功能菌剂的混合发酵研究

随着化工行业的发展，大量有毒有害难降解有机物随工业废水的排放进入环境，这些物质能够在环境中长期存在、积累和扩散，通过食物链对动植物的生存及人类的健康造成不良影响。本文以苯酚、对氯硝基苯、氯苯和十六烷为模拟污染物，以前期研制的功能菌剂为对象，经过紫外线线诱变筛选出优于出发菌株的功能菌，对诱变后功能菌的理化性能进行了研究，对菌种进行了鉴定，在此基础上，就其相互之间的微生态关系进行研究，为混合发酵提供理论基础，并就其最佳发酵条件及发酵参数进行了研究，最后对发酵产品的性能进行了检测。目前，国内外有关功能菌剂的研究还存在多方面的不足，主要包括：①由于多菌种混合发酵过程较为复杂，各菌之间存在复杂的相互作用，影响因素较多，关于菌种之间的相互关系研究得很少，环境功能菌剂的发酵方法大多采用单独发酵后混合的方式。单独发酵对原材料、设备和能源的利用率较低，对于多菌种制剂发酵，在设备、能源和原材料的方面造成的浪费更大，将会大幅增加菌剂的生产成本，影响多菌种功能菌剂的发展；②功能菌剂生产过程的质量控制方面研究得较少；③功能菌剂产品的稳定性、抗冲击性能研究得较少，对环境微生物制剂的研究主要集中在菌种选育和培养条件优化方面。 通过本论文研究，得到以下主要结论。 （1）在紫外线诱变处理中，用紫外线对发生一定程度退化的出发菌株进行诱变处理后，六株具有高效降解性能的菌株被筛选出来，诱变筛选出的菌株形态和ERIC-PCR指纹图谱与出发菌株相比发生了明显改变；而且诱变后的菌株对目标难降解底物的降解能力均得到改善，其中，FPN、FCB、F14、FEm对目标底物的降解率提高了20%以上；诱变后菌株经过7次连续传代接种后，对目标难降解底物的降解率无显著变化，具有一定的遗传稳定性。并对诱变后的功能菌进行了初步的鉴定，这6株菌都分别是芽孢杆菌。 （2）对诱变后的功能菌相互之间的微生态关系进行了研究，通过抑菌实验、生长量以及基质消耗量的比较，确定它们之间的生长关系是无害共栖关系，可以进行混合发酵。 （3）对该功能菌剂进行发酵培养条件研究，结果表明发酵培养基的最佳成分（g/L）：葡萄糖 31.0g/L、玉米粉10.0g/L、磷酸氢二钾1.0g/L、硫酸铵1.1g/L、硫酸镁0.55g/L。通过研究不同的培养条件对菌体生长和降解性能的影响，确定了最佳培养条件：培养基初始pH7.5；最适温度32℃；培养基装液量125mL（250 mL三角瓶），以及培养时间对降解性能的影响，培养20 h的产物对降解最为有利。通过研究添加不同目标污染物对菌体生长和降解性能的影响，确定了添加目标污染物的最佳量以及最佳时间：苯酚投加量：1.125 g/L，对氯硝基苯投加量：0.1 g/L；最佳投加时间为发酵培养开始后4 h。 （4）以摇瓶分批发酵最优条件为基础，对FPN、F10、FCB、FNa、F14 和 FEm进行了摇瓶分批发酵试验。以摇瓶分批发酵试验数据为依据，对功能菌剂分批发酵动力学进行了研究，建立了菌体生长和基质消耗的动力学模型，拟合模型能较好的反映功能菌剂分批发酵过程。 （5）功能菌剂和活性污泥协同作用，可以提高系统的生物降解能力，功能菌剂投加量为2%，新鲜活性污泥3500 mg/L，降解24 h条件下，功能菌剂和活性污泥的协同作用对COD的去除率和对照组相比，最多的提高了36.8%。功能菌剂和活性污泥协同作用以及活性污泥的单独作用，其生物降解过程均符合一级反应动力学过程，功能菌剂和活性污泥协同作用的生物降解动力学方程为：，相关系数97%。采用SBR运行方式，引入功能菌剂的SBR系统明显能够改善和提高生物降解的效率。与仅有活性污泥的系统相比，系统对COD的平均去除率可以提高27.1%，同时，系统的耐负荷冲击以及耐毒害冲击的性能比仅有活性污泥的SBR系统强，特别是负荷冲击对引入功能菌剂的SBR系统影响很小。仅有活性污泥的SBR系统经过负荷冲击和毒害冲击之后，不能恢复到冲击之前的水平，而且系统有效作用时间的周期比引入功能菌剂的SBR系统相比大大缩短，而引入功能菌剂的SBR系统处理效果较为稳定，恢复能力很强。 Along with the development of industries, many recalcitrant organic chemicals have been discharged into natural environments together with wastewaters and can exist in waters, soil and sediments for a long time without degradation. These haz-ardous substances, their byporducts and metabolizabilities can be highly toxic, mu-tagenic and carcinogenic, thereby threatening animals, plants and human health through food chain. Consequently the removal of these compounds is of significant interest in the area of wastewater treatment. In this dissertation, the phenol, hydro-quinone, chlorobenzene and hexadecane treated as the model pollutants, the func-tional microorganism agent was used as the starting strains, they treated with ultra-violet light, and then the mutant strains with high degradation ability were screened out and identified primarily, the relationship between these stains were studied, the medium composition and fermentation conditions were optimized, the degradation ability of the fermented production was tested. The literature survey indicates that the study of the microorganism agent is far from complete and more information is re-quired on following problems. 