Hemicellulosic Polymers Research Articles

Comparative study of alkali- and acidic organic solvent-soluble hemicellulosic polysaccharides from sugarcane bagasse

Two-stage treatments of sugarcane bagasse with mild alkali and acidic 1,4-dioxane were performed. Pretreatment with 1 M NaOH aqueous solution at 20, 25, 30, 35, and 40 °C for 18 h released 55.5%, 57.3%, 59.1%, 60.9%, and 62.1% of the original hemicelluloses, respectively. Post-treatment of the corresponding alkali-treated residue with 1,4-dioxane–2 M HCl (9:1, v/v) at 87 °C for 2 h, respectively, degraded 11.6%, 11.9%, 11.4%, 10.9%, and 10.6% of hemicelluloses (% dry starting material). It was found that the five alkali-soluble hemicellulosic preparations contained a much higher amounts of xylose (78.0–82.2%) and slightly higher uronic acids (4.8–5.8%), mainly 4- O-methyl-α- d-glucopyranosyluronic acid, but were lower in arabinose (9.3–11.7%) and glucose (2.2–4.1%) than those of the corresponding five acidic dioxane-degraded hemicellulosic fractions in which xylose (44.9–46.8%), arabinose (35.9–38.1%), and glucose (13.0–13.7%) were the major sugar constituents. The studies revealed that the five alkali-soluble hemicellulosic preparations were more linear and acidic, and had a large molecular weight (35,200–37,430 g mol −1) than those of the hemicellulosic fractions (12,080–13,320 g mol −1) degraded during the acidic dioxane post-treatment. This demonstrated that the post-treatment with acidic dioxane under the condition used resulted in substantial degradation of the hemicellulosic polymers. The 10 hemicellulosic samples were further characterized by FT-IR and 1H and 13C NMR spectroscopy, GPC and thermal analysis, and the results are reported.

How do cell walls regulate plant growth?

The cell wall of growing plant tissues has frequently been interpreted in terms of inextensible cellulose microfibrils 'tethered' by hemicellulose polymers attached to the microfibril surface by hydrogen bonds, with growth occurring when tethers are broken or 'peeled' off the microfibril surface by expansins. This has sometimes been described as the 'sticky network' model. In this paper, a number of theoretical difficulties with this model, and discrepancies between predicted behaviour and observations by a number of researchers, are noted. (i) Predictions of cell wall moduli, based upon the sticky network model, suggest that the cell wall should be much weaker than is observed. (ii) The maximum hydrogen bond energy between tethers and microfibrils is less than the work done in expansion and therefore breakage of such hydrogen bonds is unlikely to limit growth. (iii) Composites of bacterial cellulose with xyloglucan are weaker than pellicles of pure cellulose so that it seems unlikely that hemicelluloses bind the microfibrils together. (iv) Calcium chelators promote creep of plant material in a similar way to expansins. (v) Reduced relative 'permittivities' inhibit the contraction of cell wall material when an applied stress is decreased. Revisions of the sticky network model that might address these issues are considered, as are alternatives including a model of cell wall biophysics in which cell wall polymers act as 'scaffolds' to regulate the space available for microfibril movement. Experiments that support the latter hypothesis, by demonstrating that reducing cell wall free volume decreases extensibility, are briefly described.

Use of monoclonal antibodies to quantify the dynamics of α‐galactosidase and endo‐1,4‐β‐glucanase production by Trichoderma hamatum during saprotrophic growth and sporulation in peat

