Biomass 15 (1988)259-268

CO 2 Enrichment and its Relationship to Bioconvers ion of Cel lulosic B iomass of Sweet Potato (Ipomoea batatas L.)

into Fermentable Sugars

S. Bhattacharya, a J. F. Eatman, b P. K. Biswas b & M. E. M. Tolbert a

aCarver Research Foundation, bGeorge Washington Carver Experiment Station, Tuskagee University, Tuskagee, Alabama 36088, USA

(Received 18 August 1987; revised version received 25 January 1988; accepted 28 January 1988)

ABSTRACT

Sweet potatoes (Ipomoea batatas L. (Lam) 'Georgia-Jet') were grown in open field plots and open top chambers at CO 2 concentrations of 354, 431, 506, and 659 ~ l liter -1 for 90 days. The leaves and stems after the harvest were used as substrates for the production of fermentable sugars. Elevated CO 2 concentrations increased the cellulose content of stems, being most pronounced at 506 kt l liter- i. Hemicellulose content of leaves and stems as well as lignin content of stems decreased as a result of CO 2 enrichment. The increase in cellulosic biomass in plants grown in CO 2 enriched environment resulted in increased conversion of cellulose into fermentable sugars. The saccharification was greater in stems than in leaves. It was also found that chemical pretreatment of stems and leaves enhanced the enzymatic hydrolysis and the yields of glucose were higher than those from untreated stems and leaves.

Key words: COz, biomass conversion, sweet potato.

I N T R O D U C T I O N

The global carbon dioxide concentrat ion is gradually increasing due to fossil fuel consumption, and deforestation. The 1985 atmospheric level of CO 2 was approximately 344 pl l i ter- t of air (344 ppm), and the rate

259 Biomass 0144-4565/88/S03.50- © 1988 Elsevier Applied Science Publishers Ltd, England. Printed in Great Britain

260 S. Bhattacharya, J. F. Eatman~ P. K. Biswas, M. E. M. Tolbert

of increase is currently about 1.5 ppm per year. The increase in global atmospheric CO 2 concentration is now well documented and may have doubled by the year 2025.1 Kimball z reported that overall crop yields would increase by 33% with a doubling of atmospheric CO2 concentra- tion. Since most of the dry weight of plants is derived from the reduction of CO2 to carbohydrates by photosynthesis, the concentration of CO2 in the air can directly affect plants? Investigators have reported increases in net photosynthesis, 4,5 dry matter production, 6 and yield of plant species including field crops in response to an increase in CO2 concentration. 7 It has been shown that total dry matter of plants, leaf area and yield of tuberous roots of sweet potatoes increased at high CO2 concentrations. 8 The long-term consequences of increased availability of photosynthetic products in different plant parts caused by elevated CO2 are not yet fully understood.

Most agricultural residues, regardless of source, contain hemicellu- lose, cellulose and lignin. Biomass consists of collectible plant derived materials that are abundant, inexpensive and potentially convertible to feedstock chemicals by fermentation process. The hydrolysis of cellulose and the resulting product, glucose, plays a key role in the conversion of renewable resources to foods, fuels and chemical feedstock. The stems and leaves of sweet potatoes are an attractive source of alcohol fuel because they do not have much agricultural value and are considered as a waste at potato harvest. Numerous pretreatments have been developed in an effort to increase the efficiency of enzymatic saccharification. 9 The effectiveness of various pretreatment methods on the enzymatic hydroly- sis of sweet potato biomass has already been examined. ~°,1~ Our objec- tive in this study was to evaluate the effects of elevated levels of CO2 on the bioconversion of cellulosic biomass of sweet potato into fermentable sugars using Trichoderma viride cellulase.

MATERIALS AND METHODS

Sweet potato (Ipomoea batatas L. (Lam) 'Georgia-Jet') plants were grown in open field plots and open top chambers under different CO2 concentrations. The experiment was conducted at Tuskegee University's George Washington Carver Agricultural Experiment Station. The soil was a Norfolk sandy loam (Typic Paleudult), with a pH ranging from 6.2 to 6.9.

