Horseweed is a native annual plant that can grow to a height of over 2 m. When mature, several flowering stems appear at the apex, which branch frequently and create a multitude of tiny composite flowers. In each flower, there are numerous yellow disk florets in the center, which are surrounded by tiny white ray florets. There is no noticeable floral scent. The blooming period can occur any time from midsummer to fall, lasting about 3 wk.

The leaves alternate all around the stem (appearing almost whorled) and differ little in length, creating a columnar effect. The stout central stem is ridged and unbranched, except for the flower stems near the apex. Seeds are tiny and distribution is by wind. The preference of the plant is full sun, moist to dry conditions, and rich fertile soil. However, this plant flourishes in other kinds of soil, including those containing considerable amounts of gravel and clay. This weedy plant is easy to grow and sometimes forms large colonies in favorable disturbed sites. Drought resistance is very good. The plant dies in early September and drydown is rapid.

Stem construction consists of a moderately waxy epidermis. The xylem cylinder has a XPR of 0.92 and a typical stalk diameter of 0.9 cm. The xylem material has a density of almost 675 kg/m3, which is a little light. The pith is a solid spongy inner core that appears to be resilient to rot for many months.

According to estimates, horseweed may have one of the highest yields of all the plants presented here (see Table 1). Projections indicate that 13.3 t/acre can be taken with current cultivation techniques and no engineering of the plant. Another benefit of horseweed is that it flourishes in dry con­ditions and in rough soil such as clay and gravel. It is also an early maturing plant and can be harvested as a fuel as early as September.

Mike Himmel1 and David Wilson2

1 National Renewable Energy Laboratory, Golden, CO; and
2Cornell University, Ithaca, NY

Lignocellulosic biomass is a valuable and plentiful feedstock com­modity and its high cellulose and hemicellulose content (about 80% of total) provides considerable potential for inexpensive sugars production. However, enzymatic deconstruction of these polysaccharides remains a costly prospect. Strides in cellulase cost reduction have been made, yet further improvements are needed to reach the goal of $0.10/gal of EtOH expected to enable this new industry. Strategies to reach this goal will combine reduction in the cost to produce the needed enzymes as well as efforts to increase enzyme efficiency (specific activity). As this work pro­ceeds, the more easily attained achievements will be made first, and thus the overall difficulty increases with time.

This session focused on aspects of cellulase and xylanase biochemis­try needed for enhanced utilization of enzyme cocktails for bioconversion research. Fundamental studies of enzymatic action are critical to mid — and long-term success and were the subject of most presentations. Session speakers described advances in cellulase enzyme discovery, engineering, cocktail refinement, computer modeling, and active site biochemistry. Studies of enzyme production, discovery, synergism, engineering, and structure/function were also discussed. Work to select xylanases for treat­ment of hemicelluloses in biomass was presented as well, and these studies of relevant "accessory" enzymes are historically less well recognized than the cellulase work. Hemicellulose and lignin are now known to act as shields to cellulase action, so if hemicellulose can be degraded enzymati­cally, cellulase loading and pretreatment severity may be reduced. It was also concluded that we must continue to gain a better understanding of the relevant nature of biomass ultrastructure and anatomy. Such knowledge, gained primarily through application of new surface analysis tools, is nec­essary to design more effective pretreatments and enzyme cocktails.

The work presented by Tim Dodge (Genencor International), Elena Vlasenko (Novozymes Biotech), Brian Steer (Diversa Corporation), David Wilson (Cornell University), Tauna Rignall (Colorado School of Mines), and James Preston (University of Florida) collectively demonstrated the application of cutting-edge methodologies in biotechnology to reducing enzyme cost.

Copyright © 2004 by Humana PressInc.

