A Comparative Analysis of in vitro cellulose synthesis from cell-free extracts of mung bean (Vigna radiata,Fabaceae) And Cotton (Gossypium hirsutum,Malvaceae)

A comparison of cellulose synthesized in vitro from primary walls of etiolated mung bean (Vigna radiata) seedlings and secondary walls of cotton fibers (Gossypium hirsutum) was made by applying conditions found to be essential for in vitro cellulose I assembly from cotton (Kudlicka et al., 1995, Plant Physiology, vol. 107, pp. 111–123). Mung bean fractions including the plasma membrane (PM), the first solubilized fraction (SE₁), and the second solubilized fraction (SE₂), incorporated more radioactive UDP-Glc into the total product than the same fractions from secondary walls. A significant difference was found with the mild digitonin solubilized fraction (SE₁), which produced eight times more total product than the SE₁ fraction of cotton. However, the SE₁ fraction from cotton produced a larger quantity of cellulose (32.1%) than from mung bean (6.9%). Treatment of the in vitro product by acetic/nitric acid reagent (AN) for varying periods of time demonstrated that cellulose synthesized in vitro from mung bean was more easily degraded than cellulose from cotton fibers. This would suggest that cellulose I produced in vitro from the cotton SE₁ fraction may have a higher crystallinity and DP than cellulose I produced in vitro from mung bean. The fibrils of cellulose produced by the SE, fraction of mung bean were loosely associated and not arranged into a compact bundle as in case of cellulose I synthesized by the cotton SE₁ fraction. The electron diffraction patterns (ED) of both products show reflections characteristic for cellulose I. Products from the SE₂ fraction of mung bean and cotton reveal similarities with the cellulose II allomorph synthesized, as well as abundant β-1,3-glucan.     

Viability and cellulose synthesizing ability of Gluconacetobacter xylinus cells under high-hydrostatic pressure

The effect of pressure on viability and the synthesis of bacterial cellulose (BC) by Gluconacetobacter xylinus ATCC53582 were investigated. G. xylinus was statically cultivated in a pressurized vessel under 0.1, 30, 60, and 100 MPa at 25°C for 6 days. G. xylinus cells remained viable and retained cellulose producing ability under all the conditions tested, though the production of cellulose decreased with increasing the pressure. The BCs produced at each pressure condition were analyzed by field emission scanning electron microscopy (FE-SEM) and Fourier Transform Infrared (FT-IR). FE-SEM revealed that the widths of BC fibers produced under high pressure decreased as compared with those produced under the atmospheric pressure. By FT-IR, all the BCs were found to be of Cellulose type I, as the same as typical native cellulose. Our findings evidently showed that G. xylinus possessed a piezotolerant (barotolerant) feature adapting to 100 MPa without losing its BC producing ability. This was the first attempt in synthesizing BC with G. xylinus under elevated pressure of 100 MPa, which corresponded to the deep sea at 10,000 m.     

Comparative degradation of cellulose and sugar formation by three newly isolated mesophilic anaerobes and Clostridium thermocellum

Summary To develop a coculture for a single step conversion of cellulose to fuels and chemicals, the cellulose degradation by three newly isolated mesophilic anaerobes was compared with that of Clostridium thermocellum. Degradation of cellulose obtained from cotton linters, and delignified ball-milled pulp and steam-exploded aspen wood by mesophilic anaerobes was comparable to that by Cl. thermocellum. However mesophilic anaerobes produced larger amounts of sugars. In media containing 50 g/l of cellulose suspensions, the sugars produced by mesophilic anaerobes were 10.5 to 14.5 g/l, about 50% of the cellulose used.     