Open top chambers ~2-~4 were used to expose field-grown plants to elevated concentrations of CO2. Each chamber consisted of a structural

CO_~ enrichment and biomass conversion 261

aluminum frame 3 m in diameter and 2.4 m in height covered with a 0.20 mm clear polyvinyl chloride plastic film (Roll-A-Glass, Livingston Coat- ing Corporation, Charleston, NC). The opening at the top of the chamber was partially reduced by a 45 degree frustrum. The bottom half of each chamber was double-walled; the inside wall was perforated and served to distribute air uniformly into the chamber. Ambient air was supplied to this duct by a fan, mounted in a sheet metal box. Carbon dioxide was injected into the box ahead of the fan.

The system for dispensing CO2 and monitoring CO2 in the open top chambers was the same as used in other similar studies. ~2-15 Carbon dioxide was dispensed to the open top chambers from a liquid CO2 storage unit through a dispensing manifold that allowed the flow of CO2 to each chamber to be adjusted independently. Air samples were drawn continuously from each chamber and open field plot to a sampling manifold by applying a vacuum pump. A timer and a separate sample pump were used to divert one sample at a time through two absolute CO2 infrared gas analyzers (Binos Model 43-200 1/2, Inficon Leybold- Heraeus, East Syracuse, NY). Each plot was sampled every 32 min. Twice each day the gas analyzers were calibrated against a series of tanks of known CO2 concentration, and the CO 2 concentrations in the chambers were readjusted.

Season-long mean concentrations of CO2 inside the chambers were calculated using a sub-sample of the CO2 data consisting of readings taken every 2 h during 24-h periods which were taken every 9 days throughout the experiment. The values reported here are daytime (0700 to 1700 CST) means. Sweet potatoes were exposed to 5 treatments in a randomized block design with 3 replicate blocks. The blocks were arranged in rows running perpendicular to the direction of the slope of the field in order to randomize the distribution of the replicates with respect to soil drainage. The 5 treatments consisted of 4 plots with open top chambers containing mean daytime CO2 levels of 354 (ambient), 431,506 and 659/A liter-~ and an open plot with no chamber.

Plants that were 3 months old and about 20-25 cm in length were transplanted into the field. Plants were placed 30 cm apart in rows that were 90 cm apart. The rows were raised 20 cm above the surrounding soil. The open top chambers were placed in the field after planting such that there were 2 rows of 10 plants each inside each chamber.

Water was applied to the field by drip irrigation as required, to supple- ment natural rainfall; fertilizer was applied at the rate of 14.7 kg of N, 14.7 kg of P and 22"0 kg K per ha. One-half of the N and all of the P and K were applied at the time of planting; the other half of the N was side-

262 S. Bhattacharya, J. F. Eatman, P. K. Biswas, M. E. M. Tolbert

dressed 4 weeks after planting. Weeding was done by hand. No insecti- cide was needed. Nematode counts, taken at the middle and at the end of the growing period, were insignificant.

Harvest of sweet potato plants

The vegetative portion of sweet potato plants from 15 plots were harvested 90 days after planting when tuberous roots were fully developed. Ten plants from each plot were randomly harvested and leaves were separated from runners. These were dried in an oven at 70°C for 48 h and ground to a fine powder and seived. The fraction passing the 250 micron openings of spectramesh was designated as the standard substrate.

Chemical analysis

Cellulose content of stems and leaves was determined according to the procedure of Garg & Neelakantan ~6 with minor modifications. Cellulose content was obtained from a cellulose standard prepared from cellulose carried out through the same procedure. Lignin content was determined by the ultra violet acetyl bromide method 17 and hemicellulose according to the method of AdamsJ 8

Pretreatment of sweet potato biomass

Ground and sieved substrates were suspended in 10 ml of 100% methanol (stems) or a 1:1 ratio of methanol and acetone (leaves) for delignffication and removal of pigments and kept for 3 h at room temperature. The residue was washed successively with water and suspended in 1% (v/v) H2S Q and kept on a water bath at 80°C for one hour to remove hemicellulose. The residue was washed until neutral and was then heated with 75% ZnCI 2 (w/v) in 0"5% (v/v) HC1. After heating, the resultant solution was cooled and cellulose was precipitated with acetone, 19 and stored in moist form at 4°C until used.