All rights of any nature whatsoever reserved. 0273-2289/04/113/0115-0124/$25.00

Richard G. Nelson,*1 Marie Walsh,3 John J. Sheehan,2
and Robin Graham3

1 Kansas State University, 133 Ward Hsaa,

Manhattan, KS 66506, E-maia: rneason@ksu. edu;

2Nationaa Renewabae Energy Laboratory,

1617 Coae Bouaevard, Goaden, CO 80401; and
3Oak Ridge Nationaa Laboratory, Oak Ridge, TN 37831-6194

Abstract

A methodology was developed to estimate quantities of crop residues that can be removed while maintaining rain or wind erosion at less than or equal to the tolerable soil-loss level. Six corn and wheat rotations in the 10 largest corn-producing states were analyzed. Residue removal rates for each rota­tion were evaluated for conventional, mulch/reduced, and no-till field operations. The analyses indicated that potential removable maximum quan­tities range from nearly 5.5 million dry metric t/yr for a continuous corn rotation using conventional till in Kansas to more than 97 million dry metric t/yr for a corn-wheat rotation using no-till in Illinois.

Index Entries: Corn stover; wheat straw; rainfall erosion; wind erosion; tolerable soil loss.

Introduction

Current US primary energy consumption is about 102 exajoules (EJ) (97 Quads) and is expected to increase to >137 EJ (130 Quads) by 2020. Transportation fuels produced from oil are projected to account for nearly one-third of the projected energy use by 2020, with nearly 68% of the oil imported from unstable and/or unfriendly countries, resulting in a trade imbalance of more than $206 billion (US $ in $2001). Additionally, the use of fossil fuels for transportation and electricity is a significant contributor of greenhouse gasses such as carbon dioxide, nitrogen oxides, and carbon monoxide (1-4).

*Author to whom all correspondence and reprint requests should be addressed. Appaied Biochemistry and Biotechnoaogy 13 Voa. 113-116, 2004

An important component of becoming less dependent on fossil-based resources is to produce bioenergy and bioproducts from renewable energy resources such as biomass. Domestically produced bioenergy and bio­products have lower environmental impacts, have a higher energy-profit ratio (ratio of renewable energy output to total energy inputs) than tradi­tional fossil fuel technologies, and provide for economic development and enhanced energy security.

Among potential biomass resources that can be used to produce bioenergy and bioproducts are agricultural residues such as corn stover and wheat straw. Corn, soybeans, and wheat are the three largest crops produced in the United States, in terms of both acres and total production. Total production (billion bushels), residue quantities (million dry Mg), and energy density (EJ) produced from corn for grain and spring and winter wheat for the period of 1997-2001 are presented in Table 1.

While residue quantities produced are substantial, only a percentage of them can be collected for bioenergy and bioproduct use. Agricultural residues play an important role in controlling erosion and maintaining soil carbon, nutrients, and soil tilth. Removal of agricultural residues for bioenergy and bioproduct use will require consideration of the quantities that must be left to maintain soil quality. A recent analysis has demon­strated that under appropriate conditions, removal of agricultural residue can potentially occur (5).

The time courses of the polysaccharide, lignin with extractives, and ash contents for experiments conducted at 44.0 mg of P. ostreatus/g of stems and 1.60 g of H2O/g of stems are presented in Fig. 2. Time courses such as these were used to estimate xylan and glucan conversions for separate replicate samples, which were then averaged. The xylan and glucan conversions and AX/AG ratios for 23.0-149 mg of P. ostreatus/g of stems and 1.10-2.24 g of H2O/g of stems are presented in Table 4; refer to Fig. 1 for the complete set of inoculum and moisture combinations tested. Of the moisture and inoculum combinations shown in Fig. 1, the (mois­ture, inoculum) combinations (0.77, 21.0), (0.90, 34.0), and (1.20, 41.0) were performed early in the study and displayed visually uneven growth of

fungus in the stems, indicating inhomogeneous distribution of the mycelia over the straw stem surface. Samples taken from the tops and bottoms of these columns also displayed widely variable degradation results, indi­cating poor distribution of the inoculum. The methods were modified, and the uneven distribution of fungal mycelia on the straw was minimized in future experiments. These three data sets were thus not considered further in analyses of the data. The ЛХ/ AG ratios for the preliminary tests con­ducted at 0.0-10.9 mg of P. ostreatus/g of stems and 0.40-0.77 g of H2O/g of stems are given in Table 3, while the xylan and glucan conversions for those preliminary experiments are available elsewhere (15).

