Synergistic effects of cellulolytic and pectinolytic enzymes in degrading sugar beet pulp

Three cellulases, one hemicellulase and three pectinases were used, separately or in binary and ternary combinations, to hydrolyze dried beet-pulp, a by-product of the sugar industry. By IE-HPLC the compositions and concentrations of the sugars released were determined. The results obtained by enzymatic saccharification were compared to those obtained by acid hydrolysis. The synergistic action of cellulolytic and pectinolytic enzymes in release of total monosaccharides, and of glucose, arabinose and galacturonic acid was also studied. The combination of cellulase, hemicellulase and pectinase, commercially available, was as effective in degrading the beet pulp as the acid hydrolysis. Pectinase appeared to be the most important enzyme, since by hydrolyzing the pectic surface of the lignocellulosic substrate, it favoured the degradation of cellulose and hemicellulose by the respective enzymes.     

Enzymatic hydrolysis of beer brewers' spent grain and the influence of pretreatments

The enzymatic saccharification of plant material has been shown to be of interest in various fields, such as the production of fruit juices(1,2) and the utilization of biomass.(3) A combination of cellulase, pectinase, and hemicellulases is usually used because of the chemical composition of the matrix of plant cell walls.For apples, beet pulp, and potato fiber, almost a complete hydrolysis of polysaccharides is obtained by combining cellulose and pectinase. For nonparenchymatic tissue, the situation is somewhat different: pectin is a minor component and the hemicellulose content is much higher. Enzyme action is restricted by the lignin barrier and by the high crystallianity of cellulose in this material. For such materials, mechanical, thermal, or chemical pretreatments are necessary to achieve hydrolysis.(4,5)This communication describes various enzymatic treatements and chemical and physical pretreatemtn, using brewers' spent grain as substrate. Spent grain is the residue of malt and grain which remains in the mash-kettle after the liquefied and saccharified starch has been removed by filtration.     

Influence of different fibre sources on digestibility and nitrogen and energy balances in growing pigs

The present study was undertaken to investigate how three different fibre sources, sugar beet pulp, soya bean hulls and pectin residue, constituting 15% of diets for growing pigs, influenced daily body gain, feed conversion, apparent faecal digestibility and nitrogen and energy balances. Eight castrated crossbreed pigs (30-80 kg live weight) were used in a replicated 4 x 4 Latin-square design with one control diet and three fibre containing diets. Daily body weight gain and feed conversion were not affected by the dietary treatments. The apparent faecal digestibility of organic matter (OM) and energy were significantly lower for the fibre diets (OM: 0.81-0.85; energy: 0.78-0.83) compared to the control diet (OM: 0.88; energy: 0.86). The apparent faecal digestibility of crude protein (CP) was lower for the fibre diets (0.71-0.78) compared to the control diet (0.83), although it was only significantly lower for the sugar beet pulp and pectin residue diets. The pectin residue diet, which contained the highest amount of dietary fibre, lignin and insoluble non-starch polysaccharides, had the lowest digestibility of OM, CP and energy. There was a tendency (p = 0.07) for a diet effect on retained nitrogen in proportion to digested nitrogen, where the sugar beet pulp and pectin residue diets had numerically the highest values. Heat production and retained energy in proportion to metabolizable energy intake were not affected by fibre inclusion. It was concluded that the inclusion of sugar beet pulp, soya bean hulls and pectin residue in diets for growing pigs decreased the apparent faecal digestibility and in the diets with sugar beet pulp and pectin residue higher utilization of digested nitrogen for retention compensated for the lower amount of digested nitrogen.     

Improving the efficiency of enzyme utilization for sugar beet pulp hydrolysis

Sugar beet pulp (SBP) is a carbohydrate-rich residue of table sugar processing. It shows promise as a feedstock for fermentable sugar and biofuel production via enzymatic hydrolysis and microbial fermentation. This research focused on the enzymatic hydrolysis of SBP and examined the effects of solid loading (2–10?%, dry basis), enzyme preparation, and enzyme recycle on the production of fermentable sugars. The enzyme partitioning to the solid and liquid phases during SBP enzymatic hydrolysis and loss during recycling were investigated using SDS-PAGE and Zymogram analysis. Without considering product inhibition, the cellulase added initially to the SBP hydrolysis lost only 6?% filter paper activity and negligible carboxymethyl cellulose activity upon multiple cycles of SBP hydrolysis. It was found that enzyme dosage can be reduced by 50?% while maintaining similar, and in some cases higher fermentable sugar yield. The removal of hydrolysis products will further improve enzymatic hydrolysis of SBP for biofuel production.     