1, Because of the complexity of relationship in mixed fermentation and the complicated factors, the study is hardly to process.2, There is a lack of information on the quality control of the producing process .3, And there is a lack of information on the stability about the microorganism agent. In this dissertation, the main results of the present study could be summarized as follows: (1)The degenerate starting strains were treated with the ultraviolet light, and six mutant strains with high biodegradation ability were screened out by using the me-dium with selective pressure of model pollutants. The mutant strains had great changes in colonialmorphology and ERIC-PCR fingerprinting. And the mutant strains got obvious advantages over the starting strains in degradation ability and over 20% improvement of removal rates was achieved for FPN、FCB、F14 and FEm. The de-gradation ability of the mutant strains was stable after seven generations. After that, the mutant strains were primarily identified as bacillus respectively. (2) The relationship between these mutant strains was studied. By the compari-son of antibiosis effect, biomass and consumption of substrate, the relationships were neutralism and they could be mixed fermented. (3) The optimized cultivation conditions were as follows: glucose 31.0 g/L, corn power 10 g/L, K2HPO4 1.0 g/L, (NH4)2SO4 1.1 g/L, MgSO4 0.55 g/L, initial pH7.5, temperature 32℃, working volume 125 mL/250 mL, and cultivation time 20h (con-sidering the time effect on degradation ability), adding pollutants phenol (1.125 g/L) and hydroquinone (0.1 g/L) into the broth at 4 h after cultivation. (4) Based on the above optimum condition, the batch fermentation was per-formed with strains FPN, F10, FCB, FNa, F14 and FEm in shake flask. The batch fermentation kinetics was studied based on the experimental data. Two kinetic models were constructed which could reflect the regularity of growth and substrate consump-tion in the process of batch fermentation. (5) The co-operation of functional microorganism agent and activated sludge could raise biodegradation of system by adding some microorganism agent and 3500 mg/L fresh activated sludge. Bioaugumentation by the addition of high effective deg-radation culture enhanced the treatment effect of SBR system and the COD removal rate was increased by 20%-36.8%. Its biodegradation matched first-order dynamical reaction equation, and the reaction equation was ln0.2327.391ct=−+. The micro-organism agent had the effect of optimization to activated sludge micro-ecosystem. The SBR system adding 2% microorganism agent, the average COD removal rate of that was increased by 27.1% and stronger anti-shock ability to load and toxicant were achieved (compared with SBR system just adding activated sludge). Especially the load-shock has barely effect to the SBR system adding microorganism agent. After the load and toxicant shock, the SBR system just adding activated sludge couldn’t come back to original level and the activated sludge micro-ecosystem was frustrated. The applying of microorganism agent increased biological activity and system’s re-sistance ability to load shock and toxicant shock.

