Brittle Culm1, a COBRA-Like Protein,Functions in Cellulose Assembly through Binding Cellulose Microfibrils

Cellulose represents the most abundant biopolymer in nature and has great economic importance. Cellulose chains pack laterally into crystalline forms, stacking into a complicated crystallographic structure. However, the mechanism of cellulose crystallization is poorly understood. Here, via functional characterization, we report that Brittle Culm1 (BC1), a COBRA-like protein in rice, modifies cellulose crystallinity. BC1 was demonstrated to be a glycosylphosphatidylinositol (GPI) anchored protein and can be released into cell walls by removal of the GPI anchor. BC1 possesses a carbohydrate-binding module (CBM) at its N-terminus. In vitro binding assays showed that this CBM interacts specifically with crystalline cellulose, and several aromatic residues in this domain are essential for binding. It was further demonstrated that cell wall-localized BC1 via the CBM and GPI anchor is one functional form of BC1. X-ray diffraction (XRD) assays revealed that mutations in BC1 and knockdown of BC1 expression decrease the crystallite width of cellulose; overexpression of BC1 and the CBM-mutated BC1s caused varied crystallinity with results that were consistent with the in vitro binding assay. Moreover, interaction between the CBM and cellulose microfibrils was largely repressed when the cell wall residues were pre-stained with two cellulose dyes. Treating wild-type and bc1 seedlings with the dyes resulted in insensitive root growth responses in bc1 plants. Combined with the evidence that BC1 and three secondary wall cellulose synthases (CESAs) function in different steps of cellulose production as revealed by genetic analysis, we conclude that BC1 modulates cellulose assembly by interacting with cellulose and affecting microfibril crystallinity.     

不同培养方式对细菌纤维素产量和结构性质的影响

考察了自行筛选的Acetobacter xylinum NUST4.2在静置培养和发酵罐培养获得的细菌纤维素(BC)的产量、基本结构和性能的差异。结果表明:静置培养时产纤维素7.5g/L,产率为0.052g/L/h,在机械搅拌发酵罐中培养3d产量达3.13g/L,产率达0.043g/L/h;SEM分析显示静置培养和发酵罐培养得到的纤维素均具有网状结构,但静置获得的纤维素丝带相互缠绕且层状重叠,更加致密,丝带更细;FT-IR分析知搅拌不改变纤维素的化学结构,但能减弱分子间氢键,和XRD结合分析可知静置培养的纤维素具有更高结晶指数,更高Iα含量和更大晶粒尺寸,但不改变晶型,仍为纤维素I型,说明搅拌会干扰纤维素初始纤丝的结晶,有利于形成更小的晶粒和较Iα稳定的Iβ。与棉纤维素相比,静置培养获得的纤维素的热稳定性更好,而发酵罐培养获得的纤维素则阻燃性更好。     

Modification of microstructure and selected physicochemical properties of bacterial cellulose produced by bacterial isolate using hydrocolloid-fortified Hestrin–Schramm medium

Bacterial cellulose (BC) is a biopolymer with applications in numerous industries such as food and pharmaceutical sectors. In this study, various hydrocolloids including modified starches (oxidized starch—1404 and hydroxypropyl starch—1440), locust bean gum, xanthan gum (XG), guar gum, and carboxymethyl cellulose were added to the Hestrin-Schramm medium to improve the production performance and microstructure of BC by Gluconacetobacter entanii isolated from coconut water. After 14-day fermentation, medium supplemented with 0.1% carboxymethyl cellulose and 0.1% XG resulted in the highest BC yield with dry BC content of 9.82 and 6.06 g/L, respectively. In addition, scanning electron microscopy showed that all modified films have the characteristic three-dimensional network of cellulose nanofibers with dense structure and low porosity as well as larger fiber size compared to control. X-ray diffraction indicated that BC fortified with carboxymethyl cellulose exhibited lower crystallinity while Fourier infrared spectroscopy showed characteristic peaks of both control and modified BC films.     