Effects of cellulose on growth,enzyme production,and ultrastructure of a Cellulomonas species

Cellulomonas sp. (NRCC 2406) was grown on complex medium (peptone-tryptone-yeast extract) alone, or with the addition of different celluloses (solka floc, avicel, CF 11 cellulose or Whatman No. 1 filter paper) and/or glucose. Cultures growing on the complex medium without cellulose produced low levels of endo- and exo-cellulases and very little -glucosidase. Adding cellulose stimulated growth, as measured by cellular protein or by viable counts, and also stimulated production of cellulases. Adding glucose in the prescene of cellulose inhibited growth and cellulose breakdown. Glucose also inhibited attachment of growing cells to cellulose fibres. Electron microscope studies showed that Cellulomonas sp. adhered to the cellulose fibers. In the presence of cellulose in the media, the cells developed a thicker outer layer which probably helps in the adhesion process.Abbreviations PTYE peptone, tryptone, yeast extract medium - DNS dinitrosalicylic acid - CMC carboxymethyl cellulose - cfu/ml colony-forming units per ml     

Production of caproic acid by cocultures of ruminal cellulolytic bacteria and Clostridium kluyveri grown on cellulose and ethanol

Ruminal cellulolytic bacteria (Fibrobacter succinogenes S85 or Ruminococcus flavefaciens FD-1) were combined with the non-ruminal bacterium Clostridium kluyveri and grown together on cellulose and ethanol. Succinate and acetate produced by the cellulolytic organisms were converted to butyrate and caproate only when the culture medium was supplemented with ethanol. Ethanol (244 mM) and butyrate (30 mM at pH 6.8) did not inhibit cellulose digestion or product formation by S85 or FD-1; however caproate (30 mM at pH 6.8) was moderately inhibitory to FD-1. Succinate consumption and caproate production were sensitive to culture pH, with more caproic acid being produced when the culture was controlled at a pH near neutrality. In a representative experiment under conditions of controlled pH (at 6.8) 6.0 g cellulose 1^–1 and 4.4 g ethanol 1^–1 were converted to 2.6 g butyrate 1^–1 and 4.6 g caproate 1^–1. The results suggest that bacteria that efficiently produce low levels of ethanol and acetate or succinate from cellulose should be useful in cocultures for the production of caproic acid, a potentially useful industrial chemical and bio-fuel precursor.Mention of specific products is intended only to provide information and does not contitute an endorsement by the U.S. Department of Agriculture over other products not mentioned.     

Homogeneous phase synthesis of cellulose carbamate silica hybrid materials using 1-n-butyl-3-methylimidazolium chloride ionic liquid medium

Cellulose carbamate silica hybrid materials can be prepared in 78–84% yield using the homogeneous phase reaction of 3-(triethoxysilyl)propyl isocyanate with cellulose dissolved in 1-n-butyl-3-methylimidazolium chloride ionic liquid and then using NH₄OH catalyzed hydrolysis of triethoxysilyl groups and the sol–gel process. New cellulose carbamate silica hybrid materials produced were characterized by elemental analysis, FT-IR, and TG-DTA. The hydrophilic affinity of these materials is shown to decrease with the degree of substitution of the cellulose hydroxyl groups with carbamate groups.     

Characteristics of the binding of a bacterial expansin (BsEXLX1) to microcrystalline cellulose

Plant expansin proteins induce plant cell wall extension and have the ability to extend and disrupt cellulose. In addition, these proteins show synergistic activity with cellulases during cellulose hydrolysis. BsEXLX1 originating from Bacillus subtilis is a structural homolog of a β‐expansin produced by Zea mays (ZmEXPB1). The Langmuir isotherm for binding of BsEXLX1 to microcrystalline cellulose (i.e., Avicel) revealed that the equilibrium binding constant of BsEXLX1 to Avicel was similar to those of other Type A surface‐binding carbohydrate‐binding modules (CBMs) to microcrystalline cellulose, and the maximum number of binding sites on Avicel for BsEXLX1 was also comparable to those on microcrystalline cellulose for other Type A CBMs. BsEXLX1 did not bind to cellooligosaccharides, which is consistent with the typical binding behavior of Type A CBMs. The preferential binding pattern of a plant expansin, ZmEXPB1, to xylan, compared to cellulose was not exhibited by BsEXLX1. In addition, the binding capacities of cellulose and xylan for BsEXLX1 were much lower than those for CtCBD3. Biotechnol. Bioeng. 2013; 110: 401–407. © 2012 Wiley Periodicals, Inc.     