The influence of pretreatment and enzyme loading on the effectiveness of batch and fed-batch hydrolysis of corn stover

To try to improve hydrolysis yields at elevated solids loadings, a comparison was made between batch and fed-batch addition of fresh substrate at the initial and later phases of hydrolysis. Both ethanol (EPCS) and steam-pretreated corn stover (SPCS) substrates were tested at low (5 FPU) and high (60 FPU) loadings of cellulase per gram of cellulose. The fed-batch addition of fresh substrate resulted in a slight decrease in hydrolysis yields when compared with the corresponding batch reactions. A 72-h hydrolysis of the SPCS substrate resulted in a hydrolysis yield of 66% compared with 51% for the EPCS substrate. When the enzyme adsorption and substrate characteristics were assessed during batch and fed-batch hydrolysis, it appeared that the irreversible binding of cellulases to the more recalcitrant original substrate limited their access to the freshly added substrate. After 72-h hydrolysis of the SPCS substrate at low enzyme loadings, ～40-50% of the added cellulases were desorbed into solution, whereas only 20% of the added enzyme was released from the EPCS substrate. Both simultaneous and sequential treatments with xylanases and cellulases resulted in an up to a 20% increase in hydrolysis yields for both substrates at low enzyme loading. Simons' stain measurements indicated that xylanase treatment increased cellulose access, thus facilitating cellulose hydrolysis.     

Influence of different fibre sources on digestibility and nitrogen and energy balances in growing pigs

Abstract

The present study was undertaken to investigate how three different fibre sources, sugar beet pulp, soya bean hulls and pectin residue, constituting 15% of diets for growing pigs, influenced daily body gain, feed conversion, apparent faecal digestibility and nitrogen and energy balances. Eight castrated crossbreed pigs (30 – 80 kg live weight) were used in a replicated 4 × 4 Latin-square design with one control diet and three fibre containing diets. Daily body weight gain and feed conversion were not affected by the dietary treatments. The apparent faecal digestibility of organic matter (OM) and energy were significantly lower for the fibre diets (OM: 0.81 – 0.85; energy: 0.78 – 0.83) compared to the control diet (OM: 0.88; energy: 0.86). The apparent faecal digestibility of crude protein (CP) was lower for the fibre diets (0.71 – 0.78) compared to the control diet (0.83), although it was only significantly lower for the sugar beet pulp and pectin residue diets. The pectin residue diet, which contained the highest amount of dietary fibre, lignin and insoluble non-starch polysaccharides, had the lowest digestibility of OM, CP and energy. There was a tendency (p = 0.07) for a diet effect on retained nitrogen in proportion to digested nitrogen, where the sugar beet pulp and pectin residue diets had numerically the highest values. Heat production and retained energy in proportion to metabolizable energy intake were not affected by fibre inclusion. It was concluded that the inclusion of sugar beet pulp, soya bean hulls and pectin residue in diets for growing pigs decreased the apparent faecal digestibility and in the diets with sugar beet pulp and pectin residue higher utilization of digested nitrogen for retention compensated for the lower amount of digested nitrogen.     

Anaerobic hydrolysis of beet pulp — Discontinuous experiments

Pressed beet pulp has been fermented in batch experiments by adapted mixed cultures under anaerobic conditions. Hydrolysis of the constituents proceeds in two stages, first pectin and pentosans, then cellulose are degraded. These reactions limit the overall process, whereas the formation of organic acids, mainly acetic, propionic and butyric acid, proceeds more readily.     

Extraction,characterization and spontaneous emulsifying properties of pectin from sugar beet pulp

The effects of organic acid extractants on the yield and characteristics of pectin from sugar beet pulp were investigated with citric acid, malic acid and lactic acid at different pH (1.5 and 2.0) and time (1 h and 2 h). The results demonstrated that the yields of pectins were directly correlated with the decrease of pH and reaction time, and the optimum yield of 17.2% was obtained at pH 1.5 and 2 h. Furthermore, the acid type also affected the physicochemical characteristics of pectin, especially on the esterification degree (42–71), galacturonic acid content (60.2–77.8%), emulsion activity (35.2–40.1%) and emulsion stability (62.1–79.4%), and a relatively single pectin mainly consisted of homogalacturonan could be obtained under a suitable reaction condition, which was an excellent crude material for the production of emulsion activity.     

