ELSEVIER P I I : S 0 9 6 0 - 8 5 2 4 ( 9 7 ) 0 0 0 0 3 - 5

Bioresource Technology 60 (1997) 9-13 © 1997 Elsevier Science Limited

All rights reserved. Printed in Great Britain 0960-8524/97 $17.00

PRESERVATION OF SUGAR CONTENT IN ENSILED SWEET SORGHUM

J. Schmidt , a J. Sipocz, a I. Kaszfis, a G. Szakfics, b A. Gyepes h & R. P. Tenge rdy ~*

a Department of Animal Nutrition, Pannon University of Agricultural Sciences, H-9200 Mosonmagyarrvdr, Hungary bDepartment of Agricultural Chemical Technology, Technical University of Budapest, H-1521 Budapest, Hungary

"Department of Microbiology, Colorado State University, Fort Collins, CO 80523-1677 USA

(Received 1 November 1996; accepted 18 December 1996)

Abstract Ensiling in the presence of 0"5% formic acid preserved the sugar content of sweet sorghum, while in enzyme- assisted ensiling (ENLAC) with in situ produced enzymes, the sugar loss in 30days was 28.6%. The ENLAC silage contained 1.5% lactic acid and 0.6% ethanol which might be recovered as value-added secondary products, in addition to the high quality residue as animal feed. Overall the formic acid ensiling is the better choice for sugar preservation, storage and prolonged bioprocessing for biofuel production. © 1997 Elsevier Science Ltd.

Key words: Sweet sorghum, ensiling, sugar preserva- tion, formic acid, in situ enzymes.

INTRODUCTION

Sweet sorghum is a promising energy crop that can be cultivated worldwide in temperate climates (Smith et al., 1987). It is genetically diversified and drought tolerant, with an average biomass yield of 40-60 metric ton (MT)/ha and about 4-5-6-5 MT/ha sugar yield, approaching that of sugar beet. On irri- gated, good quality soil in Greece, Dalianis et al. (1995) reported a 140 MT/ha green biomass yield potential with 12 MT/ha sugar yield. The production costs of sweet sorghum are only about 60% of the sugar-beet production costs. In the European Com- munity, sweet sorghum is one of the preferred crops for the development of renewable energy sources (Gosse, 1995). The extracted sugars may be fermen- ted to ethanol or acetone-butanol for use as fuel additives.

The bioprocessing of sweet sorghum to biofuel is hampered by the short harvest time and the poor storability of the green crop (up to 50% post harvest sugar loss in 4-6 days; Eiland et al., 1983), requiring

*Author to whom correspondence should be addressed.

large capacity presses or extractors for immediate processing (Coble et al., 1983).

Preservation of the sugar content is a major con- cern for the efficient and economical bioprocessing of sweet sorghum. In this paper two methods are compared for sugar preservation. One method, enzyme-assisted ensiling (ENLAC) that may reduce sugar losses to about 30%, makes possible the more efficient extraction of sugars in sugar industry-type diffusers and produces valuable secondary products such as lactic acid, ethanol and high-quality silage (Linden et al., 1987; Schmidt et al., 1995; Tengerdy et al., 1995). This process can be economical only if inexpensive in situ produced enzymes are used in ensiling (Tengerdy et al., 1996). The other method is ensiling with formic acid. Formic acid is a selective inhibitor of certain bacterial populations, including lactic acid bacteria and Clostridia but not of yeast (Lindgren et al., 1983; Narasimhalu et al., 1992). Formic acid preserves the sugar content but does not enhance its extractability or the nutritional value of the treated crop. Henk & Linden (1996) used formic acid to inhibit bacterial action in the solid- substrate fermentation of sweet sorghum to ethanol.

METHODS

The chemical composition of the sweet sorghum, Honey-Marion hybrid, used for the ENLAC experi- ment, on dry weight (DW) basis was: reducing sugar (after inversion) 446.1g/kg, cellulose 229-1g/kg, hemicellulose 273.7 g/kg, lignin 30-2 g/kg, crude pro- tein 62.2 g/kg, ash 40.6 g/kg.