一株产细菌纤维素菌株的筛选鉴定及其产物分析

从红茶菌液中筛选获得一株产细菌纤维素的菌株BC-41,经生理生化分析和分子生物学鉴定,现证实该菌株为中间葡糖酸醋杆菌(Gluconacetobacter intermedius)。对该菌株所产生的细菌纤维素进行了物理特性的表征和分析,获得以下数据:BC-41所产的纤维素纯度达到91.32%,湿纤维素膜含水率达99.16%,每克干纤维素膜能吸水28.59 g;扫描电子显微镜观察,显示该纤维素具有网状结构,且纤维束宽度分布在40-100 nm之间;X射线衍射分析,证实该纤维素的晶型为纤维素I型,结晶指数为48.8%;通过黏度测定法,得出该纤维素的平均聚合度达2 100。     

Recent developments in the production and applications of bacterial cellulose fibers and nanocrystals

Cellulosic nanomaterials provide a novel and sustainable platform for the production of high performance materials enabled by nanotechnology. Bacterial cellulose (BC) is a highly crystalline material and contains pure cellulose without lignin and hemicellulose. BC offers an opportunity to provide control of the products’ properties in-situ, via specific BC production methods and culture conditions. The BC potential in advanced material applications are hindered by a limited knowledge of optimal BC production conditions, efficient process scale-up, separation methods, and purification methods. There is a growing body of work on the production of bacterial cellulose nanocrystals (BCNs) from BC fibers. However, there is limited information regarding the effect of BC fibers’ characteristics on the production of nanocrystals. This review describes developments in BC and BCNs production methods and factors affecting their yield and physical characteristics.     

细菌纤维素发酵培养基的优化及超微观结构分析

为了提高细菌纤维素的产量, 本研究对一株氧化葡糖杆菌菌株J2液体发酵生产细菌纤维素的培养基进行了优化, 并对其代谢的细菌纤维的超微观结构进行了观察。运用Plackett-Burman试验设计法对8个相关影响因素的效应进行了评价, 筛选出了有显著效应的3个因素: 酵母膏、ZnSO4、无水乙醇, 其他5个因素的影响未达到显著水平(P>0.05)。然后采用Box-Behnken的中心组合试验设计和响应面分析方法(RSM)确定了上述三个因素的最佳浓度, 并且以棉纤维为对照, 运用扫描电镜观察了细菌纤维素的超微观结构, 结果表明: 菌株J2利用优化后的发酵培养基生产细菌纤维素的产量为11.52 g/100 mL, 是优化前的1.35倍, 电镜照片显示细菌纤维素微纤维丝直径<0.1 mm, 比棉纤维细很多, NaOH处理可以除去纤维网络结构中的菌体。     

细菌纤维素发酵培养基的优化及超微观结构分析

为了提高细菌纤维素的产量, 本研究对一株氧化葡糖杆菌菌株J2液体发酵生产细菌纤维素的培养基进行了优化, 并对其代谢的细菌纤维的超微观结构进行了观察。运用Plackett-Burman试验设计法对8个相关影响因素的效应进行了评价, 筛选出了有显著效应的3个因素: 酵母膏、ZnSO4、无水乙醇, 其他5个因素的影响未达到显著水平(P>0.05)。然后采用Box-Behnken的中心组合试验设计和响应面分析方法(RSM)确定了上述三个因素的最佳浓度, 并且以棉纤维为对照, 运用扫描电镜观察了细菌纤维素的超微观结构, 结果表明: 菌株J2利用优化后的发酵培养基生产细菌纤维素的产量为11.52 g/100 mL, 是优化前的1.35倍, 电镜照片显示细菌纤维素微纤维丝直径<0.1 mm, 比棉纤维细很多, NaOH处理可以除去纤维网络结构中的菌体。     

Investigation into the structural, morphological, mechanical and thermal behaviour of bacterial cellulose after a two-step purification process

Bacterial cellulose (BC) is a natural hydrogel, which is produced by Acetobacter xylinum (recently renamed Gluconacetobacter xylinum) in culture and constitutes of a three-dimensional network of ribbon-shaped bundles of cellulose microfibrils. Here, a two-step purification process is presented that significantly improves the structural, mechanical, thermal and morphological behaviour of BC sheet processed from these hydrogels produced in static culture. Alkalisation of BC using a single-step treatment of 2.5 wt.% NaOH solution produced a twofold increase in Young's modulus of processed BC sheet over untreated BC sheet. Further enhancements are achieved after a second treatment with 2.5 wt.% NaOCl (bleaching). These treatments were carefully designed in order to prevent any polymorphic crystal transformation from cellulose I to cellulose II, which can be detrimental for the mechanical properties. Scanning electron microscopy and thermogravimetric analysis reveals that with increasing chemical treatment, morphological and thermal stability of the processed films are also improved.     