Four silages harvested in fall 2002 were retrieved from the silo in March 2003 for the sequential water separation. The silages were selected to represent four mechanical harvest treatments: short chop and unproc­essed, long chop and unprocessed, short chop and processed, and long chop and processed. Processing involved crushing and shearing the chopped whole plant through a pair of toothed rolls operating at small clearance and differential speed (10). Silages came from two experimental farms (Arlington, Prairie-du-Sac) and two commercial farms (Binversie, Ziegler) in Wisconsin. A measured mass of 1 kg of fresh silage was placed in a water basin containing initially 7 L of water. After 1 min of manual gentle mixing, the material still floating on the water surface was removed by hand. The rest of the basin contents was poured gently over a screen made of 0.40-mm (1/64-in.) thick wire spaced 1.59 mm (1/16 in.) center to center in a square grid with about 50% open area. The screen separated material into two components, the effluent water and the suspended sol­ids, and a third component was the sunk material that remained at the bottom of the basin after pouring. The latter two components were spread onto separate paper cloths to partially dry in ambient air. The floating material was then deposited again in the water basin with the same efflu­ent water. After 1 min, the floating and suspended solids were set aside for the next water separation and the sunk material was put on a cloth to dry. This process was repeated until eight water separations had been com­pleted. The eight-step sequential separation was replicated three times for each of the four silages.

The sunk material from each of the eight separations, the suspended solids from the first separation, and the residual floating material after the eighth separation (i. e., 10 components) were oven-dried at 103°C for 24 h to estimate the proportions of DM at each step. A well-mixed amount of 2 kg of water effluent was also measured after the eighth separation and oven-dried to estimate the total DM in the effluent.

For each replication (four silages x three replications), the 10 dried components were hand sorted to separate grain from stover. Sorted grain included full and broken grains, grain hull, and grain endosperm pieces that were large enough (1 to 2 mm) to be clearly identified as starch. The rest was considered to be stover. Because sorting occurred over a period of several weeks after oven-drying, rehydration occurred and component masses were corrected to a DM basis. Grain concentration was estimated as the proportion of sunk grain over the total of sunk grain and sunk stover.

Six pretreatments were done to compare the effect of drying or sieving on subsequent grain and stover separation:

1. A fresh untreated silage.

2. Silage that was partially dried until it lost 10 percentage units of moisture.

3. Silage that was partially dried until it lost 20 percentage units of moisture content.

4. Silage that was oven-dried to approx 0% moisture.

5. Silage that was sieved by a standard method (11) and whose material from only screen no. 3 was hydrodynamically separated (particle size between 9.0 and 18.0 mm).

6. The same sieved material as in item 5 that was also partially dried to lose 10 percentage units of moisture.

The silage for all six pretreatments was unprocessed and came from a commercial farm (Manthe) in south-central Wisconsin.

A single water separation was done with these treated silages. Using the same amounts of silage (1 kg) and water (7 L) as in the second experi­ment, the material was separated into three components: sunk, suspended, and floating material. DM in the effluent was estimated by mass balance. The three measured components were further subdivided into grain and stover by hand sorting after oven-drying. The water separation was repli­cated three times for each of the six silage treatments. In the case of sieved material, 1 kg was placed in the separator, and only the fraction retained on screen no. 3 was separated by water.

Although whey and almonds are not considered feedstocks in this study, they might be recognized as potential, alternative feedstock choices. In 2002, there were 525,000 acres of bearing almond trees in Cali­fornia. In 2001, these alternate-bearing trees produced 450 million tons of unprocessed almonds. Almond hulls have a high sugar and protein con­tent. Currently, they are used as a feedstock for cattle because of the protein content. For this use, almond hulls received an average market price of $73/t between 1990 and 2000.