The chemical composition of the sweet sorghum Honey Marion hybrid used in the formic acid ensil- ing experiment was: reducing sugar (after inversion) 499-6 g/kg, cellulose 201.5 g/kg, hemicellulose 270.5 g/kg, lignin 27.0 g/kg, crude protein 60.1 g/kg, ash 39.3 g/kg.

10 J. Schmidt et al.

Ensiling Sweet sorghum was ensiled immediately after har- vest, chopped to about 0.5-1.0cm particle size, mixed with additives, and 650 g was packed into a 1 1 jar, equipped with a gas vent that prevented air access. The moisture content of the substrate was 75%. Twenty jars were packed per treatment group. The jars were incubated at 25+2°C. On days 7, 30, and 60, 5 jars per treatment were opened for assays. Each assay was done by duplicate determinations.

Silage additives Lactic acid bacteria (LAB) were added in the form of a commercial preparation (SILAFERM, Monor, Hungary), containing 95% Lactobacillus plantarum and 5% Streptococcus faecium with a total count of 2"5 X 10 9 colony-forming units (CFU) per g. The level of addition was 1.0 x 105 CFU/g material.

The commercial enzymes Celluclast 1.5 L and Viscozyme 120 L (NOVO, Denmark) were added at 0.025% level each (wet basis); this corresponded to a cellulase activity of 0.02 filter paper units (FPU) per g wet material. The in situ enzyme (ISE) was prepared on extracted sweet sorghum silage by solid- substrate fermentation with a Gliocladium sp. (TUB F-498, Collection of G. Szakfics, Hungary) as described elsewhere (Tengerdy et al., 1996). The cel- lulase activity of the fermented substrate was 1.3 FPU/g DW and xylanase activity 540 IU/g DW. This material was added to the silo at the 5% level (0.016 FPU/g) and the 10% level (0.032 FPU/g) on wet basis. Formic acid was added at concentrations of 0.5, 0.75 and 1.0% (wet basis).

Assays The dry weight, crude protein, crude fiber, crude lipids and crude ash content of green sorghum and sorghum silage was determined by standard methods (Association of Official Analytical Chemists, 1995). Fiber analysis was performed as described by van Soest & Wine (1968). The lactic acid, acetic acid and ethanol content of silages was determined by gas chromatography (Boettcher, 1982). Reducing sugar was determined by the dinitrosalicylic acid method (Miller, 1959). NH3 was determined with an NH3-sensitive electrode (Radelkis OP 264/2, Hungary). Detailed sugar analysis was performed by HPLC (Biotronic 2000, Biotronik Wissenschaftliche Ger~ite GmbH, Frankfurt/Main, Germany). Plant material was extracted with 0.01 N NaOH, centri- fuged, and the supernatant was injected onto a 250 x 4 mm Aminex HPX-87 C-type column, eluted with a mixture of acetonitrile:water = 80:20. Cellu- lase activity was determined by the filter-paper assay method (Ghose, 1987) and xylanase activity as described by Bailey et al. (1992). Statistical analysis of data was performed by the program Statistica of StatSoft, Inc. (2325 East 13th Street Tulsa, OK 74104). The 0 h control was always the respective treatment mixture.

RESULTS AND DISCUSSION

Lactic acid fermentation The LAB silage additive was beneficial for the fer- mentation, because it initially provided a large number of homofermentative LAB and competi- tively suppressed non-desirable microorganisms (Table 1). The presence of enzymes or enzyme- enriched fungi did not interfere with the fermentation; the fungi were inactive under anaero- bic conditions. The enzymes further helped the fermentation in-the homofermentative direction; the best lactic acid:acetic acid ratio (4:1) was achieved in the 10% ISE+LAB supplemented 30 day silage with the lowest ethanol content (0.56%). The relatively high N content of the ISE additive slightly increased the amount of NH3 in the silage.

