Fermentations of pectin-rich biomass with recombinant bacteria to produce fuel ethanol

Pectin-rich residues from sugar beet processing contain significant carbohydrates and insignificant amounts of lignin. Beet pulp was evaluated for conversion toethanol using recombinant bacteria as biocatalysts. Hydrolysis of pectin-rich residues followed by ethanolic fermentations by yeasts has not been productive because galacturonic acid and arabinose are not ferm entable toethanol by these organisms. The three recombinant bacteria evaluated in this study, Escherichia coli strain KO11, Klebsiella oxytoca strain P2, and Erwinia chrysanthemi EC 16 pLOI 555, ferment carbohydrates in beet pulp with varying efficiencies. E. coli KO11 is able to convert pure galactu ronic acid to ethanol with minimal acetate production. Using an enzyme loading of 10.5 filter paper un its of cellulase, 120.4 polygalactu ronase units of pectinase, and 6.4 g of cellobiase (per gram of dry wt sugar beet pulp), with substrate addition after 24 h of fermentation, 40 g of ethanol/L was produced. Other recombinants exhibited lower ethanol yields with increases in acetate and succinate production.     

Optimization of cellouronic acid synthesis by TEMPO-mediated oxidation of cellulose III from sugar beet pulp

Microfibrillated cellulose from purified sugar beet pulp was converted into cellulose III by immersion in liquid ammonia. When freed from ammonia, this product was oxidized in water at pH-10 using NaBr, NaOCl and 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO) under various conditions and concentrations. The resulting water-soluble cellouronic acid—i.e. cellulose oxidized at the C6 position- was analyzed by high performance size exclusion chromatography (HPSEC) together with ¹³C NMR spectroscopy. The oxidation parameters, namely reaction time, temperature, NaBr and TEMPO concentrations were varied to determine the optimum reaction conditions. A low TEMPO concentration, a rather fast reaction time and the conducting of the oxidation at 0 °C were critical to obtain pure cellouronic acid in high yield, high purity and high DP.     

Optimization of Bioethanol Production from Enzymatic Treatment of Argan Pulp Feedstock

Argan pulp is an abundant byproduct from the argan oil process. It was investigated to study the feasibility of second-generation bioethanol production using, for the first time, enzymatic hydrolysis pretreatment. Argan pulp was subjected to an industrial grinding process before enzymatic hydrolysis using Viscozyme L and Celluclast 1.5 L, followed by fermentation of the resulting sugar solution by Saccharomyces cerevisiae. The argan pulp, as a biomass rich on carbohydrates, presented high saccharification yields (up to 91% and 88%) and an optimal ethanol bioconversion of 44.82% and 47.16% using 30 FBGU/g and 30 U/g of Viscozyme L and Celluclast 1.5 L, respectively, at 10%_w/v of argan biomass.     

Ethanol production from olive oil extraction residue pretreated with hot water

The olive pulp fraction contained in the residue generated in olive oil extraction by a two-step centrifugation process can be upgraded by using the cellulose fraction to produce ethanol and recovering high value phenols (tyrosol and hydroxytyrosol). Olive pulp was pretreated in a laboratory scale stirred autoclave at different temperatures (150–250°C). Pretreatment was evaluated regarding cellulose recovery, enzymatic hydrolysis effectiveness ethanol production by a simultaneous saccharification and fermentation process (SSF), and phenols recovery in the filtrate. The pretreatment of olive pulp using water at temperatures between 200°C and 250°C enhanced enzymatic hydrolysis. Maximum ethanol production (11.9 g/L) was obtained after pretreating pulp at 210°C in a SSF fed-batch procedure. Maximum hydroxytyrosol recovery was obtained in the liquid fraction when pretreated at 230°C.     

