Synthesis And Characterization Of Microbial Cellulose And Its Oxidation Derivative

This study belongs to part of Chinese National High-tech R&D Program (863 program), Serial No.2002AA302212.The main theme of this study was to synthesis and develop microbial cellulose (MC) biomaterials from three aspects. First, the synthesis and application of MC pellicle produced by Acetobacter xylinum (A. xylinum) using coconut water as culture medium were studied. The optimized inclined plane strain culture medium and synthsis culture medium were prepared. The high yield of MC was achieved at low cost with short culture period. Second, the natural mutation A. xylinum strain which could produce nano size short fiber with high yield was bred selectively named A. xylinum HN001. The biological method of preparing nano cellulose materials was developed. And the third, dialdehyde cellulose(DAC) was prepared by selective oxidation of MC. DACs could use as medical adsorbent material. Thus, the three expected MC biomaterials were obtained.Firstly, Macromolecular MC pellicle was synthesized using Hainan coconut water as culture medium by A. xylinum strain selectively bred in our own laboratory. Nutrition components of fresh coconut water and their changes after natural fermentation were measured. The optimization of the inclined plane strain culture medium, the optimization of synthesis culture medium, the synthesis process conditions, the static synthesis dynamics, and partial substitution of the coconut water by molasses wastewater to produce MC pellicle were investigated in detail. All these studies were to get high yield and good physical properties of MC at low cost.Coconut water richly contained carbohydrates, amino acids, inorganic salts, vitamins, etc. Several kinds of organic acids, such as acetic acid, citric acid and succinic acid, were produced after its natural fermentation. They formed a natural buffer system with some metal ion in coconut water which could guarantee the A. xylinum growing in stable environment. These organic acids could also serve as carbon source or energy substance by the A. xylinum. Therefore the fermented coconut water was more beneficial to the A. xylinum growing and synthesis of MC. This was proposed firstly in the study. In order to breed strong active A. xylinum tube inclined plane strain, the influence of the coconut water fermentation time and phosphate added in the system on reproduces of A. xylinum as well as the yield of MC were both investigated. The optimum culture medium was ammonium sulfate 0.2%, magnesium sulfate 0.05%, agar 2%, sufficient fermented 2-day-long coconut water, the pH value of the medium 5.0. The bred tube inclined plane strain with the optimum culture was strong active, and not easy to degenerate in transferring generation.Optimization of the synthesis culture medium composition was investigated, such as, amount of fermented coconut water, various carbon sources, various nitrogen sources, promoting factor, initial pH of the system. The effects of these factors on yield of MC pellicle were in order:coconut water amount> sodium acetate (promoting factor)> ammonium sulfate> sucrose>potassium dihydrogen phosphate. The effects of these factors on dried cellulose content of MC pellicle were in order:coconut water> sodium acetate >sucrose> potassium dihydrogen phosphate and ammonium sulfate. The optimized culture medium composition was fermented coconut water 50%, sucrose 4%, ammonium sulfate 0.5%, sodium acetate 0.2%, potassium dihydrogen phosphate 0.05%, initial pH 4.2, strain age 3-day-long, inoculation volume 5%, the culture period 7 days, optimum culture temperature 30℃. The optimum yield was achieved about 737g/L of pellicle wet weight and 4.5g/L of dried cellulose weight. The yield was higher and the cost was lower than other culture medium. This showed that the coconut water was an excellent culture medium.The culture process of A. xylinum static was researched, and growing dynamics model of the A. xylinum strain, dynamics model of MC producing, dynamics model of the culture medium consumption were established. The optimum process conditions, such as, bacterium concentration, culture medium concentration, and pH of the system in the process, could be simulated and determined by these models.MC could also be synthesized throgh partly replacing the coconut water by 30% molasses wastewater. This was helpful to develop a