Enzymatic hydrolysis of cellulosic substrates

The treated or untreated cellulosic substrates were added to commercial cellulase enzyme (activity of 4 units mg - l solid) derived from Tr icoderma viride obtained from the United States Biochemical Corporation, Cleveland, Ohio. The enzyme digest contained treated or untreated substrates, 2% (w/v) cellulase in 0"05 M sodium acetate buffer

CO, enrichment and biomass conversion 263

(pH 4"8) in a final volume of 6 ml (240 enzyme units). The enzyme digests were incubated in a shaking water bath (150 rpm) at 48°C for 48 h and 72 h. The samples were taken out at the end of each incubation period, placed in an ice bath to inactivate the enzymes and centrifuged. Supernatants were diluted to constant volume with distilled water. A 0.1 ml aliquot of each sample was assayed against reagent blank as well as enzyme blanks for reducing sugar as glucose using 1,5-dinitrosalicylic acid (DNS) reagent according to the procedure of Miller. 2° The cellulose residue after saccharification was washed two times with acetate buffer and was recovered by centrifugation. The residues were resuspended in 6 ml (240 units) of 2% enzyme and were incubated further for 48 h to ensure complete hydrolysis. Samples were taken out and assayed for glucose as described earlier. Percent saccharification (accumulative value) was calculated as the amount (mg) of glucose released from 100 mg of untreated dry sample.

Statistical analysis of data

Statistical analysis was performed using standard analysis of variance techniques 2~ and by utilizing SAS (Statistical Analysis System, SAS Institute, Cary, NC). In the analysis, a complete randomized block design was used with five treatments (four open top chambers at 354, 431,506 and 659/~1 liter-~ and an open field plot) in each block. There were three replicate blocks and three replicates of samples were taken from each block for the biochemical assay of samples.

RESULTS AND DISCUSSION

Chemical composition of leaves and stems

As can be seen from Figs 1, 2 and 3, stems contained more cellulose and lignin but less hemicellulose than the leaves of sweet potatoes grown in either ambient or different CO2 concentrations. While CO2 enrichment resulted in increased cellulose contents in stems, hemicellulose and cellulose contents were not affected much in leaves (Figs 1 and 2). Most of the increases in cellulose content in stems occurred with the first increment of atmospheric CO2 above ambient (431 /zl liter-I). The increase in cellulose content in stems of 506 ktl liter- ~ CO2 grown plants was about 31% more than in plants grown in ambient (open field plots) CO2 concentrations (Fig. 1). However, cellulose content of stems and leaves was more in plants grown at 354 /A liter -~ CO2 in open top

264 S. Bhattacharya, J. F. Eatman, P. K. Biswas, M. E. M. Tolbert

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open field plots.

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Effect of different CO 2 concent- rations on hemicellulose content at 90 day

harvest. Key as for Fig. 1.

14

1 3

d

z

8

i i I

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I I I I 3 5 0 4 5 0 5 5 0 6 5 0

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Fig. 3. Effect of different CO 2 concent- rations on lignin content at 90 day harvest.

Key as for Fig. 1.

chambers than at 354 ~1 liter-~ CO2 in open field plots. Previous reports indicated that elevated CO2 concentrations increased dry matter content in potatoes, 22 and sweet potatoes. 8,23 It has also been shown that CO2 enrichment results in faster development of tubers s because of transloca- tion of proportionately more photosynthate into the roots than into the shoot during later stages of growth.

CO, enrichment and biomass conversion 265

Results show that lignin content of stems (Fig. 3) increased slightly in plants grown in ambient CO2 concentrations in open top chambers as compared with open field plots. As atmospheric CO2 concentration was increased to 506/al liter-~, lignin content of stems decreased by about 10%. In contrast to this, plants grown in open top chambers irrespective of CO 2 concentrations produced more lignin in leaves than those plants grown in open field plots (354 /A liter-l). Lu et al. reported that insoluble dietary fiber (lignin, cellulose, hemicellulose, etc.) significantly decreased in sweet potato tuberous roots in response to CO 2 enrich- ment. 23