Homogenous reactions of cellulose from different natural sources

Two different homogenous reactions on bacterial cellulose (BC), kenaf fiber (KF) and microcrystalline cellulose (MC) were performed to monitor their chemical reactivity. The first reaction was selective oxidation of the primary hydroxyl group with sodium chlorite in the presence of catalytic amount of sodium chloride. While, the second was the formation of triester hypoiodous cellulose using potassium iodate and potassium iodide. The chemical structures of these derivatives were investigated using FT-IR and solid state ¹³C NMR spectroscopies. The BC fibrils required the shortest time among these cellulose samples for both reactions, whereas the viscosity values of BC after iodination and oxidation have the best values compared to KF and MC. FT-IR results show the absence of the hydroxy group of BC and a weak absorption band in both KF and MC. On the other hand, the crystallinity index (CI) of BC is higher than those of both KF and MC. FT-IR spectra of the oxidized different cellulose samples, confirmed the presence of a strong absorption band at around 1590 cm⁻¹ that attributed to vibration band of carbonyl group of carboxylic moiety. Moreover, in the ¹³C NMR spectrum of oxidized cellulose, the lack of signal at 62 ppm and the appearance of signal at 171 ppm indicated that the primary alcohol group is completely oxidized to carboxylic acid. These results showed that BC had a higher reactivity than other samples due to its great purity and low degree of polymerization.     

Effect of addition of sodium alginate on bacterial cellulose production by <Emphasis Type="Italic">Acetobacter xylinum</Emphasis>

Bacterial cellulose (BC) production by Acetobacter xylinum NUST4.1 was carried out in the shake flask and in a stirred-tank reactor by means of adding sodium alginate (NaAlg) into the medium. When 0.04% (w/v) NaAlg was added in the shake flask, BC production reached 6.0 g/l and the terminal yield of the cellulose was 27% of the total sugar initially added, compared with 3.7 g/l and 24% in the control, respectively. The variation between replicates in all determinations was less than 5%. During the cultivation in the stirred-tank reactor, the addition of NaAlg changed the morphology of cellulose from the irregular clumps and fibrous masses entangled in the internals to discrete masses dispersing into the broth, which indicates that NaAlg hinders formation of large clumps of BC, and enhances cellulose yield. Because the structure of cellulose is changed depending on the culture condition such as additives, structural characteristics of BC produced in the NaAlg-free and NaAlg medium are compared using scanning electron microscopy (SEM), fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD). SEM photographs show some differences in reticulated structures and ribbon width and FT-IR spectra indicate that there is the hydrogen bonding interaction between BC and NaAlg, then X-ray diffraction (XRD) analysis reveals that BC produced with NaAlg-added has a lower crystallinity and a smaller crystalline size. The results show that enhanced yields and modification of cellulose structure occur in the presence of NaAlg.     

Acid hydrolysis of bacterial cellulose reveals different modes of synergistic action between cellobiohydrolase I and endoglucanase I.

Intact and partially acid hydrolyzed cellulose from Acetobacter xylinum were used as model substrates for cellulose hydrolysis by 1,4-beta-D-glucan-cellobiohydrolase I (CBH I) and 1,4-beta-D-endoglucanase I (EG I) from Trichoderma reesei. A high synergy between CBH I and EG I in simultaneous action was observed with intact bacterial cellulose (BC), but this synergistic effect was rapidly reduced by acid pretreatment of the cellulose. Moreover, a distinct synergistic effect was observed upon sequential endo-exo action on BC, but not on bacterial microcrystalline cellulose (BMCC). A mechanism for endo-exo synergism on crystalline cellulose is proposed where the simultaneous action of the enzymes counteract the decrease of activity caused by undesirable changes in the cellulose surface microstructure.     