Ethanol/water pulps from sugar cane straw and their biobleaching with xylanase from Bacillus pumilus

The influence of independent variables (temperature and time) on the cooking of sugar cane straw with ethanol/water mixtures was studied to determine operating conditions that obtain pulp with high cellulose contents and a low lignin content. An experimental 2(2) design was applied for temperatures of 185 and 215 degrees C, and time of 1 and 2.5 h with the ethanol/water mixture concentration and constant straw-to-solvent ratio. The system was scaled-up at 200 degrees C cooking temperature for 2 h with 50% ethanol-water concentration, and 1:10 (w/v) straw-to-solvent ratio to obtain a pulp with 3.14 cP viscosity, 58.09 kappa-number, and the chemical composition of the pulps were 3.2% pentosan and 31.5% lignin. Xylanase from Bacillus pumilus was then applied at a loading of 5-150 IU/g dry pulp in the sugar cane straw ethanol/water pulp at 50 degrees C for 2 and 20 h. To ethanol/water pulps, the best enzyme dosage was found to be 20 IU/g dry pulp at 20 h, and a high enzyme dosage of 150 IU/g dry pulp did not decrease the kappa-number of the pulp.     

Plant-mediated asymmetric reduction of 1-(3,4-dimethylphenyl)ethanone

Bioreduction mediated by plants is a highly selective and environmentally friendly approach to synthesise optically active alcohols. Herein the bioreduction of 1-(3,4)-dimethylphenyl)ethanone to the corresponding chiral alcohol 1-(3,4-dimethylphenyl)ethanol has been evaluated using nine different vegetables roots as biocatalysts. The chiral alcohol was prepared in yields ranging from 44.1% to 88.2%, and with enantiomeric excess up to 97.2% for the (S)-enantiomer. Sugar beet was the most promising among the tested vegetables roots. Therefore, sugar beet cell cultures (normal and transformed) were obtained and tested for the reduction as well, yielding the corresponding alcohol in the range from 62.1% to 88.2% yield and with enantiomeric excesses of >99%. Based on these results reported, there are no profound differences in the reductive capacity of undifferentiated cells and organs of the same plant species (sugar beet). Due to overall advantages of using cell cultures, we have highlighted this approach as a promising method for preparation of enantiomerically pure 1-(3,4-dimethylphenyl)ethanol.     

Determination of the 13C/12C ratio of ethanol derived from fruit juices and maple syrup by isotope ratio mass spectrometry: collaborative study

A collaborative study of the carbon-13 isotope ratio mass spectrometry (13C-IRMS) method based on fermentation ethanol for detecting some sugar additions in fruit juices and maple syrup is reported. This method is complementary to the site-specific natural isotope fractionation by nuclear magnetic resonance (SNIF-NMR) method for detecting added beet sugar in the same products (AOAC Official Methods 995.17 and 2000.19), and uses the same initial steps to recover pure ethanol. The fruit juices or maple syrups are completely fermented with yeast, and the alcohol is distilled with a quantitative yield (>96%). The carbon-13 deviation (delta13C) of ethanol is then determined by IRMS. This parameter becomes less negative when exogenous sugar derived from plants exhibiting a C4 metabolism (e.g., corn or cane) is added to a juice obtained from plants exhibiting a C3 metabolism (most common fruits except pineapple) or to maple syrup. Conversely, the delta13C of ethanol becomes more negative when exogenous sugar derived from C3 plants (e.g., beet, wheat, rice) is added to pineapple products. Twelve laboratories analyzed 2 materials (orange juice and pure cane sugar) in blind duplicate and 4 sugar-adulterated materials (orange juice, maple syrup, pineapple juice, and apple juice) as Youden pairs. The precision of that method for measuring delta13C was similar to that of other methods applied to wine ethanol or extracted sugars in juices. The within-laboratory (Sr) values ranged from 0.06 to 0.16%o (r = 0.17 to 0.46 percent per thousand), and the among-laboratories (SR) values ranged from 0.17 to 0.26 percent per thousand (R = 0.49 to 0.73 percent per thousand). The Study Directors recommend that the method be adopted as First Action by AOAC INTERNATIONAL.     

Detection of added beet or cane sugar in maple syrup by the site-specific deuterium nuclear magnetic resonance (SNIF-NMR) method: collaborative study