Trichoderma species are ubiquitous soil and peat-borne saprotrophs that have received enormous scientific interest as biocontrol agents of plant diseases caused by destructive root pathogens. Mechanisms of biocontrol such as antibiosis and hyperparasitism are well documented and the biochemistry and molecular genetics of these processes defined. An aspect of biocontrol that has received little attention is the ability of Trichoderma species to compete for nutrients in their natural environments. Trichoderma species are efficient producers of polysaccharide-degrading enzymes that enable them to colonize organic matter thereby preventing the saprotrophic spread of plant pathogens. This study details the use of monoclonal antibodies (mAbs) to quantify the production of two enzymes implicated in the saprotrophic growth of Trichoderma species in peat. Using mAbs specific to the hemicellulase enzyme alpha-galactosidase (AGL) and the cellulase enzyme endo-1,4-beta-glucanase (EG), the relationship between the saprotrophic growth dynamics of a biocontrol strain of Trichoderma hamatum and the concomitant production of these enzymes in peat-based microcosms was studied. Enzyme activity assays and enzyme protein concentrations derived by enzyme-linked immunosorbent assay (ELISA) established the precision and sensitivity of mAb-based assays in quantifying enzyme production during active growth of the fungus. Trends in enzyme activities and protein concentrations were similar for both enzymes, during a 21-day sampling period in which active growth and sporulation of the fungus in peat was quantified using an independent mAb-based assay. There was a sharp increase in active biomass of T. hamatum 3 days after inoculation of microcosms with phialoconidia. After 3 days there was a rapid decline in active biomass which coincided with sporulation of the fungus. A similar trend was witnessed with EG activities and concentrations. This showed that EG production related directly to active growth of the fungus. The trend was not found, however, with AGL. There was a rapid increase in enzyme activities and protein concentrations on day 3, after which they remained static. The reason for the maintenance of elevated AGL probably resulted from secretion of the enzyme from conidia and chlamydospores. ELISA, immunofluoresence and immunogold electron microscopy studies of these cells showed that the enzyme is localized within the cytoplasm and is secreted extracellularly into the surrounding environment. It is postulated that release of oligosaccharides from polymeric hemicellulose by the constitutive spore-bound enzyme leads to AGL induction and could act as an environmental cue for spore germination.

Characteristics of degraded hemicellulosic polymers obtained from steam exploded wheat straw

The fractionation of wheat straw was studied using a two-stage process based on a steam explosion pre-treatment followed by alkaline peroxide post-treatment. Straw was steamed at temperatures comprised between 200 °C for 10 and 33 min and 220 °C for 3, 5, and 8 min. The steamed straw was washed with water to yield a solution rich in hemicelluloses-derived mono- and oligosaccharides (20.5–28.5%) together with small amounts of degraded hemicellulosic polymers (2.4–6.2%) and minor quantities of lignin (1.9–2.1%). The washed fibre was post-treated by 2% alkaline peroxide (pH 11.5) at 50 °C for 5 h. The alkali-soluble lignin (13.7–15.0%) and hemicelluloses (8.4–13.3%) were recovered by precipitation. The post-treatment was sufficient to remove lignin and survived hemicellulosic polymers and increase the brightness of the exploded straw significantly. The two-stage treatments together degraded 77.0–87.6 of the total original hemicelluloses and 92.3–99.4% of the total original lignin. This paper examined these two groups of six hemicellulosic polymer preparations degraded during the various steam explosion pre-treatments (H 1a–6a) and released during the sequential alkaline peroxide post-treatment (H 1b–6b). The effect of the most important variables in steam explosion pre-treatment (temperature, residence time, and water to straw ratio) on physico-chemical properties and structural features of the degraded hemicellulosic polymers were investigated. Thermoanalysis of these degraded hemicellulosic polymer fractions indicated that hemicellulose degradation products were also condensed to form more thermostable substrates during the steam explosion.

Alterations in cell wall polysaccharides during ripening in distinct anatomical tissue regions of the fig (Ficus carica L.) fruit

Abstract During the last stage of fig ( Ficus carica L.) fruit development, profound cell wall modification processes occur as indicated by increase in fruit size and tissue softening. In this study, we characterized the changes in cell wall polysaccharides taking place within the distinct and separate tissues of the receptacle and the pulpy drupelets during sequential ripening in fig fruit. The pectic extracts had high uronic acid contents in addition to high amounts of Ara, Gal and Rha. The gel filtration profile of the water-soluble polymers at the ripening onset in drupelets were different when compared to those of the receptacle, even though in both tissues, these polymers underwent increased solubilization and depolymerization during ripening. The molecular downshift of the CDTA-soluble polymers and the decrease in the amounts of both the uronic acid and total sugars were more pronounced in the drupelets than in the receptacle. Major difference in the neutral sugar composition between the two tissues was only observed in the Na 2 CO 3 -soluble fraction. The xyloglucan polymers in 4% KOH fraction exhibited a molecular size downshift accompanied by a decline in Xyl and increase in Glc. In the 24% KOH fractions of both tissues, the total sugar and xyloglucan components decreased in amount and also exhibited a molecular size downshift during ripening. These data suggest that even though quantitative and qualitative changes in cell wall polysaccharides occurred during ripening in both tissues, qualitative variations between tissues occurred only in the pectic polymers but not in the hemicellulosic polymers.