CO 2 enrichment and biomass conversion

The percent saccharification (% glucose) of leaf and stem substrates (treated and untreated) of sweet potato plants grown under various CO2 concentrations (354 ktl liter-1 open field plots; 354, 431,506 and 659 ktl liter- 1 within chamber) are presented in Figs 4 and 5. The results show that both untreated stems and untreated leaves yielded more glucose when those plants were grown in ambient CO2 concentrations in open top chambers as compared with those in open field plots. Increasing the CO2 concentration in the chambers resulted in the accumulation of carbohydrates in the stems. As a result, percent saccharification was greater in the stems which were obtained from plants grown in a CO2 enriched environment. The results of saccharification yields were similar in leaves. The glucose yield increased with an increase in CO2 concentra- tion up to 506/~1 liter- 1. CO2 but declined in 659/~1 liter- i CO2_grown plants. However, the concentration of glucose was more pronounced in 659 ~1 liter-~- than in 354/~1 liter-i CO2_grown plants. There was not much difference in saccharification values between two ambient concen- trations (open field plots and open top chambers). There was, however, a significant difference in saccharification values between substrates collected from ambient as well as from 431,506 and 659 ~1 liter-~ CO2 environments. The trends of results were similar in leaves and stems at 48 h and 72 h of incubation, therefore, only data from 72 h of incuba- tion are given (Figs 4 and 5).

Increasing the period of incubation from 48 h to 72 h also increased the conversion of cellulose to glucose. In general, magnitude of cellulosic conversion was more pronounced in stems than in leaves, which further confirms the presence of high concentrations of cellulose in stems than in the leaves. The increase in fermentable sugar content was probably associated with an increase in dry matter production due to an increase in photosynthetic rate in sweet potato plants in a CO2-enriched environ-

36 I

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266 S. Bhattacharya, J. F. Eatman, P. K. Biswas, M. E. M. Tolbert

o L , I I I 150 4 5 0 5 5 0 6 5 0

CO 2 lal 1-1

Fig. 4. Enzymatic saccharification of untreated ( ~ ) and pretreated ( ~ ) leaves of sweet potato grown in different CO2 concentrations for 90 days. Values for CO 2 are day time means. Vertical bars represent _+ SE, n = 9. Untreated leaves (*) and pretreated leaves (4) in open field

plots.

, r !

45

40

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350 450 550 6 0 CO 2 IJ I 1-1

Fig. 5. Enymatic saccharification of untreated ( ~ ) and pretreated (---or---) stems of sweet potato grown in different CO2 concentrations for 90 days. Values for CO 2 are day time means. Vertical bar represent _+ SE, n -- 9. Untreated stem ( • ) and pretreated stems (x7) in open field

plots.

ment (unpublished data). Bhattacharya et al? reported an increase in starch content of leaves of sweet potato (Ipomoea batatas L. 'Georgia- Jet') at elevated CO2 concentrations. Several investigators 24,25 have also reported that CO2 enrichment increased starch content of soybean leaves. Sionit et al. 26 reported increases in total dry matter production in soybean, radish, sugar beet and corn at all growth stages with increased CO2 concentrations and fight intensities.

In the present study, pretreatment of stems did not increase glucose yields to a great extent as compared with untreated samples. However, pretreatment of leaves with methanol and acetone and then H2SO4 and subsequently with ZnClz-HC1 increased the extent of hydrolysis at 72 h ( 3 5 4 / d liter-~ CO2). The reason why some substrates are less suscept- ible than others to cellulase after chemical pretreatments is not known. The glucose yields were higher at 72 h than at 48 h in all CO2 treat- ments. Enzymatic hydrolysis of pretreated stems and leaves obtained from plants grown in various CO 2 concentrations showed similar trends to untreated ones, i.e. an increase with increase in CO2 concentration.

In the present investigation chemical pretreatments of sweet potato stems and leaves appear to be effective in enhancing the hydrolysis rate

co , enrichment and biomass conversion 267

to some extent. In order to enhance the enzymatic susceptibility of cellulosic substrates of sweet potato a variety of pretreatment processes ~° were used prior to cellulose hydrolysis. ~ The results presented here are in conformity with our previous report 1° and suggest that grinding of stem and leaf substrates which resulted in reduction of the particle size also served as an effective pretreatment. 27 Even when lignin levels are low, however, the hydrolysis of cellulose can be limited by the physical properties of the polysaccharide itself. In addition, removal of lignin and hemicellulose is not sufficient to achieve high rates and yield of glucose formation from cellulose. 2s Further work on the characterization of sugars in the hydrolysates, and fermentation using different strains of yeast is under way.