Synthesis of 20α- and 20β-Amino-3,5-pregnadiene Derivatives and Antimicrobial Activity

Cellulose production by Acetobacter strains is enhanced by the addition of a small amount of cellulose to the production culture. The effect of an endo-β-1, 4-glucanase from Bacillus subtilis on the cellulose production by Acebohacter xylinum BPR2001 was examined by adding various amounts of the purified glucanase to the culture. The addition of a small amount of this glucanase enhanced cellulose production. Furthermore, it reduced the amount of a polysaccharide called acetan produced. However, an active-site mutant enzyme of the glucanase, which showed no enzyme activity but still had cellulose-binding ability, had no effect on cellulose production. It was concluded, therefore, that the endoglucanase activity itself, but not the cellulose-binding ability, was essential for the enhancement of cellulose production. The structural properties of the cellulose produced in the presence of the endoglucanase were found to be almost identical to those of native bacterial cellulose.     

Molecular Size and Chain Length Distribution in Acanthamoeba Cellulose*

SYNOPSIS Evidence obtained by total hydrolysis, partial acetolysis, periodate oxidation, as well as treatment with amylase, emulsin, and Trichoderma virideβ-(1→4)-glucanase, verified that the alkali insoluble component of Acanthamoeba castellanii was pure β-(1→4)-glucan. The weight average chain length of the cellulose varied from DP = 3170 to DP = 4130 (mean DP = 3480) with polysaccharide obtained from seven seemingly identical cultures. Isolation of the cyst-wall cellulose by nondegrading means indicated that alkali extraction was not depolymerizing the polysaccharide. Fractionation of cellulose obtained from a single culture produced fractions from DP = 550 to DP = 4550 (mean DP = 3280; 98.7% of the original cellulose), indicating that the cellulose is polydisperse.     

Gas Chromatographic Determination of Optical Purity of a Chiral Derivatization Reagent,N-Acyl-l-prolyl Chloride

Two endo-type cellulases, tentatively called carboxymethyl cellulases (CMCases) I and II, were purified by gel filtration, ion-exchange chromatography, affinity chromatography, and chromato-focusing from a culture supernatant of Penicillium purpurogenum. Their homogeneity was verified by analytical polyacrylamide gel electrophoresis. The molecular weights of CMCases I and II, estimated by gel filtration, were 72,000 and 50,000, respectively. CMCases I and II contained about 12% and 8% carbohydrate, and had isoelectric points of 4.3 and 3.9, respectively. CMCase I produced cellobiose, glucose, and a trace amount of cellotriose from H₃PO₄-swollen cellulose and Avicel (microcrystalline cellulose), while CMCase II produced cellobiose and cellotriose with a small amount of glucose and cellotetraose. The products from reduced cellopentaose by both enzymes were released predominantly in the β-configuration. CMCase II appeared to act in more random fashion than I against carboxymethyl cellulose. These results suggest that both enzymes attack insoluble cellulose randomly, although there are some differences in the mode of hydrolytic action.     

Thermophilic,anaerobic co-digestion of microalgal biomass and cellulose for H<Subscript>2</Subscript> production

Microalgal biomass has been a focus in the sustainable energy field, especially biodiesel production. The purpose of this study was to assess the feasibility of treating microalgal biomass and cellulose by anaerobic digestion for H₂ production. A microbial consortium, TC60, known to degrade cellulose and other plant polymers, was enriched on a mixture of cellulose and green microalgal biomass of Dunaliella tertiolecta, a marine species, or Chlorella vulgaris, a freshwater species. After five enrichment steps at 60°C, hydrogen yields increased at least 10% under all conditions. Anaerobic digestion of D. tertiolecta and cellulose by TC60 produced 7.7 mmol H₂/g volatile solids (VS) which were higher than the levels (2.9–4.2 mmol/g VS) obtained with cellulose and C. vulgaris biomass. Both microalgal slurries contained satellite prokaryotes. The C. vulgaris slurry, without TC60 inoculation, generated H₂ levels on par with that of TC60 on cellulose alone. The biomass-fed anaerobic digestion resulted in large shifts in short chain fatty acid concentrations and increased ammonium levels. Growth and H₂ production increased when TC60 was grown on a combination of D. tertiolecta and cellulose due to nutrients released from algal cells via lysis. The results indicated that satellite heterotrophs from C. vulgaris produced H₂ but the Chlorella biomass was not substantially degraded by TC60. To date, this is the first study to examine H₂ production by anaerobic digestion of microalgal biomass. The results indicate that H₂ production is feasible but higher yields could be achieved by optimization of the bioprocess conditions including biomass pretreatment.     