Results of a collaborative study are reported for the detection of added beet or cane sugar in maple syrup by the site-specific natural isotope fractionation-nuclear magnetic resonance (SNIF-NMR) method. The method is based on the fact that the deuterium content at specific positions of the sugar molecules is different in maple syrup from that in beet or cane sugar. The syrup is diluted with pure water and fermented; the alcohol is distilled with a quantitative yield and analyzed with a high-field NMR spectrometer fitted with a deuterium probe and fluorine lock. The proportion of ethanol molecules monodeuterated at the methyl site is recorded. This parameter (D/H)I is decreased when beet sugar is added and increased when cane sugar is added to the maple syrup. The precision of the method for measuring (D/H)I was found to be in good agreement with the values already published for the application of this method to fruit juice concentrates (AOAC Official Method 995.17). An excellent correlation was found between the percentage of added beet sugar and the (D/H)I isotopic ratio measured in this collaborative study. Consequently, all samples in which exogenous sugars were added were found to have a (D/H)I isotopic ratio significantly different from the normal value for an authentic maple syrup. By extension of what is known about plants having the C4 cycle, the method can be applied to corn sweeteners as well as to cane sugar. One limitation of the method is its reduced sensitivity when applied to specific blends of beet and cane sugars or corn sweeteners. In such case, the C13 ratio measurement (see AOAC Official Method 984.23, Corn Syrup and Cane Sugar in Maple Syrup) may be used in conjunction.     

Bioconversion of secondary fiber fines to ethanol using counter-current enzymatic saccharification and Co-fermentation

This research examined several enzymatic and microbial process for the conversion of waste cellulosic fibers into ethanol. The first was a one-stage process in which pulp fines were contacted with commercial enzyme solutions. The second process used sequential, multistage saccharification. The third used sequential enzyme addition in a countercurrent mode. Experiments compared the results with various feed stocks, different commercial enzymes, supplementation with β-glucosidase, and saccharification combined with fermentation. The highest saccharification (65%) from a 4% consistency pulp and the highest sugar concentration (5.4%) from an 8% consistency pulp were attained when 5 FPU/g plus 10 IU/g of β-glucosidase were used. Sequential addition of enzyme to the pulp in small aliquots produced a higher overall sugar yield/U enzyme than the addition of the same total amount of enzyme in a singledose. In the saccharification and fermentation experiments, we produced 2.12% ethanol from a 5.4% sugar solution. This represents 78% of the theoretical maximum. This yield could probably be increased through optimization of the fermentation step. Even when little saccharification occurred, the enzyme facilitated separation of water, fiber, and ash, so cellulase treatment could be an effective means for dewatering pulp sludges.     

Ethanol production from lignocellulosic byproducts of olive oil extraction

The recent implementation of a new two-step centrifugation process for extracting olive oil in Spain has substantially reduced water consumption, thereby eliminating oil mill wastewater. However, a new high sugar content residue is still generated. In this work the two fractions present in the residue (olive pulp and fragm ented stones) were assayed as substrate for ethanol production by the simultaneous saccharification and fermentation (SSF) process. Pretreatment of fragmented olive stones by sulfuric acid-catalyzed steam explosion was the most effective treatment for increasing enzymatic digestibility; however, a pretreatment step was not necessary to bioconvert the olive pulp into ethanol. Theolive pulp and fragmented olive stones were tested by the SSF process using a fed-batch procedure. By adding the pulp three times at 24-h intervals, 76% of the theoretical SSF yield was obtained. Experiments with fed-batch pretreated olive stones provided SSF yields significantly lower than those obtained at standard SSF procedure. The preferred SSF conditions to obtain ethanol from olives stones (61% of theoretical yield) were 10% substrate and addition of cellulases at 15 filter paper units/g of substrate.     

Enhancement of the Enzymatic Digestibility and Ethanol Production from Sugarcane Bagasse by Moderate Temperature-Dilute Ammonia Treatment

In this study, sugarcane bagasse was pretreated with ammonium hydroxide, and the effectiveness of the pretreatment on enzyme hydrolysis and ethanol production was examined. Bagasse was soaked in ammonium hydroxide and water at a ratio of 1:0.5:8 for 0–4 days at 70 °C. Approximately, 14–45 % lignin, 2–6 % cellulose, and 13–22 % hemicellulose were removed during a 0.5- to 4-day ammonia soaking period. The highest glucan conversion of sugarcane bagasse soaked in dilute ammonia at moderate temperature by cellulase was accomplished at 78 % with 75 % of the theoretical ethanol yield. Under the same conditions, untreated bagasse resulted in a cellulose digestibility of 29 and 27 % of the theoretical ethanol yield. The increased enzymatic digestibility and ethanol yields after dilute ammonia pretreatment was related to a combined effect of the removal of lignin and increase in the surface area of fibers.     