Increased yield and selected properties of bacterial cellulose exposed to different modes of a rotating magnetic field

Rotating magnetic field (RMF) is an interesting alternative to conventional bacterial cellulose (BC) production methods. The BC synthesis processes may be affected by RMF, which facilitates the transfer of oxygen and nutrients from the media to the microbial cells. RMF may also directly influence the various physical and chemical properties of BC. The main aim of the present study was to evaluate the impact of the RMF on the BC in regard to its yield and material properties. The correlation between the efficiency of polymer production and the different time of exposure to the RMF was also analyzed to determine the conditions of lower energy consumption during the cellulose formation process. It was found that the Gluconacetobacter xylinus cultures exposed to the RMF for a half of the time of the entire cellulose production process (72 h), considering the results obtained in controls, synthesized BC more effectively than bacteria continuously exposed to the RMF for 144 h. Furthermore, the application of the RMF, regardless of the exposure mode, did not negatively affect the polymer material properties. It was concluded that the use of the RMF may provide a novel technique for altering cellulose biogenesis and may be used in multiple biotechnological applications.     

Production of bacterial cellulose from Komagataeibacter saccharivorans strain BC1 isolated from rotten green grapes

Abstract

Bacterial cellulose (BC) is one of the prominent biopolymers that has been acquiring attention currently due to its distinctive properties and applications in various fields. The current work presents the isolation of Komagataeibacter saccharivorans strain BC1 isolated from rotten green grapes, followed by biochemical and genotypic characterization, which confirmed that the strain is capable of synthesizing cellulose. Further, production media was designed and certain variables such as carbon, nitrogen sources, pH, and temperature were optimized in order to obtain the maximum concentration of cellulose production. We found mannitol to be the ideal carbon source and yeast extract as the ideal nitrogen source with a highest BC dry yield of 1.81?±?0.25?g/100?mL at pH 5.76 for a week at 30?°C.The charcterization of pellicles by FTIR spectrum depicted similar functional groups present in synthesized BC as that of the commercial cellulose. X-ray diffraction revealed that BC showed 82% crystallinity. Surface morphology of the dried pellicle was studied by SEM image which showed that the BC surface was tightly packed with thin fibers with less porosity. Hence the study demonstrates that the isolates of K.saccharivorans could be used to produce a biopolymer in a short period of time using a modified production medium.     

Multi-Mode Binding of Cellobiohydrolase Cel7A from Trichoderma reesei to Cellulose

Enzymatic hydrolysis of recalcitrant polysaccharides like cellulose takes place on the solid-liquid interface. Therefore the adsorption of enzymes to the solid surface is a pre-requisite for catalysis. Here we used enzymatic activity measurements with fluorescent model-substrate 4-methyl-umbelliferyl-β-D-lactoside for sensitive monitoring of the binding of cellobiohydrolase TrCel7A from Trichoderma reesei to bacterial cellulose (BC). The binding at low nanomolar free TrCel7A concentrations was exclusively active site mediated and was consistent with Langmuir''s one binding site model with K _d and A _max values of 2.9 nM and 126 nmol/g BC, respectively. This is the strongest binding observed with non-complexed cellulases and apparently represents the productive binding of TrCel7A to cellulose chain ends on the hydrophobic face of BC microfibril. With increasing free TrCel7A concentrations the isotherm gradually deviated from the Langmuir''s one binding site model. This was caused by the increasing contribution of lower affinity binding modes that included both active site mediated binding and non-productive binding with active site free from cellulose chain. The binding of TrCel7A to BC was found to be only partially reversible. Furthermore, the isotherm was dependent on the concentration of BC with more efficient binding observed at lower BC concentrations. The phenomenon can be ascribed to the BC concentration dependent aggregation of BC microfibrils with concomitant reduction of specific surface area.     

Bacterial cellulose production by Acetobacter xylinum in a 50-L internal-loop airlift reactor

Bacterial cellulose (BC) production was realized in a batch cultivation of Acetobacter xylinum subsp. sucrofermentans BPR2001 in a 50-L internal-loop airlift reactor. When the bacterium was cultivated with air supply, 3.8 g/L of BC was produced after 67 hours. When oxygen-enriched gas was supplied, the concentration of BC was doubled and the production rate of BC was 0.116 g/L. h, which was two times higher than that of air-supplied culture and comparable to that in a mechanically agitated stirred-tank fermentor. Bacterial cellulose produced by the airlift reactor formed a unique ellipse pellet (BC pellet), different from the fibrous form which was produced in an agitated stirred-tank fermentor. The BC-pellet suspension was demonstrated to have a higher volumetric oxygen transfer coefficient than the fibrous BC suspension in a 50-L internal-loop airlift reactor. The mixing time of BC-pellet suspension in the airlift reactor was also shorter than that in water.     