Structural investigation of hemicellulosic polysaccharides from Argania spinosa: characterisation of a novel xyloglucan motif

Hemicellulose polymers were isolated from Argania spinosa leaf cell walls by sequential extractions with alkali. The structure of the two main polymers, xylan and xyloglucan, was investigated by enzyme degradation with specific endoglycosidases followed by analysis of the resulting fragments by high performance anion exchange chromatography (HPAEC) and matrix-assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF MS). The results show that A. spinosa xylan is composed of a β-(1 → 4)-linked- d-xylopyranose backbone substituted with 4- O-methyl- d-glucuronic acid residues. Xyloglucan oligosaccharide subunits were generated by treatment with an endo-(1 → 4)-β- d-glucanase of the xyloglucan-rich hemicellulosic fractions. MALDI-TOF mass spectra and HPAE-PAD chromatography of the pool of endoglucanase-generated xyloglucan oligomers indicated that A. spinosa cell wall contains a XXXG-type xyloglucan. In addition to XXXG, XXFG, XLXG/XXLG, XLFG fragments previously characterised in various plants, a second group of XXXG-type fragments was detected. The primary structure of the major subunit was determined by a combination of sugar analysis, methylation analysis, post-source decay (PSD) fragment analysis of MALDI-TOF MS and 1H NMR spectroscopy. This fragment, termed XUFG, contains a novel β- d-Xyl p-(1 → 2)-α- d-Xyl p side chain linked to C-6 of the second glucose unit from the nonreducing end of the cellotetraose sequence.

Altered matrix polysaccharides in cell walls of pocket galls formed by an aphid on Distylium racemosum leaves

ABSTRACTThe present work reports the results of a study on the isolation and characterization of matrix polysaccharides in the cell walls of galls formed by an aphid (Neothoracaphis yanonis) on Distylium racemosum leaves. Cell walls were isolated from both healthy Distylium leaf and gall tissues and then extracted sequentially with cyclohexane‐trans‐1,2‐diaminetetra‐acetate (CDTA), Na2CO3, 1 m KOH, and 4 m KOH. The amount of pectin solubilized from gall cell walls was approximately 2.6‐fold higher than the pectin solubilized from leaf cell walls, whereas the amount of hemicellulose solubilized from gall cell walls was 1.4‐fold higher than that from normal leaf cell walls. When the polysaccharides were fractionated by anion‐exchange chromatography, considerable increases in arabinose and galactose were observed in CDTA‐soluble pectic polymer (fraction PI‐1) from gall cell walls, whereas the gall cell walls had less xylose in 1 m KOH‐soluble hemicellulosic polymers (fractions HI‐2, HI‐3, and HI‐4) than did the cell walls from the healthy leaf. The hemicellulosic polymers of the gall cell walls exhibited distinctly different patterns of molecular mass, compared with the healthy leaf cell walls. These results suggest that an extensive change occurs in the matrix polysaccharide structure of the cell walls of Distylium galls formed by an aphid. In addition, many glycosylhydrolase activities were detected in the protein fraction solubilized with strong saline solution from the gall cell walls, and the activities of β‐galactosidase, β‐xylosidase and α‐l‐arabinofuranosidase were considerably increased under gall formation.

Cellulosomes from mesophilic bacteria.

Plant cell wall-degrading enzymes have become increasingly important, since the development of efficient biomass degradation methods and the conversion of sugars to valuable products such as butanol and amino acids and utilizable forms of energy such as ethanol and methane could lead to less dependence on imported petroleum as a fuel and chemical source. Plant biomass is an abundant renewable resource. Since cellulose and hemicellulose comprise about 40 to 50% of plant cell walls and are considered to be the largest components of the earth's biomass, efficient conversion of this material by engineered enzymes and/or microorganisms would be highly desirable. The rate-limiting step in biomass degradation is the conversion of the cellulose and hemicellulose polymers to sugars.