Ethanol from lignocellulosic wastes with utilization of recombinant bacteria

This article presents the advanced technology that has been developed by BioEnergy International of Gainesville, Florida, utilizing novel recombinant strains of bacteria developed by Lonnie Ingram of the University of Florida. The first commercial applications of these unique fermenting organisms convert 5-carbon sugars, as well as 6-carbon sugars, and oligomers of cellulose (e.g., cellobiose and cellotriose) directly to ethanol. The proposed systems that will be utilized for conversion of agricultural wastes, mixed waste papers, and pulp and paper mill waste in forthcoming commercial installations are now under design. This involves the extensive experience of Raphael Katzen Associates International, Inc. in acid hydrolysis, enzyme production, enzymatic hydrolysis, large-scale fermentation engineering, and distillation/dehydration. Specific examples of this advanced technology will be presented in different applications, namely:

Improvement of paper strength via surface application of sugar beet pectin

The influence of surface-applied original and enzymatically-modified sugar beet pectin on strength properties of fluting, coating base paper, and core board was investigated. The effect was compared with the application of commercial strength-increasing agents. With increasing the polymer uptake, measured strength of paper increased. Original sugar beet pectin increased the strength properties of papers, at the same uptake of polymer, to a higher extent than oxidised potato starch or modified grain flour, while the effect of enzymatically-modified sugar beet pectin was the lowest. For the same increase of paper strength, a several times higher uptake of enzymatically-modified sugar beet pectin was required when compared with the original pectin, oxidised potato starch, or modified grain flour.     

Dilute acid hydrolysis of softwoods

Whole tree chips obtained from softwood forest thinnings were converted to ethanol via a two-stage dilute acid hydrolysis followed by yeast fermentation. The chips were first impregnated with dilute sulfuric acid, then pretreated in a steam explosion reactor to hydrolyze, more than 90% of the hemicellulose and approx 10% of the cellulose. The hydrolysate was filtered and washed with water to recover the sugars. The washed fibers were then subjected to a second acid im pregnation and hydrolysis to hydrolyze as much as 45% of the reamining cellulose. The liquors from both hydrolysates were combined and fermented to ethanol by a Saccharomyces cerevisiae yeast that had been adapted to the inhibitors. Based on available hexose sugars, ethanol yields varied from 74 to 89% of theoretical. Oligosaccharide contents higher than about 10% of the total available sugar appear to have a negative impact on ethanol yield.     

Enzymatic hydrolysis of high-moisture corn fiber pretreated by afex and recovery and recycling of the enzyme complex

Corn fiber is a grain-processing residue containing significant amounts of cellulose, hemicellulose, and starch, which is collected in facilities where fuel ethanol is currently manufactured. Preliminary research has shown that corn fiber (30% moisture dry weight basis [dwb]) responds well to ammonia-fiber explosion (AFEX) pretreatment. However, an important AFEX pretreatment variable that has not been adequately explored for corn fiber is sample moisture. In the present investigation, we determined the best AFEX operating conditions for pretreatment of corn fiber at high moisture content (150% moisture dwb). The optimized AFEX treatment conditions are defined in terms of the moisture content, particle size, ammonia to biomass ratio, temperature, and residence time using the response of the pretreated biomass to enzymatic hydrolysis as an indicator. Approximate optimal-pretreatment conditions for unground corn fiber containing 150% (dwb) moisture were found to be: temperature, 90?C; ammonia: dry corn fiber mass ratio, 1:1; and residence time 30 min (average reactor pressure under these conditions was 200 pounds per square inch [psig]). Enzymatic hydrolysis of the treated corn fiber was performed with three different enzyme combinations. More than 80% of the theoretical sugar yield was obtained during enzymatic hydrolysis using the best enzyme combination after pretreatment of corn fiber under the optimized conditions previously described. A simple process for enzyme recovery and reuse to hydrolyze multiple portions of AFEX-treated corn fiber by one portion of enzyme preparation is demonstrated. Using this process, five batches of fresh substrate (at a concentration of 5% w/v) were successfully hydrolyzed by repeated recovery and reuse of one portion of enzyme preparation, with the addition of a small portion of fresh enzyme in each subsequent recycling step.     