Fiber digestion in enzyme-assisted ensiling (ENLAC) The CeUuclast+Viscozyme enzyme treatment or the in situ enzyme treatment reduced the fiber content of the silage during 30 day ensiling (Table 2). The 10% ISE+LAB treatment reduced cellulose by 9.5% and hemicellulose by 17.8%. These results corro- borate earlier observations that in ENLAC very low enzyme levels cause an efficient cell-wall degrada- tion (Tengerdy et al., 1991; Tengerdy et al., 1995). The reason for this is that at low pH and anaerobic conditions, and with a long reaction time (30 days), cell wall hydrolysis proceeds efficiently even at very low enzyme levels, making the process economical for agrobiotechnological applications. The conse- quence of partial cell-wall hydrolysis is that the available amount of sugar is higher in ENLAC sila- ges than in conventional silages. The hydrolyzed sugars partially compensate for the sugar consump- tion by the LAB; this can be seen in Table 3. The highest reducing sugar content was observed in the 10% ISE+LAB treatment (7-4%) on day 30; the sugar loss was only 28.6%, compared with 60.8% in the LAB-treated silage. Since the respiratory sugar loss in freshly harvested sweet sorghum may reach 50% in 4-7 days, the enzyme-assisted ensiling is justified for reducing sugar losses, on the other hand it increases the time window for bioprocessing by only 30 days. After 30 days the sugar losses are high, due to the continued fermentative activity of yeasts, encouraged by the high sugar concentrations. Henk & Linden (1992) also observed high yeast activity and ethanol production up to 8 g/kg DW in enzyme- assisted ensiling of sweet sorghum.

Potentially, the ethanol and lactic acid may be recovered from silage as value-added secondary products and the remaining pulp is a medium- quality animal feed.

In these experiments, the in situ enzyme source was at least as effective as, or more effective than, commercial enzymes. Since in situ enzyme can be

Sugar in ensiled sorghum

Table 1. Fermentation parameters in ENLAC silage

11

Treatment Day pH LA, AC, EtOH, NH3, % % % mg/100 g silage

Control 7 3.78 + 0.01a 0.90 + 0.05a 0.53 + 0.09a 0.98 + 0.08a 6.49 + 0.66a 30 3.74+0.09a 1.11_0.20a 0.44+0.15a 1.85+0.10a 7.19_+ 2.24a 60 3.33_0.02a 1.53+0.57 0.86_0.12a 1.96+0.19 12.80 +0.50a

C + V 7 3.80 _ 0.04 0.82 + 0.07 0.55 + 0.08 1.36 + 0.11 6.23 ___ 0.62 30 3.58+0.04a 1.30__+0.15 0.69+0.04a 1.75+0.20 11.76_0.38a 60 3.29 + 0.07 1.92 _ 0.40 0.87 _ 0.15 1.80 + 0.33 13.61 _ 0.65

5% ISE 7 3.75 +0.01a 0.90+0.02 0.60+0.04 0.83+0.17 9.53_ 0.73a 30 3.58+0.01a 1.29+0.11 0.62+0.03 1.33+0.41 13.51_0.66a 60 3.49 _ 0.15 1.89 + 0.83 0.72 _ 0.04 1.98 + 0.43 12.30 _+ 4.52

10% ISE 7 3.75 +0.02a 0.82___0.06 0.57+0.02 0.15 +0.01a 11.23+0.21a 30 3.55 +0.01a 1.31+0.10 0.62+0.03 0.85+0.12a 15.44+ 1.01a 60 3.52+0.07a 1.95__+0.61 0.62+0.10 1.96+0.27 17.31 + 1.00a

LAB 7 3.67___ 0 .01a 1.19+0.06a 0.46___0.07 0.93-t-0.25 5.91+0.57 30 3.68 _+ 0.03 1.20 + 0.17 0.45 _ 0.06 1.58 ___ 0.32 8.94 _+ 0.59 60 3.31+0.03 1.91+0.74 0.48_0.16a 1.70+0.37 10.94_ 0.88a