Decomposition of 14C-labeled cellulose substrates in litter and soil from a beechwood on limestone

The decomposition of three different ¹⁴C-labeled cellulose substrates (plant holocellulose, plant cellulose prepared from ¹⁴C-labeled beech wood (Fagus sylvatica) and bacterial cellulose produced by Acetobacter xylinum) in samples from the litter and mineral soil layer of a beechwood on limestone was studied. In a long-term (154 day) experiment, mineralization of cellulose materials, production of ¹⁴C-labeled water-soluble compounds, and incorporation of ¹⁴C in microbial biomass was in the order Acetobacter cellulose > holocellulose > plant cellulose in both litter and soil. In general, mineralization of cellulose, production of ¹⁴C-labeled water-soluble compounds, and incorporation of ¹⁴C in microbial biomass were more pronounced, but microbial biomass ¹⁴C declined more rapidly in litter than in soil. In short-term (14 day) incubations, mineralization of cellulose substrates generally corresponded with cellulase and xylanase activities in litter and soil. Pre-incubation with trace amounts of unlabeled holocellulose significantly increased the decomposition of ¹⁴C-labeled cellulose substrates and increased cellulase activity later in the experiment but did not affect xylanase activity. The sum of ¹⁴CO₂ production, ¹⁴C in microbial biomass, and ¹⁴C in water-soluble compounds is considered to be a sensitive parameter by which to measure cellulolytic activity in soil and litter samples in short-term incubations. Shorter periods than 14 days are preferable in assays using Acetobacter cellulose, because the decomposition of this substrate is more variable than that of holocellulose and plant cellulose.Offprint requests to: S. Scheu.     

Application of cellulases from Acrophialophora nainiana and Penicillium echinulatum in textile processing of cellulosic fibres

New cellulases from the fungi Acrophialophora nainiana and Penicillium echinulatum were used in the finishing of knitted cotton fabrics (biopolishing) and compared with the well established enzymes from Trichoderma reesei. Both cellulases reduced the pilling tendency with a lower weight loss than T. reesei cellulases. Cellulases from P. echinulatum were also studied in stonewashing of denim fabrics to obtain the fashionable aged look in indigo dyed jeans ware and were found to remove more colour from denim fabrics and produce less indigo dye redeposition (back-staining) than commercial acid or neutral cellulases under the test conditions. Efficiency was found to be influenced by pH during textile processing and the substrate used for the production of cellulases. Cellulases produced by P. echinulatum grown on cellulose showed better stonewashing results (higher colour removal and less back-staining) than cellulases produced on sugar cane bagasse. The substrate used during enzyme production of P. echinulatum cellulases seems to have a significant influence on cellulose composition, which affects textile processing results.     

An amperometric enzyme biosensor for real‐time measurements of cellobiohydrolase activity on insoluble cellulose

An amperometric enzyme biosensor for continuous detection of cellobiose has been implemented as an enzyme assay for cellulases. We show that the initial kinetics for cellobiohydrolase I, Cel7A from Trichoderma reesei, acting on different types of cellulose substrates, semi‐crystalline and amorphous, can be monitored directly and in real‐time by an enzyme‐modified electrode based on cellobiose dehydrogenase (CDH) from Phanerochaete chrysosporium (Pc). PcCDH was cross‐linked and immobilized on the surface of a carbon paste electrode which contained a mediator, benzoquinone. An oxidation current of the reduced mediator, hydroquinone, produced by the CDH‐catalyzed reaction with cellobiose, was recorded under constant‐potential amperometry at +0.5 V (vs. Ag/AgCl). The CDH‐biosensors showed high sensitivity (87.7 µA mM^?1 cm^?2), low detection limit (25 nM), and fast response time (t_95% ～ 3 s) and this provided experimental access to the transient kinetics of cellobiohydrolases acting on insoluble cellulose. The response from the CDH‐biosensor during enzymatic hydrolysis was corrected for the specificity of PcCDH for the β‐anomer of cello‐oligosaccharides and the approach were validated against HPLC. It is suggested that quantitative, real‐time data on pure insoluble cellulose substrates will be useful in attempts to probe the molecular mechanism underlying enzymatic hydrolysis of cellulose. Biotechnol. Bioeng. 2012; 109: 3199–3204. © 2012 Wiley Periodicals, Inc.     