It may be possible to use the sugar in almond hulls for ethanol produc­tion, while leaving the protein for use by animals. Research and invest­ments may be required to develop a suitable production process and some time and effort may be required for market development. We recommend further study of the potential use of almond hulls for ethanol production in California.

Whey is a coproduct of cheese manufacturing. In 2000, California produced an estimated 1.5 billion lb of cheese, yielding 747,000 t of dried whey. It is costly to dispose whey in municipal water systems. Hence, an alternative use for whey would enhance the economics of cheese produc­tion. Currently, whey protein is used as a food additive, a protein supple­ment, and an animal feed. In addition, there are a few ethanol plants in California and the Midwest that use whey as a feedstock. The current Cali­fornia whey production would yield approx 4.7 million gal of ethanol.

The market price of whey used as animal feed is $340/t (7). As with almond hulls, it may be possible to utilize the sugar in whey for ethanol production, while enabling the protein in the byproduct to be used for animal feed. Hence, the net feedstock cost of whey in ethanol production may be less than $340/t of whey. Further research on the potential of expanding the use of whey as an ethanol feedstock would be helpful in evaluating the viability of this alternative.

The cockleburr is a hitchhiker. The seed pod is a burr with hooked spines. It tangles the fur of animals unfortunate enough to brush against the dying plant.

Male flower heads occur at the ends of branches, and the female flower heads occur in the lower parts of these branches. The female heads develop into hard, woody, spiny burrs. These burrs are oval shaped, brown, 20-30 mm long, and covered in hooked spines.

Cockleburr is an annual species that generally germinates from late winter to late summer. Germination often occurs after rainfall and irriga­tion. The burrs contain two seeds, one larger than the other. The larger seed has limited dormancy and usually germinates in the season it is produced or the following season. The smaller seed has a longer period of dormancy. The plant grows in a wide range of soil types.

Stem construction consists of a moderately waxy, dark brownish-red epidermis. The xylem cylinder has an XPR of 0.46 and a typical stalk diameter of 1.75 cm. Xylem material has a density of almost 390 kg/m3, which corresponds to a corkwood. The pith is a solid spongy inner core that appears to be resilient to rot for many months.

Although an SSP, cocklebur does not have good technical character­istics. In particular, the low XPR and density indicate that there is not much dry matter in this stalk. In addition, the burrs are a hazard, or at least an inconvenience, to the workers who must deal with this material. Surely the most serious disadvantage is that it is rated a Class C noxious weed, indi­cating that the introduction of burrs must be prevented.

Nora SzijArto,1 Zsolt Szengyel,1
Gunnar Liden,2 and Kati Reczey*1

1 Department of Agrtcnltnral Chemtcal Technology,
Bndapest Untversity of Technology and Economtcs,
H-1521 Bndapest, Szent Gellbrt ter 4, Hnngary,

E-matl: katt_reczey@mkt. bme. hn;
and 2Department of Chemtcal Engtneertng,

Lnnd Untversity, PO Box 124, SE-221 00 Lnnd, Sweden

Abstract

An economic process for the enzymatic hydrolysis of cellulose would allow utilization of cellulosic biomass for the production of easily ferment­able low-cost sugars. New and more efficient fermentation processes are emerging to convert this biologic currency to a variety of commodity prod­ucts with a special emphasis on fuel ethanol production. Since the cost of cellulase production currently accounts for a large fraction of the estimated total production costs of bioethanol, a significantly less expensive process for cellulase enzyme production is needed. It will most likely be desirable to obtain cellulase production on different carbon sources—including both polymeric carbohydrates and monosaccharides. The relation between enzyme production and growth profile of the microorganism is key for designing such processes. We conducted a careful characterization of growth and cellulase production by the soft-rot fungus Trichoderma reesei. Glucose — grown cultures of T. reesei Rut-C30 were subjected to pulse additions of Solka- floc (delignified pine pulp), and the response was monitored in terms of CO2 evolution and increased enzyme activity. There was an immediate and unexpectedly strong CO2 evolution at the point of Solka-floc addition. The time profiles of induction of cellulase activity, cellulose degradation, and CO2 evolution are analyzed and discussed herein.