Column cellulose hydrolysis reactor: An efficient cellulose hydrolysis reactor with continuous cellulase recycling

Summary The use of a column cellulose hydrolysis reactor with continuous enzyme recycling was demonstrated by incorporating a continuous ultrafiltration apparatus at the effluent end of the column reactor. Using this setup, over 90% (w/v) cellulose hydrolysis was achieved, resulting in an average sugar concentration of 6.8% (w/v) in the effluent stream. The output of the system was 1.98 g of reducing sugar/l/h with a ratio of 87% (w/v) of the reducing sugars being monomeric sugars. Batch hydrolysis reactors were less effective, resulting in 57% (w/v) of the cellulose being hydrolyzed. The output of the batch reactor was 1.33 g of reducing sugar/l/h with similar product concentrations and percentage of monomeric sugars. The ratio of reducing sugar/filter paper unit of cellulase activity for the column method was 69.1 mg/U as compared to only 21.2 mg/U for the batch reactor.     

Anaerobic biodegradation of sugar beet pulp

Sugar beet pulp is a by-product of sugar production and consists mainly of cellulose, hemicellulose and pectin. Its composition is suitable for biological degradation. A possible alternative for the utilization of this material (besides cattle feeding) can be anaerobic methanogenic degradation. It has an additional advantage – biogas production. Beet pulp was treated by a two-step anaerobic process. The first step consisted of hydrolysis andacidification. The second step was methanogenesis. In this paper, observation ofthe process of anaerobic degradation and determination of optimal parameters is discussed. A laboratory-scale model for sugar beet pulp anaerobic biodegradation was operated. Results of model performance have shown very good pulp digestion characteristics. In addition, high efficiency removal of organic matter was achieved. Methane yield was over 0.360 m³ kg^-1 dried pulp and excess sludge production was 0.094 g per gram COD added.     

Oxidative cross-linking of chemically and enzymatically modified sugar-beet pectin

Hot acid-soluble pectins from sugar-beet pulp which do not gel in the presence of persulfate were submitted to hydrolysis with acid under different conditions of concentration, temperature and time, or with different combinations of enzymes. All the modified pectins have been chemically characterised and tested for their gelling capacity with persulfate ions. They varied primarily in the structure of the side-chains and only those obtained from cold acid hydrolysis and from degradation by arabino-furanosidase were able to gel. Accessibility of the feruloyl groups carried by the arabinose side-chains appeared essential for the gelling of beet pectins with persulfate.     

Feruloylated pectic substances from sugar-beet pulp

Pectins have been isolated from an ethanol-insoluble residue of sugar-beet pulp by sequential extraction with water, oxalate, hot dilute acid, andcold dilute alkali in yields of 2.2, 0.53, 20, and 11%, respectively. They were purified by chromatography on DEAE-cellulose at pH 4.8,or by precipitation with copper sulphate (alkali-soluble pectin). The pectins had fairly low molecular weights, a high degree of acetylation, and relatively high contents of neutral sugars, but there were clear differences beteen the four fractions. The main neutral sugars in each pectin were arabinose and galactose, and rhamnose, fucose, xylose, mannose, and glucose were also present. The fractions were homogeneous in ion-exchange and gel-filtration chromatography. Polyphenols (1–2%) and possibly proteins (3–6%) were associated with the purified pectins. In addition, feruloyl groups (up to 0.6%) were linked mainly to the acid-soluble and alkali-soluble pectins.     