Structural confirmation of oligosaccharides newly isolated from sugar beet molasses

ABSTRACT: BACKGROUND: Sugar beet molasses is a viscous by-product of the processing of sugar beets into sugar. The molasses is known to contain sucrose and raffinose, a typical trisaccharide, with a well-established structure. Although sugar beet molasses contains various other oligosaccharides as well, the structures of those oligosaccharides have not been examined in detail. The purpose of this study was isolation and structural confirmation of these other oligosaccharides found in sugar beet molasses. RESULTS: Four oligosaccharides were newly isolated from sugar beet molasses using high-performance liquid chromatography (HPLC) and carbon-Celite column chromatography. Structural confirmation of the saccharides was provided by methylation analysis, matrix-assisted laser desorption/ionaization time of flight mass spectrometry (MALDI-TOF-MS), and nuclear magnetic resonance (NMR) measurements. CONCLUSION: The following oligosaccharides were identified in sugar beet molasses: beta-D-galactopyranosyl-(1- > 6)-beta-D-fructofuranosyl-(2 <-> 1)-alpha-D-glucopyranoside (named beta-planteose), alpha-D-galactopyranosyl-(1- > 1)-beta-D-fructofuranosyl-(2 <-> 1)-alpha-D-glucopyranoside (named1-planteose), alpha-D-glucopyranosyl-(1- > 6)-alpha-D-glucopyranosyl-(1 <-> 2)-beta-D-fructofuranoside (theanderose), and beta-D-glucopyranosyl-(1- > 3)-alpha-D-glucopyranosyl-(1 <-> 2)-beta-D-fructofuranoside (laminaribiofructose). 1-planteose and laminaribiofructose were isolated from natural sources for the first time.     

TheTalaromyces emersonii enzyme system

In typical fermentations at 45‡C on cellulose/corn steep liquor/ammonium and mineral salts medium, growth of the thermophilic fungusTalaromyces emersonii increases rapidly up to about 50 h and then decreases, presumably because of cell lysis, sporulation, or both. The accumulation of cellulase activity follows closely on growth and essentially reaches a maximum at about the same time that cell protein does. By contrast, two peaks of Β-glucosidase activity are observed, one maximal at about 36 h and the second at about 75 h. Fractionation of culture filtrates showed that the cellulase system is comprised of at least four endoglucanases (EC 3.2.1.4), four or five exoglucanases (cello-biohydrolase; EC 3.2.1.91), and three types of Β-glucosidase (cellobiase; EC 3.2.1.21). All are glycoproteins. Indeed, variation in carbohydrate content may account for some of the observed multiplicity of enzyme forms. Although none of the individual components is active against cellulose, reconstitution experiments show that appropriate mixtures of each type act synergistically to effect hydrolysis of substrate. In addition to the three extracellular Β-glucosidases I (M_r, 135,000), II (M_r, 100,000), and III (M_r, 45,700), an intracellular form, IV (M_r, 57,600), has been isolated. All exist as single polypeptides. The extracellular forms I and III are most active at 70‡C, pH 5, and have half-lives under these conditions of 6 and 3 h, respectively. By contrast, the intracellular form (IV) is most active at 35‡C and is rapidly denatured at higher temperatures. Substrate specificity and other studies provide clues to their possible roles in vivo. Β-Glucosidase III acts as an exoglucohydrolase by removing glucose residues from cellooligosaccharides arising from the action of endocellulases. Β-Glucosidase I is the major enzyme involved in cleaving cellobiose and short chain cellooligosaccharides. In doing so it relieves the inhibition by cellobiose of cellulase action. The intracellular form, Β-glucosidase IV, may have a dual role. By virtue of its transferase activity it may convert incoming cellobiose to the active inducer of cellulase synthesis, whereas by cleaving cellobiose to glucose (hydrolase action) it provides energy for the cell and a repressor of cellulase formation. Four endocellulases have been purified to apparent homogeneity as judged by electrophoresis. Preliminary results show that they all have M_r values of about 70,000 and pI values less than 4. However, they differ from one another in carbohydrate content, thermal stability, and affinity for substrate. The complete cellulase system is most active at pH 4.2, 60–65‡C, and retains about 80% of its original activity after 5 d incubation at 60‡C, pH 5. Avicel and filter paper most effectively induce synthesis of the complete cellulase system, as measured by the ability of culture filtrate to digest filter paper. Cotton, Solka floc, and α-cellulose are also effective inducers, as are “wastes” such as newspaper, straw, and beet pulp. Little or no cellulase synthesis is evident when lactose, cellobiose, or glucose replaces cellulose in growth media. From a practical viewpoint we find that saccharification of beet pulp is most readily achieved by using enzyme (i.e., culture filtrate) obtained by growing the organism on medium containing beet pulp as the source of cellulose. Of the various strains ofTalaromyces emersonii investigated for cellulase production, we found CBS 814.70 to be the best, yielding approx. 0.5 IU/mL of culture filtrate. By medium optimization and genetic manipulation we have isolated a number of mutants of this strain giving 2 IU/mL or more and enzyme productivities of 20–25 IU/L/h. Xylanase, arabinogalactanase, and pectinase activities have also been detected in culture filtrates of the organism when grown on beet pulp. Various lignocellulosic materials, including cotton, Solka floc, Avicel, filter paper, newspaper, and straw, can be degraded by the enzyme system. However, much of our effort has been directed to investigation of the saccharification of beet pulp since it is available in large quantities at central locations and because its lignin content is low. About 85% of the dry weight of this material is accounted for by cellulose, hemicellulose, and pectin in roughly equal proportions. Culture filtrates effect significant saccharification of pulp as measured by the release of reducing sugars or of glucose. Ball-milling the pulp prior to incubation with enzyme effects considerable improvement in the extent of digestion. Alkali or peracetic acid pretreatment of the ball-milled substrate facilitates enzymic hydrolysis even further. Good results are also obtained when unmilled pulp is (a) pretreated with pectinase prior to incubation with normal culture filtrates or (b) incubated with more concentrated culture filtrates with good pectinase activity. Under suitable conditions, 80% hydrolysis of beet pulp polysaccharides was achieved in 5 d at 60‡C, pH 5.     