Features of bacterial cellulose synthesis in a mutant generated by disruption of the diguanylate cyclase 1 gene of <Emphasis Type="Italic">Acetobacter xylinum</Emphasis> BPR 2001

The diguanylate cyclase 1 (DGC1) (dgc1) gene in Acetobacter xylinum BPR 2001—a bacterial cellulose (BC) producer—was cloned and sequenced, and a DGC1 gene-disrupted mutant, strain DD, was constructed. The production and structural characteristics of the BC formed by DD were compared with those of the parental strain BPR 2001. BC production by DD was almost the same as that by BPR 2001 in static cultivation and in shake flask cultivation. However, in a jar fermentor DD produced about 36% more BC than the parental strain. DD produced suspended particle materials that cannot aggregate owing to their random structural characteristics in static cultivation; more uniformly dispersed BC pellicles and smaller BC pellets are produced on average in a jar fermentor, as reflected by the higher BC production by DD than by the parental strain in a jar fermentor. Micrographs of BC produced by DD revealed that the width of cellulose ribbons assemblies decreased as a result of differences in the ultrastructure and mechanism of formation of BC between the two strains. These results reveal that disruption of the dgc1 gene, which catalyzes synthesis of c-di-GMP (an effector of BC synthase), is not fatal for BC synthesis, although it affects BC structure.     

Isolation and screening of potential actinobacteria for rapid composting of rice straw

Rice straw is produced as a by-product from rice cultivation, which is composed largely of lignocellulosic materials amenable to general biodegradation. Lignocellulolytic actinobacteria can be used as a potential agent for rapid composting of bulky rice straw. Twenty-five actinobacteria isolates were isolated from various in situ and in vitro rice straw compost sources. Isolates A2, A4, A7, A9 and A24 were selected through enzymatic degradation of starch, cellulose and lignin followed by the screening for their adaptability on rice straw powder amended media. The best adapted isolate (A7) was identified as Micromonospora carbonacea. It was able to degrade cellulose, hemicelluloses and carbon significantly (P ≤ 0.05) over the control. C/N ratio was reduced to 18.1 from an initial value of 29.3 in 6 weeks of composting thus having the potential to be used in large scale composting of rice straw.     

Engineering endoglucanase-secreting strains of ethanologenic Klebsiella oxytoca P2

Klebsiella oxytoca P2 was developed as a biocatalyst for the simultaneous saccharification and fermentation (SSF) of cellulose by chromosomally integrating Zymomonas mobilis genes (pdc, adhB) encoding the ethanol pathway. This strain contains the native ability to transport and metabolize cellobiose, eliminating the need to supplement with β-glucosidase during SSF. To increase the utility of this biocatalyst, we have now chromosomally integrated the celZ gene encoding the primary endoglucanase from Erwinia chrysanthemi. This gene was expressed at high levels by replacing the native promoter with a surrogate promoter derived from Z. mobilis DNA. With the addition of out genes encoding the type II protein secretion system from E. chrysanthemi, over half of the active endoglucanase (EGZ) was secreted into the extracellular environment. The two most active strains, SZ2(pCPP2006) and SZ6(pCPP2006), produced approximately 24 000 IU L⁻¹ of CMCase activity, equivalent to 5% of total cellular protein. Recombinant EGZ partially depolymerized acid-swollen cellulose and allowed the production of small amounts of ethanol by SZ6(pCPP2006) without the addition of fungal cellulase. However, additional endoglucanase activities will be required to complete the depolymerization of cellulose into small soluble products which can be efficiently metabolized to ethanol. Received 14 December 1998/ Accepted in revised form 04 March 1999     

