increase the profitability of fuel alcohol production from sorghum which would otherwise have limited uses. Levels of proanthocyanidins in sorghum are reduced during fermentation hence high PA cultivars may be used in VHG technology.
Fig. 8. Changes in water soluble proanthocyanidins during normal cofermentation of sweet stem sorghum juice and sorghum grain. Media components: — —, malted DC-75 meal + juice; — — , DC-75 meal + juice.
Acknowledgements Fig. 6. Changes in free amino nitrogen levels during normal cofermentation of sweet stem sorghum juice and sorghum grain. Media components: — —, malted DC-75 meal + juice; — —, DC-75 meal+ juice; — —, malted SV-2 meal + juice; — — , SV-2 meal + juice.
The work was supported by grants from the McKnight Foundation Collaborative Research Program, USA and the University of Zimbabwe Research Board. We thank Dr I. Ncube for help with G.C. analysis.
J.M. B6ochora et al. / Industrial Crops and Products 11 (2000) 11–17
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