Index Entries: Trichoderma reesei; fermentation; cellulase; growth charac­terization; cellulose hydrolysis.

*Author to whom all correspondence and reprint requests should be addressed. Applted Btochemtstry and Btotechnology 115 Vo!. 113-116, 2004

The accelerating accumulation of CO2 and other greenhouse gases in the atmosphere may lead to adverse climate changes that would seriously endanger the sensitive ecologic balance of Earth (1). Energy shortages in the world coupled with environmental considerations have directed applied research toward the development of novel processes to produce renewable fuels with a special emphasis on fuel ethanol production from cellulosic materials (2). Even though CO2 is released during the bioprocess of fuel ethanol production and also during its combustion, the CO2 is reuti­lized to grow new biomass, replacing that harvested for ethanol produc­tion. As a result, the net produced CO2 is small in comparison with that released by the utilization of fossil fuels, thus reducing the hazards of a global climate change (1,3).

The potential for using cellulosic materials to produce fermentable sugars for biotechnological processes—including bioethanol production— is enormous (4,5). Ethanol production from cellulose comprises hydrolysis of cellulosic raw materials to sugars and the subsequent anaerobic fermen­tation of sugar compounds by yeast to produce ethanol. Although enzy­matic hydrolysis is superior, in several aspects, to acid hydrolysis, its economic realization is highly hindered by the presently too high produc­tion cost of cellulose-degrading enzymes.

Cellulases are inducible enzymes, which are synthesized by many microorganisms during their growth on cellulosic materials. Example microorganisms known to produce cellulases include bacterial species of Clostridium and Bacillus and species of filamentous fungi from Penicillium, Aspergillus, and Trichoderma (6). Complete enzymatic degradation of native cellulose requires the synergistic action of three general types of cellu­lolytic enzymes, traditionally classified as endoglucanases, cellobio — hydrolases, and P-glucosidases (7). Endoglucanases preferentially hydro­lyze the amorphous regions of the fibrils by randomly cleaving P-gluco — sidic bonds; cellobiohydrolases are exoglucanases releasing cellobiose, the repeat unit of cellulose from the chain ends; while P-glucosidases complete the degradation process by hydrolyzing cellobiose and other cellodextrins with a low degree of polymerization to glucose units. The high level of synergy among cellulase enzymes results from their different, but comple­mentary, mode of action. This synergy increases the degree of hydrolysis by more than twofold over that achieved with individual enzymes (8).

Because of its ability to produce and secrete the complete set of cellu­lolytic enzymes, thus making it particularly potent in hydrolyzing the cellulose polymer to glucose monomers, the soft-rot fungus Trichoderma, in particular T. reesei has been the focus of cellulase research for decades (8). The preferred substrates used by most researchers for cellulase pro­duction are pure celluloses such as Avicel, Solka-floc, and cotton (9). Cel — lulase production by Trichoderma is controlled by a complex metabolic regulation (10-12). Cellulose acts (indirectly) as an inducer for the produc­tion of cellulases. Expression of cellulases is furthermore subject to repres­sion by the end product of the hydrolysis—glucose. Cellulose-derived inducers, sophorose being the most potent, are likely to provide an effec­tive induction during cultivation on cellulose, but the concentration of the end product, glucose, may negatively affect cellulase production. Glucose concentration is determined by the dynamic balance between the rates of glucose generation (by cellulose hydrolysis) and consumption (by micro­bial uptake). At low concentrations of cellulase and/or cellulose, glucose generation may be too slow to meet the need of active cell growth and function. On the other hand, cellulase synthesis can be halted by glucose repression when glucose generation is faster than its consumption. Glu­cose repression of enzyme expression is an obvious target for strain improvement. Many of the high-producing strains of T. reesei that have been isolated have also been shown to be partly glucose derepressed. This is the case for, e. g., the strain T. reesei Rut-C30 (6), which is used in the present study.