These results lead us to conclude that sweet potato vines can serve as a potential source of renewable lignocellulosic biomass for conversion into alcohol fuels. If CO2 concentration in the atmosphere continues to increase at the present rate of 1.5/A liter- ~ year- 1, the cellulosic biomass of sweet potato vines would also increase, thus increasing available carbohydrate sources for bioconversion into ethanol fuel.

ACKNOWLEDGEMENTS

This research was sponsored through grants from US Department of Agriculture-CSRS, ALX-SP1, Martin Marietta Energy Systems, No. 19X-89651V; and Department of Energy~ No. DE-A505-838R60166; and Alabama Research Institute, ARI-84-628. The authors are grateful to Dr N. Bhattacharya for critically reviewing the manuscript.

REFERENCES

1. Clark, W. C., Cook, K. H., Moreland, G., Weinberg, A. K., Rotty, R. M., Bell, P. R., Cooper, L. J. & Cooper, C. L., In Carbon Dioxide Review, Clarendon Press, Oxford, 1982, 3-43.

2. Kimball, B. A., Agron. J., 75 (1979), 779-88. 3. Waggoner, E E., American Sci., 72 (1984), 179. 4. Sionit, N., Rogers, H. H., Bingham, G. E. & Strain, B. R., Agron. J., 76

(1984), 447-51. 5. Wittwer, S. H., Hort. Sci., 18 (1983), 667-73. 6. Krizek, D. T., Cotton Physiology -- A Treatise, State of the Art Mono-

graph, Department of Energy, Washington, DC, 1984. 7. Strain, B. R., Sionit, N. & Cure, J., A Bibliography, Dept. of Botany, Duke

University, Durham, NC, 1984.

268 S. Bhattacharya, J. F. Eatman, P. K. Biswas, M. E. M. Tolbert

8. Bhattacharya, N. C., Biswas, E K., Bhattacharya, S., Sionit, N. & Strain, B. R., Crop Science, 25 (1985), 975-81.

9. Grethlein, H. E., Biotechnol. Adv., 2 (1984), 43. 10. Bhattacharya, S., Biswas, E K. & Tolbert, M. E. M., Biol. Wastes, 19 (1987),

215-26. 11. Tolbert, M. E. M., Bhattacharya, S. & Biswas, E K., Biomass, 11 (1986),

205-13. 12. Biswas, E K. et aL, Response of vegetation to carbon dioxide: field studies

of sweet potatoes and cowpeas in response to elevated carbon dioxide. Report 022, US Dept. of Energy, Carbon Dioxide Research Division, Office of Energy Research, Washington, DC, 1985.

13. Heagle, A. S., Body, D. E. & Heck, W. W., J. Environ. Qual., 2 (1973), 365-8.

14. Heagle, A. S., Philbeck, R. B. & Letchworth, M. B., Phytopathology, 69 (1979), 15-20.

15. Rogers, H. H., Heck, W. W. & Heagle, A. S., J. AirPollut. ControlAssoc., 33 (1983), 42-4.

16. Garg, S. K. & Neelakantan, S., Biotechnol. Bioeng., 24 (1982), 2407. 17. Bagby, M. O., Cunningham, R. L. & Maloney, R. L., Tappi, 56 (1973), 162. 18. Adams, G. A., In Methods in carbohydrate chemistry, Academic Press, New

York and London, 1965, 142-260. 19. Chen, L. E & Gong, C. S., Biotechnol. Bioeng. S~mp., 5 (1982), 163. 20. Miller, G. L., Anal. Chem., 31 (1959), 426. 21. Snedecor, G. W. & Cochran, W. G., Statistical methods, Iowa University

Press, Ames, Iowa, 1967. 22. Collins, W. B., Hort. Sci., 11 (1976), 467-9. 23. Lu, J. Y., Biswas, E K. & Pace, R. D., J. Food Sci., 51 (1986), 358. 24. Mauney, J. R., Guinn, K. E., Fry, K. E. & Hesketh, J. D., Photosynthetica, 13

(1979), 260-6. 25. Finn, G. A. & Brun, W. A., Plant Physiol., 69 (1982), 327-31. 26. Sionit, N., Hellmers, H. & Strain, B. R., Agron. J., 74 (1982), 721-5. 27. Bertran, M. S. & Dale, B. E., Biotechnol. Bioeng., 27 (1985), 177. 28. Lipinsky, E. S., Adv. Ser., 181 (1979), 1.