C+V LAB 7 3.66_0.01a 1.06 +0.12a 0.42__+0.01 1.03+0.09 6.25+ 11.82 30 3.68 + 0.06 1.38 _ 0.24 0.45 ___ 0.05 1.71 + 0.23 11.32 + 1.74a 60 3.60+0.05a 1.77+0.49 0.35+0.11a 2.18+0.49 7.27+2.69a

5% ISE LAB 7 3.62+0.02a 1.07+0.04a 0.39+0.07 1.30___0.35 7.18__+0.43 30 3.58_0.01a 1.31+0.11 0 . 4 2 _ + 0 . 0 8 1 . 8 0 _ + 0 . 3 1 11.65_+0.83a 60 3 .60_+0.16a 1.54_+0.39 0.40_+ 0 .09a 2.56_+0.59 10.81 _+3.09

10% ISE LAB 7 3.58+0.01a 1 .33-+0.03a 0 .38-+0 .02a 0.43+0.07a 10.62_+0.64a 30 3.56_0.01a 1 .47-+0.17a 0 . 3 7 - + 0 . 0 3 0.56-+0.18a 14.72_+ 0.33a 60 3 .51_+0.06a 2.15+0.60 0.48-+ 0 .16a 2 .04-1-0 .24 12.97_+0.38

LA = lactic acid; AC = acetic acid; C = 0.025% Celluclast; V = 0.025% Viscozyme; ISE = in situ enzyme; LAB = lactic acid bacteria 1.0 x 105 CFU/g. Values followed by the same letters indicate significant differences from the control at P < 0.05. Data are on wet weight basis.

produced on recycled process residues on site by farm-level technology, the enzyme costs of the pro- cess may be reduced about 100-fold (Tengerdy et al., 1996), making the application of in situ enzymes very economical for agrobiotechnological operations.

Ensiling in the presence of only 0.5% formic acid completely preserved the original sugar content of green sorghum, even increased it slightly, possibly by partial hydrolysis of starch in the crop (Table 4). Formic acid suppressed fermentation activity by lac- tic acid bacteria and yeast, only a minimal amount of lactic acid was formed and no ethanol (Table 5).

The fiber content of the silage did not differ from that of green sorghum (data not shown).

The presence of formic acid up to 0.3% did not inhibit the alcoholic fermentation of the extracted juice. Since in the extraction process the juice is diluted about two-fold, the 0.5% treatment level would not inhibit the fermentability of the extracted juice.

The economic evaluation of the two processes shows that in the bioprocessing of an average sweet sorghum crop with 10% sugar content by formic acid ensiling about 100kg reducing sugar or 50kg

Table 2. Fiber contents of ENLAC silages

Treatment Dry matter, NDF, g/kg ADF, g/kg ADL, g/kg Cellulose, g/kg Hemicellulose, g/kg g/kg wet wet weight wet weight wet weight wet weight wet weight weight

Green sweet sorghum 233.57 124.48 60.57 7.06 53.51 Green sorghum +5% ISE 233.33 126.30 62.22 8.00 54.22 Green sorghum +10% ISE 233.12 134.03 66.77 9.30 57.47 Control 228.69 121.89 59.92 6.98 52.94 C + V 220.90 115.15 56.61 7.28 49.33 5% ISE 205.02 117.21 59.55 8.17 51.38 10% ISE 215.41 124.41 63.10 9.61 53.49 LAB 220.20 120.66 59.87 6.85 53.02 C+V LAB 198.56 113.12 56.53 8.26 48.27 5% ISE LAB 196.07 112.42 58.18 7.90 50.28 10% ISE LAB 201.00 117.13 61.85 9.87 51.98

63.91 64.08 67.26 61.97 58.54 57.66 61.31 60.79 56.59 54.24 55.28

C = 0.025% Celluclast; V = 0.025% Viscozyme; ISE = in situ enzyme. LAB = lactic acid bacteria 1.0 x 105 CFU/g. Ensiling for 60 days. NDF = neutral detergent fiber; ADF = acid detergent fiber; ADL = acid detergent lignin.