Linking Foliar Chemistry to Forest Floor Solid and Solution Phase Organic C and N in Picea abies [L.] Karst Stands in Northern Bohemia

Dissolved organic carbon and nitrogen (DOC and DON) produced in the forest floor are important for ecosystem functions such as microbial metabolism, pedogenesis and pollutant transport. Past work has shown that both DOC and DON production are related to litterfall and standing stocks of C and N in the forest floor. This study, conducted in spring, 2003, investigated variation in forest floor water extractable DOC (WEDOC) and DON (WEDON) and forest floor C and N as a function of lignin, cellulose and N contained in live canopy foliage across eight Picea abies [L.] Karst stands in northern Bohemia. Based on Near Infrared Spectroscopy (NIR) analysis of foliar materials, lignin:N and cellulose:N content of the youngest needles (those produced in 2002) were positively and significantly related to WEDOC (R² = 0.82–0.97; P<0.01) and to forest floor C:N ratio (R = 0.72–0.78; P<0.01). Foliar N was strongly and negatively related to WEDOC and C:N ratio (R = −0.91 and 0.72; P<0.05) among our study sites. WEDON was positively correlated to foliar lignin:N (R = 0.48; P<0.05; n=40). Forest floor C pools were not positively correlated with foliar lignin and cellulose and forest floor N pools were not positively correlated with foliar N. Instead, a significant negative correlation was found between forest floor N pools and foliar cellulose (R=−0.41; P<0.05), and between forest floor C pools and foliar N (R = −0.44; P<0.05). From a remote sensing standpoint, our results are important because canopy reflectance properties are primarily influenced by the most recent foliage, and it was the chemistry of the most recently produced needles that showed a stronger relationship with forest floor WEDOC and C:N ratio suggesting forest floor production of WEDOC can be calculated regionally with remote sensing.     

Enzymatic degradation of steam-pretreated Lespedeza stalk (Lespedeza crytobotrya) by cellulosic-substrate induced cellulases

The cellulase production by Trichoderma viride, cultivated on different substrates, namely steam-pretreated Lespedeza, filter paper, microcrystalline cellulose (MCC) or carboxymethyl cellulose (CMC), was studied. Different cellulase systems were secreted when cultivated on different substrates. The cellulolytic enzyme from steam-pretreated Lespedeza medium performed the highest filter paper activity, exoglucanase and endoglucanase activities, while the highest β-glucosidase activity was obtained from the enzyme produced on filter paper medium. The hydrolytic potential of the enzymes produced from different media was evaluated on steam-pretreated Lespedeza. The cellulase from steam-pretreated Lespedeza was found to have the most efficient hydrolysis capability to this specific substrate. The molecular weights of the cellulases produced on steam-pretreated Lespedeza, filter paper and MCC media were 33, 37 and 40 kDa, respectively, and the cellulase from CMC medium had molecular weights of 20 and 43 kDa. The degree of polymerization, crystallinity index and micro structure scanned by the scanning electron microscopy of degraded steam-pretreated Lespedeza residues were also studied.     

The curdlan-type exopolysaccharide produced by <Emphasis Type="Italic">Cellulomonas flavigena</Emphasis> KU forms part of an extracellular glycocalyx involved in cellulose degradation

The genus Cellulomonas is comprised of a group of Gram-positive, soil bacteria capable of utilizing cellulose as their sole source of carbon and energy. Cellulomonas flavigena KU was originally isolated from leaf litter and subsequently shown to produce large quantities of a curdlan-type (-1,3-glucan) exopolysaccharide (EPS) when provided with an excess of glucose or other soluble carbon-source. We report here that curdlan EPS is also produced by Cellulomonas flavigena KU when growing on microcrystalline cellulose in mineral salts-yeast extract media. Microscopic examination of such cultures shows an adherent biofilm matrix composed of cells, curdlan EPS, and numerous surface structures resembling cellulosome complexes. Those Cellulomonas species that produce curdlan EPS are all non-motile and adhere to cellulose as it is broken down into soluble sugars. These observations suggest two very different approaches towards the complex process of cellulose degradation within the genus Cellulomonas.