Bioengineering bacterial cellulose/poly(ethylene oxide) nanocomposites

By adding poly(ethylene oxide) (PEO) to the growth medium of Acetobacter xylinum, finely dispersed bacterial cellulose (BC)/PEO nanocomposites were produced in a wide range of compositions and morphologies. As the BC/PEO w/w ratio increased from 15:85 to 59:41, the cellulose nanofibers became smaller but aggregated in larger bundles, indicating that PEO mixed with the cellulose on the nanometer scale. Fourier transform infrared spectroscopy suggested intermolecular hydrogen bonding and also preferred crystallization into cellulose Ibeta in the BC/PEO nanocomposites. The fine dispersion of cellulose nanofibers hindered the crystallization of PEO, lowering its melting point and crystallinity in the nanocomposites although remaining bacterial cell debris also contributed to the melting point depression. The decomposition temperature of PEO also increased by approximately 15 degrees C, and the tensile storage modulus of PEO improved significantly especially above 50 degrees C in the nanocomposites. It is argued that this integrated manufacturing approach to fiber-reinforced thermoplastic nanocomposites affords a good flexibility for tailoring morphology and properties. These results further pose the question of the necessity to remove bacterial cells to achieve desirable materials properties in biologically derived products.     

Factors affecting the yield and properties of bacterial cellulose

Acetobacter xylinum E₂₅ has been applied in our studies in order to find optimal culture conditions for effective bacterial cellulose (BC) production. The strain displays significantly higher stability in BC production under stationary culture conditions. In contrast, intensive agitation and aeration appear to drastically reduce cellulose synthesis since such conditions induced formation of spontaneous cellulose nonproducing mutants (Cel−), which dominated in the culture. Mutation frequency strictly depends on the medium composition in agitated cultures. Enrichment of the standard SH and Yamanaka media with 1% ethanol significantly enhanced BC production in stationary cultures. Horizontal fermentors equipped with rotating discs or rollers were successfully applied in order to improve culture conditions. Relatively slow rotation velocity (4 rpm) and large surface area enabling effective cell attachment are optimal parameters for cellulose production. Physical properties of BC samples synthesized either in stationary cultures or in a horizontal fermentor revealed that cellulose from stationary cultures demonstrated a much higher value of Young's modulus, but a much lower value of water-holding capacity. Journal of Industrial Microbiology & Biotechnology (2002) 29, 189–195 doi:10.1038/sj.jim.7000303 Received 01 March 2002/ Accepted in revised form 18 July 2002     

Effects of alcohols on bacterial cellulose production by <Emphasis Type="Italic">Acetobacter xylinum</Emphasis> 186

To improve the yield of cellulose production in bacteria, we investigated the stimulatory effects of six different alcohols during fermentation of Acetobacter xylinum 186. Our study showed that after static fermentation at 30°C for 6 days, bacterial culture with 1.0% (v/v) of methanol added in the medium produced the highest bacterial cellulose (BC) yield at 103.5 mg/100 ml, which was 21.8% higher than the control group. Addition of 0.5% of ethylene glycol in the culture yielded 105.5 mg/100 ml BC, 24.1% higher than the control group. Adding 0.5% of n-propanol yielded 96.4 mg/100 ml BC, 13.4% higher; 3.0% of glycerol yielded 108.3 mg/100 ml BC, 27.4% higher; 0.5% of n-butanol yielded 132.6 mg/100 ml BC, 56.0% higher; and 4.0% of mannitol in the culture yielded 125.2 mg/100 ml BC, 47.3% higher, respectively. The rate of bacterial cellulose production increased with the growth rate of the bacteria. The stimulatory effects of these alcohols that we observed were significant in the later stage of fermentation, which was considered to be important for the biosynthesis of bacterial cellulose.     

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.     

细菌纤维素的合成与调控进展

细菌纤维素是1种天然的高纯度生物多聚物,与木质纤维素相比,其生产和加工过程更为方便和环保,因此已成为1种极有潜力的生物材料。葡糖酸醋杆菌是目前已知的产纤维素能力最高的菌株。综述了葡糖酸醋杆菌的细菌纤维素合成和调控机制以及为提高产量所进行的基因工程手段和培养方法。