synthesizing biomaterial path using wastewater source.IR test confirmed theβ-pyranose structure of MC. The crystal cell parameters were calculated by XRD analysis, and the results indicated that the crystal form of synthesized MC was mainly cellulose I form.Secondly, Natural mutation A. xylinum strain which could produce nano size short fiber with high yield was bred selectively named A. xylinum HN001 by the Institute of Microbiology, Chinese Academy of Science. The strain shape, the main physiological biochemistry characteristic, and functional gene quantities were investigated. The quantities of HN001 GC base pairs were remarkably more than the degenerated strain. This explained for more functional gene quantities and strong activity of producing nano microbial cellulose (NMC).Using coconut water as the culture medium, NMC was biosynthesized by A. xylinum HN001 with static culture, which was water soluble and low relative molecular mass microbial cellulose. The 1.2g/L dried cellulose of NMC yield was achieved when the optimum conditions followed as, culture time 72h, culture temperature at 33℃, culture medium initial pH 4.According to different physical and chemical properties of the system components, the physical separation and the chemical separation methods were combined to separate and purify NMC. The suitable processe was founded.The relative molecular weight (MW) of NMC and its molecular weight distribution(MWD) were determined by Gel Filtration Chromatograph (GFC). The results indicated that the MW were hardly influenced when culture medium initial pH value ranged from 3.5 to 5.5. And the MWD does hardly affected by the variation of pH and the MWD index is almost same 1.3, which showed their MWs is extremely uniform.TEM image of NMC in water solution showed that the appearance was nearly sphere and its size was about 20nm. Dried NMC powder grain shape also appeared nearly sphere. The granularity D5o was 85.6nm, and the distribution range was about 40-500nm. This may be from the aggregation among particles in the drying process.The IR spectrum proved that NMC was glucosanβ-1,4-bond. The TG curves indicated that the total weight loss of NMC and microcrystalline cellulose(MCC) were about 89.6% and 84.6% respectively. The peak weight loss temperatures were about 336.93℃and 358.12℃respectively. The crystal forms of NMC is almost the same with that of MC, and some different with MCC at 14-17 degree of 2θaccording to the X-ray diffraction profiles. The crystallinity was higher than MCC. The thermal decomposition products of NMC were mainly carbon dioxide and water.Thirdly, in order to develop more applications of MC in biomaterial field, MC was oxidized to synthesize dialdehyde derivative which could be used as medical absorbent material.The selective oxidation of MC with sodium periodate oxidant in heterogeneous phase system was studied to synthesize dialdehyde cellulose(DAC). The experiment results showed that, the acidic environment was helpful to form aldehyde group. The optimum conditions were:temperature 35℃, pH 2, reaction time 20h, oxidant concentration 0.2 mol/L, and oxidant amount 1:1.IR test confirmed aldehyde group of the DAC. The adsorption capacity of MC, MCC, and DAC were determined. Their optimum absorption water quantity was about 12.0g/g, 6.6g/g,3.6g/g, and the max adsorption iron ion amount was about llmg/g,8.7mg/g, 6.9mg/g, respectively. The max adsorption of DAC with hexanediamine and p-phenylenediamine was 686mg/g and 460mg/g respectively. These indicated DACs possessed expected properties of low adsorption amount of water and iron ion, and high adsorption amount of amino compounds.The IR tests showed that adsorption of DAC with hexanediamine was belong to physical adsorption, and adsorption of DAC with p-phenylenediamine was not only physical adsorption but also chemical reaction.When aldehyde content of DACs increased up to 0.75mmol/g, the crystallinity degree decreased to 56% compared to that of 66% of MC. Because the introduction of aldehyde group breaks up the sugar residue ring, molecular ordered crystallization arrangement, accumulation and crystallization degree were decreased. The pyrolysis temperatures of DACs were decreased from 230℃of MC to 180℃, a'nd the peak weight loss rate temperature was decreased from 340℃of MC to 290℃with the aldehyde content increased up to 0.75mmol/g. These results showed Athat DACs were easier to pyrolyze.