12 J. Schmidt et al.

e thanol may be obta ined per M T of green crop with a value of about $100/MT ($2/kg E t O H ) , c o m p a r e d to the value of the green sorghum ($25/MT) and the es t imated processing cost ($25/MT), thus represent- ing a net gain of $50/MT. In the E N L A C process only about 35 kg e thano l /MT may be obta ined with

a value of $70/MT, assuming a 30% sugar loss. The E N L A C process, in contrast , would be economical , if the 2% lactic acid p roduced (a value of $70/MT, $3.5/kg lactic acid) could be recovered. The ensiled residue represents a value of about $5/MT, giving a potent ia l net gain of $95/MT.

Table 3. Sugar contents of ENLAC silages

Treatment Day Reducing Xylose, Fructose, Glucose + mannitol, sugar, % % % %

Control 0 10.42" 7 3.14 ___ 0.31a 0.04 +- 0.009a 1.02 + 0.21a 4.57 +- 0.67

30 2.44 4- 0.27a 0.04 +- 0.008a 1.07 4- 0.26 4.28 + 0.46a 60 1.60 +- 0.19a 0.08 4- 0.018a 0.37 + 0.12 3.10 +- 0.53

C + V

5% ISE

10% ISE

LAB

C + V LAB

5% ISE LAB

10% ISE LAB

7 3.63 +0.47 0.05 +- 0.011 0.97 +-0.24 3.564-0.51 30 4.08 4- 0.40a 0.06 +- 0.017 1.02 4- 0.23 4.57 ___ 0.48 60 2.61 + 0.31a 0.13 4- 0.042a 0.34 4- 0.09 3.78 + 0.69

7 5.53 +- 0.44a 0.15 +- 0.033a 1.38 +- 0.39 5.78 ± 0.58a 30 5.35 +- 0.48a 0.23 +- 0.051 a 0.87 +- 0.38 5.84 + 0.51 a 60 2.27 _ 0.27a 0.26 + 0.055a 0.26 +- 0.13 3.49 4- 0.43

7 5.63 +- 0.46a 0.15 +- 0.029a 1.20 4- 0.28 5.81 + 0.52a 30 6.06 4- 0.5 la 0.29 +- 0.055a 1.06 +- 0.25 6.34 _+ 0.57a 60 3.08 _ 0.25a 0.32 + 0.069a 0.35 +- 0.09 3.72 _ 0.46

7 4.96 + 0.51a 0.06 + 0.019 1.55 +- 0.26a 3.91 _+ 0.43 30 4.08 4- 0.37a 0.06 +__ 0.021 0.90 +- 0.27 3.91 +- 0.38 60 3.08 +- 0.35a 0.07 4- 0.027 1.01 + 0.29 3.51 + 0.36

7 4.37 _ 0.47a 0.07 _ 0.034 1.53 +- 0.29a 3.74 _ 0.39 30 4.49 +- 0.42a 0.07 -+ 0.026 0.93 4- 0.31 4.04 _+ 0.38 60 3.74 +- 0.40a 0.09 + 0.013 0.97 +- 0.22 3.20 +- 0.25

7 5.58 4- 0.36a 0.06 ± 0.021 1.59 + 0.28a 3.66 + 0.42 30 6.62 4- 0.71a 0.21 +- 0.043a 1.59 + 0.27a 4.29 4- 0.39 60 3.80 +- 0.47a 0.25 4- 0.067a 0.86 -+ 0.21 3.09 + 0.28

7 6.87 + 0.62a 0.13 + 0.016a 2.57 +- 0.39a 5.77 4- 0.53a 30 7.44 4- 0.66a 0.25 -+ 0.054a 2.03 4- 0.35a 5.95 +- 0.57a 60 3.52 +- 0.3 la 0.31 + 0.069a 0.95 4- 0.17 3.61 +- 0.27

C = 0.025% Celluclast; V = 0.025% Viscozyme; ISE = in situ enzyme; LAB = lactic acid bacteria 1.0 x 105 CFU/g. Mean values are shown on wet weight basis. Values followed by the same letters indicate significant differences at P < 0.05. *The 0 h reducing sugar data were taken after inversion.