Hemicellulosic polymer from Vetiver grass and its physicochemical properties

Chemical composition of two ecotypes of the Vetiver grass ( Vetiveria zizanioides nash) leaves has been investigated using the gravimetric method. The principal polysaccharides found in samples were hemicelluloses (ca. 38%), followed by cellulose (ca. 27%). Protein content determined by bicinchoninic acid assay using bovine serum albumin (BSA) as a standard was approximately 5%. Lignin content determined by acid chlorite method was approximately 10%. Ash content was 3% consisting of mainly silica (ca. 50%). The obtained hemicelluloses with the molecular weight of 30,000 showed the decomposition temperature at 310 °C. In order to determine an optimum condition for extraction of hemicelluloses, effects of process parameters: alkaline type (NaOH, KOH, Ca(OH) 2 and Ba(OH) 2), alkaline concentration (0.025–4 M), extraction time (2–18 h) and temperature (25–60 °C) were studied. It was found that all parameters influenced on the hemicellulose yield and properties in various degrees. Finally, an optimum condition giving a maximum yield of 35% hemicelluloses was recommended as 4 M NaOH at ambient temperature for 8 h.

Degradation of wheat straw lignin and hemicellulosic polymers by a totally chlorine-free method

Wheat straw lignin and hemicelluloses were degraded in a totally chlorine-free medium containing 80% acetic acid and 0.92–13.5% nitric acid. Effect of treatment time and temperature as well as nitric acid concentration on the efficiency of degradation of lignin and hemicellulosic polymers and on the yield of cellulose were comparatively investigated. The treatment with 80% acetic acid and 0.92% nitric acid as a catalyst at 120 °C for 20 min resulted in over 81% original hemicelluloses and 92% original lignin degradation. As the nitric acid concentration raises to 8.5%, over 96% original hemicelluloses and approximately 98% original lignin were degraded, yielding the cellulose approaching 96% purity. The characterisation of isolated cellulosic preparations was performed by their intrinsic viscosity, molecular weight, FT-IR, CP-MAS 13C-NMR, and thermal studies. The results showed that the treatment using 80% acetic acid and nitric acid as a catalyst under the conditions given also resulted in somewhat acetylation of the cellulose and decreased the degree of crystallinity of cellulose except for significant degradation of lignin and hemicellulosic polymers. The thermal stability of the cellulose declined with a decrease in its intrinsic viscosity

Cocoa bean carbohydrates: roasting-induced changes and polymer interactions

Roasting induced change to carbohydrates and cell wall polysaccharides was investigated in three varieties of cocoa beans. The concentrations of glucose and fructose decreased after roasting but levels of the non-reducing sugars, sucrose, raffinose, stachyose and verbascose, were not markedly affected. Approximately 10% of the arabinose content of the polysaccharides was degraded but, overall, the pectic and hemicellulosic polymers remained intact after roasting. The degree of esterification and acetylation of the pectic polysaccharides were unaffected by roasting. Roasting did promote an interaction between polysaccharides, proteins, polyphenolics and Maillard products. This led to the formation of insoluble complexes which co-purified with, and augmented, the levels of cell wall material isolated from roasted compared to unroasted beans. The implications of the results are discussed in relation to the role that “Klason lignin” plays in the formation of these chemical amalgams during roasting.

α-L-Arabinofuranosidase Activity during Development and Ripening of Normal and ACC Synthase Antisense Tomato Fruit

α-l-Arabinofuranosidases (α-Af) are plant enzymes that have the capacity to release terminal arabinofuranosyl residues from a wide variety of pectic and hemicellulosic polymers, as well as different glycoconjugates. Our interest in α-Af is related to its potential role in ripening-related loss of arabinose from tomato fruit cell walls. Using both control (cv. VF 36) and ACC synthase antisense (A11.1) tomatoes (Lycopersicon esculentum Mill.), we demonstrate that tomato α-Af activity is present during the entire ontogeny of the fruit. Immature 10-day-old fruit displayed 6-fold more α-Af activity on a per gram fresh weight basis, than mature green fruit. In VF 36 fruit, α-Af activity increased 45% from mature green 4 (48 days post anthesis) to light red stages (55 days) when fruit ripened on the vine. In contrast, no similar increase was detected in ACC synthase antisense fruit that do not ripen in the same time frame. However, when A11.1 fruit were detached at 48 days after anthesis and treated continuously with 100 mL·L-1 ethylene the fruit ripened and α-Af increased, as in ripening normal fruit. The α-Af activity pattern is similar to that reported for tomato β-galactosidases. The increasing α-Af activity during ripening and the decreased activity in antisense ACC synthase fruit after reaching the mature green stage suggest a role for ethylene in the ripening-related synthesis or activation of this enzyme.

Organization of cell walls in Sandersonia aurantiaca floral tissue.