Fractional extraction and physico-chemical characterization of hemicelluloses and cellulose from sugar beet pulp

Four hemicelluloses and cellulose fractions were extracted with 10% KOH or 7.5% NaOH at 15°C for 16 h and with 24% KOH or 17.5% NaOH at 15°C for 2 h from defatted, protein and pectin free, lignified or delignified sugar beet pulp (SBP). There was no significant difference in the yield and sugar composition of isolated hemicelluloses and cellulose obtained from four different procedures. 7.5% NaOH extraction at 15°C for 16 h from lignified SBP gave a slightly higher yield of hemicelluloses (10.96%), while 24% KOH extraction at 15°C for 2 h from delignified SBP produced the highest yield of cellulose (18.35%). Molecular-average weights ranged from 88 850 to 91 330 Da for the hemicelluloses obtained from lignified SBP, and 21 620–21 990 Da for the hemicelluloses isolated from delignified SBP. The neutral sugar composition of the hemicelluloses consisted of glucose, arabinose, galactose, xylose, and minor quantities of rhamnose and mannose. The infrared spectra showed an absorption band at 900 cm⁻¹, indicating some amounts of β-linked polysaccharides. Besides ferulic and p-coumaric acids, six other phenolics were also identified in the mixture of alkaline nitrobenzene oxidation of associated lignin in the isolated hemicelluloses and cellulose fractions.     

Effect of storage of apple on the enzymatic hydrolysis of cell wall polysaccharides

Cell wall materials in the form of water-insoluble solids (WIS) and water-soluble fractions (WSF) were prepared from apples stored at 4 °C for 30 weeks. During storage, the WIS content decreased whereas the WSF content remained unchanged. The total amount of polysaccharides decreased, in particular the pectic polymers which decreased by 10%. In contrast, the soluble pectic fraction increased by 40% whilst its degree of methoxylation remained constant. The arabinose and galactose content progressively declined. The enzymatic treatment of the apple tissues was more effective the longer the storage; yields correlated well with the enzyme hydrolysis of WIS. The accessibility of pectin to poly-galacturonases in apple tissues is discussed since it was higher at the end of storage, whereas the solubilisation of pectins from WIS by polygalacturonases remained constant. On the other hand, with liquefying enzymes, the yield of pectin solubilisation from apple tissues or WIS were well correlated and increased with storage time.     

Optimization of reaction conditions for enzymatic viscosity reduction and hydrolysis of wheat arabinoxylan in an industrial ethanol fermentation residue

This study examined enzyme-catalyzed viscosity reduction and evaluated the effects of substrate dry matter concentration on enzymatic degradation of arabinoxylan in a fermentation residue, "vinasse", resulting from industrial ethanol manufacture on wheat. Enzymatic catalysis was accomplished with a 50:50 mixture of an enzyme preparation from Humicola insolens, Ultraflo L, and a cellulolytic enzyme preparation from Trichoderma reesei, Celluclast 1.5 L. This enzyme mixture was previously shown to exhibit a synergistic action on arabinoxylan degradation. The viscosity of vinasse decreased with increased enzyme dosage and treatment time at pH 5, 50 degrees C, 5 wt % vinasse dry matter. After 24 h of enzymatic treatment, 76-84%, 75-80%, and 43-47%, respectively, of the theoretically maximal arabinose, xylose, and glucose releases were achieved, indicating that the viscosity decrease was a result of enzyme-catalyzed hydrolysis of arabinoxylan, beta-glucan, and cellulose. In designed response surface experiments, the optimal enzyme reaction conditions with respect to pH and temperature of the vinasse, the vinasse supernatant (mainly soluble material), and the vinasse sediment (mainly insoluble substances) varied from pH 5.2-6.4 and 41-49 degrees C for arabinose release and from pH 4.9-5.3 and 42-46 degrees C for xylose release. Even though only limited hydrolysis of the arabinoxylan in the vinasse sediment fraction was obtained, the results indicated that the same enzyme activities acted on the arabinoxylan in the different vinasse fractions irrespective of the state of solubility of the substrate material. The levels of liberated arabinose and xylose increased with increased dry matter concentration during enzymatic hydrolysis in the vinasse and the vinasse supernatant, but at the same time, increased substrate dry matter concentrations gave corresponding linear decreases in the hydrolytic efficiency as evaluated from levels of monosaccharide release per weight unit dry matter. The study thus documents that enzymatic arabinoxylan hydrolysis of the vinasse significantly decreases the vinasse viscosity and that a compromise in the dry matter must be found if enzymatic efficiency must be balanced with monosaccharide yields.     