Optimizing ammonia pressurization/depressurization processing conditions to enhance enzymatic susceptibility of dwarf elephant grass

An ammonia pressurization/depressurization process was investigated to evaluate the potential of producing reducing sugars from dwarf elephant grass, a warm-season forage. Moisture, temperature, and ammonia loading affected sugar yield (p<0.0001). At optimal conditions, ammonia processing solubilized 50.9% of the hemicellulose and raised the sugar yield (percentage of theoretical) from 18 to 83%. Glucose and xylose production were increased 3.2-and 8.2-fold, respectively. The mild processing conditions of the ammonia treatment (90–100°C, 5 min), the low enzyme loading (2 international filter paper units/g), and the short hydrolysis time (24 h), greatly enhance the potential of using for ages to produce sugars valuable for several applications.     

Preliminary results on optimization of pilot scale pretreatment of wheat straw used in coproduction of bioethanol and electricity

The overall objective in this European Union-project is to develop cost and energy effective production systems for coproduction of bioethanol and electricity based on integrated biomass utilization. A pilot plan reactor for hydrothermal pretreatment (including weak acid hydrolysis, wet oxidation, and steam pretreatment) with a capacity of 100 kg/h was constructed and tested for pretreatment of wheat straw for ethanol production. Highest hemicellulose (C5 sugar) recovery and extraction of hemicellulose sugars was obtained at 190°C whereas highest C6 sugar yield was obtained at 200°C. Lowest toxicity of hydrolysates was observed at 190°C; however, addition of H₂O₂ improved the fermentability and sugar recoveries at the higher temperatures. The estimated total ethanol production was 223 kg/t straw assuming utilisation of both C6 and C5 during fermentation, and 0.5 g ethanol/g sugar.     

Flame retarded composite panels from sugar beet residues

Sugar beet residues and various additives were used to prepare panels by forming the semi-dry or wet mixtures and pressing the composites at a temperature of 150?°C and pressures of 100?C5410?kPa for up to 105?min. The highest panel density and second highest thickness were observed when a combination of calcium hydroxide and boric acid were used as additives using the semi-dry procedure. SEM images revealed that at pressures over 1000?kPa the cell wall structure of sugar beet was completely unrecognizable. The FTIR results indicated that the non-cellulosic polysaccharides contributed significantly to the properties of the panels by acting as adhesives. The best flame retardant parameters were also obtained with the calcium hydroxide/boric acid formulation. In comparison to composites prepared from recycled paper and mixtures of sugar beet shreds with recycled paper, higher density panels were prepared at lower pressure from sugar beet sources.