Table 4. Sugar content of formic acid silage

Treatment Day Reducing Xylose, % Fructose, % Glucose, % Glucose+mannitol, sugar, % %

Green sorghum 0 13.83" Control 15 5.61 +0.62a 0.09+0.022 1.41 +0.31a - -

30 4.86+0.51a 0.10+0.020 0.79+0.18a - - 60 3.36 + 0.32a 0.06 + 0.014 0.39 ___ 0.09a - -

0.50% formic acid 15 12.82+ 1.15a 0.08+0.011 6.27+1.24a 6.16+0.92 30 13.52+1.24a 0.094-0.027 6.16+1.31a 6.57+__0.86 60 14.25+1.56a 0.16+_0.062 6.16+1.07a 6.63+1.17

0.75% formic acid 15 13.24 +- 1.48a - - 7.04_ 1.53a 6.85 +- 0.81 30 13.80 +- 1.35a 0.05 4- 0.012 6.44 + 0.96a 6.78 +- 0.97 60 14.83 + 1.47a 0.06_+0.017 6.02 +- 1.35a 6.43+-0.73

1.00% formic acid 15 14.55+1.75a - - 6.90+-1.11a 6.92+-0.76 30 14.73 +_ 1.66a - - 6.42 +- 1.39a 6.74 +- 0.07 60 15.96 +- 1.59a 0.06 +- 0.016 5.88 + 0.88a 6.34 ± 1.22

7.01 +0.91 6.86 + 0.98 3.69 _ 0.75

Data are on wet weight basis. Values followed by the same letters indicate significant differences at P < 0.05. *The reducing sugar data were taken after inversion.

Sugar in ensiled sorghum

Table 5. Fermentation parameters in formic acid silage

13

Treatment Day pH LA, % AC, % EtOH, % NH3, mg/100 g silage

Control 15 3.70 _ 0.042a 0.92 + 0.062a 0.76 + 0.085a 0.33 + 0.063 11.75 + 2.07a 30 3.62__+ 0.0036a 1.11 _0.066a 0.77_+0.041a 0.53+0.122 18.01 +0.80a 60 3.61 _ 0.041a 1.18 + 0.099a 0.86 + 0.176a 1.09+0.262 15.39_+0.33a

0.50 % formic acid 15 3.72+0.048 0.08__+0.015a 0.10+0.005a - - 4.39___0.46a 30 3.70-t-0.042 0.15_ 0.024a 0.10 +0.005a - - 7.08+0.13a 60 3.74+0.031 0.13 +_0.037a 0.10 + 0.006a - - 8.78+0.42a

0.75% formic acid 15 3.42_0.053a 0.14+0.045a 0.08 __+ 0.010a - - 3.49+0.08a 30 3.48 +0.014a 0.15 +0.031a 0.09 + 0.008a - - 5.86+0.06a 60 3.50-t-0.082 0.14 +0.029a 0.11 + 0.005a - - 6.74+0.51a

1.00% formic acid 15 3.31 _ 0.024a 0.11 +0.029a 0.08+0.001a - - 2.84+0.03a 30 3.33__+0.011a 0.16+0.031a 0.10+0.012a - - 4.90+0.09a 60 3.37_ 0.005a 0.10 _+ 0.014a 0.10___ 0.006a - - 5.41 ___ 0.02a

LA = lactic acid; AC = acetic acid. Mean values are shown on wet weight basis. Values followed by the same letter indicate significant differences at 0.05%.

If the purpose of bioprocessing is primarily bio- fuel production, the formic acid ensiling is preferable for its technological simplicity and higher sugar yield.

ACKNOWLEDGEMENTS

This research was supported by a U S A - H u n g a r y Science Cooperat ion Grant by NSF-INT-9214903, by NSF grant BCS-9115515, by a U S A - H u n g a r y Joint Board Grant No. 307/92 and by a N A T O Linkage Grant L G 920571.

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