The objective of the current work was to characterize carefully the dynamics of cellulase production and metabolic activity following cellu­lose addition in a batch cultivation of the strain T. reesei Rut-C30. Cells were initially grown on glucose as the carbon source, and after its depletion, cellulose was added. Since it is difficult to follow the growth directly after addition of a solid substrate, on-line measurements of CO2 evolution were used to follow the metabolic activity of the cells. Frequent samples were also taken to measure enzyme activity and sugar concentrations.

Removal of agricultural residues for bioenergy and bioproduct use is directly influenced by a number of factors including grain yield, crop rota­tion, field-management practices within a rotation (e. g., tillage), climate, and physical characteristics of the soil such as erodibility and topology.

The goal of the analysis is to develop and apply a methodology to estimate quantities of agricultural crop residues that can be removed for bioenergy and bioproduct use from both continuous crop and multi-crop rotations, while maintaining rain and/or wind erosion rates (Mg/[ha-yr]) at or below the tolerable soil-loss level, T. T is the maximum rate of soil erosion that will not lead to prolonged soil deterioration and/or loss of productivity as defined by the United States Department of Agriculture’s Natural Resource Conservation Service (USDA-NRCS). For the purpose of this article, the methodology developed is applied to the top 10 corn-pro­ducing states (Iowa, Illinois, Indiana, Kansas, Minnesota, Missouri, Nebraska, Ohio, South Dakota, Wisconsin) based on total production (bush­els) between 1997-2001. Three of these states—Kansas, Minnesota, and South Dakota—are among the top 10 wheat-producing states as well.

For each county in the 10 states evaluated, all cropland soil types in land capability classes (LCCs) I-VIII are identified. For each individual soil type, acres of that particular soil type, field topology characteristics (per­centage low and high slopes), erodibility, and tolerable soil-loss limit are obtained from the USDA. These data are used in the rain and wind erosion equations described later. In each of the states analyzed, the following crop rotations are considered (where applicable): continuous corn, corn-soy­bean, corn-winter wheat, corn-spring wheat, continuous winter wheat, winter wheat-soybeans.

For each of these crop rotations, three tillage scenarios (conventional, reduced/mulch, and no-till) are considered. Conventional tillage scenarios consist mainly of moldboard plowing and/or heavy disking, reduced/ mulch tillage scenarios include light disking and chisel plowing, and the no-till scenarios use field operations that provide little or no disturbance to the field surface. Harvest, planting, tillage, and chemical application dates for each field operation are adjusted to reflect the most likely time of year and month that they are expected to occur within each of the 10 states. Tables 2 and 3 describe field operations for each crop rotation and tillage combination analyzed.

Treatment time is an important process variable because large pro­cess footprints such as those required for this type of treatment require large amounts of land, and thus long treatment times can have a negative influence of the economics of the process, depending on land use require­ments (16). In addition, depending on the sensitivity of the treatment process to initial and transient conditions, widely variable product com­positions produced in time-sensitive degradation processes could have a large effect on the next step of the manufacturing process. Longer treat­ments gave progressively smaller gains in xylan removal and larger gains in glucan removal (Table 4). This observation is consistent with the fun­gus first utilizing the easily degraded hemicellulose and amorphous cel­lulose fractions, beginning with the hemicellulose.

In 4 wk, the maximum conversions observed were about 27% xylan and 19% glucan. At 8 wk, the maximum conversions observed were about 40% xylan and 34% glucan, while at 12 wk they were about 48% xylan and 41% glucan. At very low inoculum levels, the initial degradation (at 4 wk) was generally nonselective and thus primarily owing to indigenous organisms (Table 3). Above 10.9 mg of P. ostreatus/g of stems, the early degradation was much more selective for xylan vs glucan, indicating sig­nificant activity of the inoculated fungus. Maximum selectivities for xylan removal (ЛХ/AG) for inoculum levels exceeding 10.9 mg of P. ostreatus/g of stems were observed earlier in the treatments, with all moisture and inoculum combinations showing similar selectivities after 12 wk of treatment.