Influence of steam pretreatment severity on post‐treatments used to enhance the enzymatic hydrolysis of pretreated softwoods at low enzyme loadings

It is recognized that some form of post‐treatment will usually be required if reasonable hydrolysis yields (>60%) of steam pretreated softwood are to be achieved when using low enzyme loadings (5 FPU/g cellulose). In the work reported here we modified/removed lignin from steam pretreated softwood while investigating the influence that the severity of pretreatment might have on the effectiveness of subsequent post‐treatments. Although treatment at a lower severity could provide better overall hemicellulose recovery, post‐treatment was not as effective on the cellulosic component. Pretreatment at medium severity resulted in the best compromise, providing reasonable recovery of the water soluble hemicellulose sugars and the use of post‐treatment conditions that significantly increased the enzymatic hydrolysis of the water insoluble cellulosic component. Post‐treatment with alkaline hydrogen peroxide or neutral sulfonation resulted in 62% cellulose hydrolysis at an enzyme loading of 5 FPU/g cellulose, which was four times greater than was obtained when the cellulosic fraction was not post‐treated. When the enzyme loading was increased to 15 FPU/g cellulose, the post‐treated cellulosic fraction was almost completely hydrolyzed to glucose. Despite the higher lignin content (44%) of the sulfonated substrate, similar hydrolysis yields to those achieved after alkaline peroxide post‐treatment (14% lignin content) indicated that, in addition to lignin removal, lignin modification also plays an important role in influencing the effectiveness of hydrolysis when low enzyme loadings are used. Biotechnol. Bioeng. 2011;108: 2300–2311. © 2011 Wiley Periodicals, Inc.     

Effect of various nitrogen salts in beet pulp medium on polygalacturonase activity of Penicillium sp. 7/4B/EI 1 mutant.

The highest PG activity was obtained in beet pulp with (NH4)2HPO4 in amount equivalent to 0.14--0.28% N after 7 days of growth; At optimal concentration, (NH4)2HPO4 did not influence the biomass production, but increased the pH of the culture media to above 6.0. The PG was an extracellular enzyme, acting mainly on highly esterified pectin. The attack was of a random manner, characteristic for the endo-enzymes. The enzyme was active at temperature 20 degrees C--40 degrees C and pH 5.0--6.0.     

Depolymerization of starch and pectin using superporous matrix supported enzymes

Immobilized enzyme catalyzed biotransformations involving macromolecular substrates and/or products are greatly retarded due to slow diffusion of large substrate molecules in and out of the typical enzyme supports. Slow diffusion of macromolecules into the matrix pores can be speeded up by use of macroporous supports as enzyme carriers. Depolymerization reactions of polysaccharides like starch, pectin, and dextran to their respective low molecular weight products are some of the reactions that can benefit from use of such superporous matrices. In the present work, an indigenously prepared rigid cross-linked cellulose matrix (called CELBEADS) has been used as support for immobilizing alpha amylase (1,4-alpha-D-glucan glucanohydrolase, EC 3.2.1.1.) and pectinase (endo-PG: poly(1,4-alpha-galactouronide) glycanohydrolase, EC 3.2.1.15). The immobilized enzymes were used for starch and pectin hydrolysis respectively, in batch, packed bed and expanded bed modes. The macroporosity of CELBEADS was found to permit through-flow and easy diffusion of substrates pectin and starch to enzyme sites in the porous supports and gave reaction rates comparable to the rates obtained using soluble enzymes.     

Organosolv pretreatment for enzymatic hydrolysis of poplars: I. Enzyme hydrolysis of cellulosic residues

Aspen (Populus tremuloides) and black cottonwood (Populus trichocarpa) organosolv pulps produced in a wide range of solvent composition (between 30 and 70% by volume of methanol) and catalysts (H(2)SO(4) and H(3)PO(4)) such that the cooking liquor pH 相似文献   

Pectin substances of the callus culture of Silene vulgaris (M.) G

Pectin-protein fraction SVC was isolated from the callus culture of the bladder campion (Silene vulgaris). The main components in it were residues of D-galacturonic acid, galactose, arabinose, rhamnose, and protein. Using ion-exchange chromatography, ultrafiltration, and acid and enzymatic hydrolysis, it was shown that SVC contained a mixture of molecules of linear pectin, branched pectin polysaccharide, and pectin-protein polymer. A fragment of the linear chain of galacturonan amounted to more than half of the entire carbohydrate silenan chain. The branched area of the macromolecule is represented by rhamnogalacturonan I. The pectin-protein polymer consisted mainly of protein and weakly branched pectin fragments with molecular mass of more than 300 kDa.