Visual symptoms of the onset of senescence in Sandersonia aurantiaca flowers begin with fading of flower colour and wilting of the tissue. When fully senescent, the flowers form a papery shell that remains attached to the plant. The cell walls of these flowers have been examined to determine whether there are wall modifications associated with the late stages of expansion and subsequent senescence-related wilting. Changes in the average molecular size of pectin were limited through flower opening and senescence, although there was a loss of neutral sugar-containing side-branches from pectins in opening flowers, and the total amounts of pectin and cellulose continued to rise in cell walls of fully senescent sandersonia flowers. Xyloglucan endotransglycosylase activity increased in opening and mature flowers, but declined sharply as flowers wilted. Concomitantly, the proportion of hemicellulose polymers of increasing molecular weight increased from flower expansion up to the point at which wilting occurred. Approximately 50% of the non-cellulosic neutral sugar in mature flower cell walls was galactose, primarily located in an insoluble cell wall fraction. Total galactose in this fraction increased per flower with maturity, then declined at the onset of wilting. Beta-galactosidase activity was low in expanding tepals, but increased as flowers matured and wilted.

Cell-wall polysaccharides from the fruits of Limonia acidissima: isolation, purification and chemical investigation

Polysaccharides were isolated from the fruits of Limonia acidissima by sequential extraction with water, and 1 M and 4 M KOH. The water extract contained pectic polymers substituted with side chains comprising mainly of 1,5-, 1,3,5-linked arabinose together with 1,4-, 1,6-, 1,3,6-linked galactose, and lesser amounts of 1,2,4- and 1,3-linked galactose residues. Galactosyl and arabinofuranosyl groups terminated most of the branched residues. The alkaline extracts contained both pectic and hemicellulosic polymers. The insoluble material consists mainly of cellulose-rich material.

Hydrolytic Processing of Rice Husks in Aqueous Media: A Kinetic Assessment

The kinetics of hemicellulose decomposition in the non-isothermal rice husk autohydrolysis was experimentally assessed. Experimental data on the time course of the concentrations of hemicellulosic polymers (xylan, araban and acetyl groups) and their autohydrolysis products (including sugar oligomers, monosaccharides, furfural and acetic acid) were fitted to several kinetic models based on multiple pseudohomogeneous first-order reactions. Sugar oligomers were the major reaction products. The hydrolytic conversion of xylan, araban and acetyl groups was well interpreted by kinetic models based on consecutive and parallel reactions.

Characteristics, hydrolysis of cell wall polymers, and response to calcium deficiency of a cell wall-associated β-galactosidase from carrot cells

Summary The activity of β-galactosidase (EC 3.2.1.23) in the protein fraction solubilized with 3 mol/L LiCl from cell walls of suspension-cultured carrot ( Daucus carota ) cells has been found to be higher than those of the other glycosyl-hydrolytic enzymes. The cell wall-associated β-galactosidase/exo-galactanase was purified to electrophoretic homogeneity. Analysis by denaturing PAGE revealed a single polypeptide chain with a molecular mass of 50 kDa, similar to 52 kDa estimated for the native protein by size-exclusion chromatography. The enzyme contained carbohydrate and protein in a ratio of 1 : 10 (w/w), and was rich in Gly and Phe, followed by Ser, Ala and Thr. The isoelectric point was pH 6.5, and pH and temperature optima 4.4 and 45-50 °C, respectively. The β-galactosidase activity was inhibited by Mg 2+ , Ag + , Hg 2+ , p -chloromercuribenzoate and D-galactono-1,4-lactone. The K m and V max values for p -nitrophenyl-β-D-galactopyranoside were 0.77 mmol/L and 0.32 mmol (mg protein) −1 h −1 , respectively. The enzyme hydrolyzed citrus galactan in an exo-fashion and was capable of hydrolysing an acidic pectic polymer containing galactosyl and arabinosyl residues from carrot cell walls and, therefore, is an exo-galactanase. However, even after an exhaustive reaction, the enzyme had no effect on a galactose-rich hemicellulosic polymer from carrot cell walls. Under calcium deficiency the activities of a cell wall-associated α-galactosidase (EC 3.2.1.22) and β-galactosidase increased until 15 days of culture and then decreased gradually, whereas β-glucosidase (EC 3.2.1.21) activity increased markedly with time despite poor cell growth.

Interpretation of deacetylation and hemicellulose hydrolysis during hydrothermal treatments on the basis of the severity factor

Experimental and reported data on the cleavage of acetyl groups and hemicellulose removal during hydrothermal treatments of several lignocellulosic raw materials were interpreted using a generalised calculation procedure based on the severity factor. In the mathematical equations describing the hydrolysis of xylan (the main hemicellulosic polymer) and acetyl groups, the correction of the potential concentrations of both fractions to consider the presence of non-reactive portions of the substrates led to an improved reproduction of the experimental data. The generalised analysis of data allowed the calculation of activation energies for the two reactions considered, providing a fair interpretation of results obtained under a variety of experimental conditions.

Separation, characterization and hydrogel-formation of hemicellulose from aspen wood

Hemicellulose from aspen ( Populus tremula) was isolated by an alkali extraction method, which was followed by hydrogen peroxide treatment, ultrafiltration and recovery by spray drying. The sugar composition and lignin content were monitored with HPLC at each step of the separation procedure. Size-exclusion chromatography showed a polymeric hemicellulose of relatively high molar mass. The product was characterized by 1H and 13C NMR spectroscopy and was found to be composed of a linear (1→4)-β-linked d-xylose main chain with a 4- O-methyl-α- d-glucuronic acid substituting the 2-position of approximately every eighth xylose unit. Lignin and O-acetyl groups had largely been removed in the separation process. The xylan was soluble in hot water, and the film forming properties were examined at various mixtures of the hemicellulose and chitosan. These films formed hydrogels with a high swelling capacity at certain compositions. The morphologies of the films were examined with wide angle X-ray spectroscopy, and a pure xylan film was found to be crystalline, which was suggested to be a consequence of the lack of O-acetyl groups. The crystallinity of the films was found to decrease with an increasing amount of chitosan, and the film of chitosan alone showed no crystallinity. The cohesive forces of the hydrogels are suggested to be the result of the crystalline arrangement of the polymers and of electrostatic interactions between acidic groups in the hemicellulose and amine groups in the chitosan.

A cross-polarization, magic-angle-spinning, 13C-nuclear-magnetic-resonance study of polysaccharides in sugar beet cell walls

Solid-state nuclear magnetic resonance relaxation experiments were used to study the rigidity and spatial proximity of polymers in sugar beet (Beta vulgaris) cell walls. Proton T1rho decay and cross-polarization patterns were consistent with the presence of rigid, crystalline cellulose microfibrils with a diameter of approximately 3 nm, mobile pectic galacturonans, and highly mobile arabinans. A direct-polarization, magic-angle-spinning spectrum recorded under conditions adapted to mobile polymers showed only the arabinans, which had a conformation similar to that of beet arabinans in solution. These cell walls contained very small amounts of hemicellulosic polymers such as xyloglucan, xylan, and mannan, and no arabinan or galacturonan fraction closely associated with cellulose microfibrils, as would be expected of hemicelluloses. Cellulose microfibrils in the beet cell walls were stable in the absence of any polysaccharide coating.

Enteric bacterial catalysts for fuel ethanol production.

The technology is available to produce fuel ethanol from renewable lignocellulosic biomass. The current challenge is to assemble the various process options into a commercial venture and begin the task of incremental improvement. Current process designs for lignocellulose are far more complex than grain to ethanol processes. This complexity results in part from the complexity of the substrate and the biological limitations of the catalyst. Our work at the University of Florida has focused primarily on the genetic engineering of Enteric bacteria using genes encoding Zymomonas mobilis pyruvate decarboxylase and alcohol dehydrogenase. These two genes have been assembled into a portable ethanol production cassette, the PET operon, and integrated into the chromosome of Escherichia coli B for use with hemicellulose-derived syrups. The resulting strain, KO11, produces ethanol efficiently from all hexose and pentose sugars present in the polymers of hemicellulose. By using the same approach, we integrated the PET operon into the chromosome of Klebsiella oxytoca to produce strain P2 for use in the simultaneous saccharification and fermentation (SSF) process for cellulose. Strain P2 has the native ability to ferment cellobiose and cellotriose, eliminating the need for one class of cellulase enzymes. Recently, the ability to produce and secrete high levels of endoglucanase has also been added to strain P2, further reducing the requirement for fungal cellulase. The general approach for the genetic engineering of new biocatalysts using the PET operon has been most successful with Enteric bacteria but was also extended to Gram positive bacteria, which have other useful traits for lignocellulose conversion. Many opportunities remain for further improvements in these biocatalysts as we proceed toward the development of single organisms that can be used for the efficient fermentation of both hemicellulosic and cellulosic substrates.