克拉维酸产生菌的诱变育种与发酵工艺优化研究

以克拉维酸产生菌棒状链霉菌Streptomyces clavuligerus CCRC11518（ATCC 27064）III50为出发菌株, 首先比较各种物理和化学诱变剂处理对其克拉维酸生物合成的影响, 确定了亚硝基胍为棒状链霉菌诱变育种的诱变剂及其处理剂量: 2mg/ml、40min. 经浓度为2mg/ml的亚硝基胍处理40min后, 采用新颖理性化筛选方法, 通过逐步筛选自身代谢产物抗性突变株、克拉维酸抗性突变株和链霉素抗性突变株, 最终得到一株克拉维酸高产菌VI118(效价633μg/ml), 其克拉维酸效价是出发菌株(效价377μg/ml)的167.9%. 该高产突变株在琼脂斜面培养基上连续传接10代, 克拉维酸效价保持稳定. 通过单因子和多因子摇瓶正交试验, 对高产菌株VI118的发酵条件进行了研究, 确定最佳发酵条件: 甘油60g, 水解植物蛋白 60g, KH2PO4 0.5 g, 玉米浆 7.5g, MnSO4•H2O 0.34g, MgSO4•7H2O 0.99g, FeSO4•7H2O 0.56g, 蒸馏1000ml, pH 7.0, 发酵培养基装量20ml/250ml三角瓶, 接种量10％, 培养温度28ºC, 220r/min摇床培养72h后测定效价. 在最佳发酵条件下克拉维酸效价达到651μg/ml, 同时把初始发酵培养基的昂贵成分替换为廉价的工业原料. 通过摇瓶分批补料试验, 得到最佳补料物质和补料方式：在上述最佳发酵条件下, 分别在发酵培养48h、56h、64h、72h时补加4ml无菌水, 80h发酵结束, 克拉维酸效价达到905μg/ml. 在不增加原料成本的情况下通过摇瓶补料方式克拉维酸效价为未补料的139.0％, 总产量为未补料的264％. By a novel rational screening method, mutant Streptomyces clavuligerus CCRC11518（ATCC 27064）III50（titres 377μg/ml）, as the clavulanic acid-producing parent strain, was treated by NTG (2mg/ml) for 40min, and the self-generated metabolites resistant mark, the clavulanic acid resistant mark and the streptomycin resistant mark were added step by step. Finally, the mutant VI118（titres 633μg/ml）with the three marks was obtained. The clavulanic acid productivity of this mutant was increased by 167.9% compared with the parent strain. After reproducing 10 generations on the agar medium slant, the productivity of this mutant was stable. The optimum fermentation conditions were established as followings: glycerol 60g, acid hydrolyzed vegetable protein 60g, KH2PO4 0.5g, corn steep liquor 7.5g, MnSO4•H2O 0.34g, MgSO4•7H2O 0.99g, FeSO4•7H2O 0.56g, distilled water 1 liter, pH 7.0, 20ml in 250ml shake-flask, inoculation 10%(v/v), fermentation temperature 28ºC, rotation speed 220 r/min, time 72h. The clavulanic acid productivity was 651μg/ml, while used the low-priced industrial raw materials. After studying on fed-batch in the shake-flask, the optimum fed-batch manner was obtained: under optimum fermentation conditions, at 48h, 56h, 64h and 72h, adding 4ml distilled water into each flask, fermentation ending at 80h. The clavulanic acid productivity was increased by 139% compared with no fed-batch, meanwhile the total yield was increased by 264%.

红发夫酵母发酵虾青素的研究

红发夫酵母分离于北美西部高山地区和日本一些岛屿上落叶树的渗出液中，因其所产主要色素为在水产养殖、食品和医药工业有广阔应用前景的虾青素而成为研究的热点。本论文对红发夫酵母Phaffia rhodozyma 的生长特性、培养参数与培养基组分对生长和虾青素积累的影响及其优化、虾青素合成的调节控制、虾青素的提取测定及红发夫酵母耐高温菌种的诱变进行了系统的研究。 虾青素是红发夫酵母的胞内色素，要对其进行分析首先要对红发夫酵母进行破壁处理，实验发现二甲亚砜是最有效的破壁溶剂，用氯仿和丙酮可以有效地把类胡萝卜素从二甲亚砜破壁后的红发夫酵母细胞中提取出来。 在固定摇床转速为200 rpm，温度为20 ℃的条件下，当种龄为36 h，以10％的接种量接入装液量为30 mL的250 mL三角瓶，初始pH为5.5时最有利于红发夫酵母的生长及类胡萝卜素的合成。 本实验中红发夫酵母最佳利用碳、氮源分别为蔗糖和蛋白胨，但蛋白胨价格昂贵，不适宜作单一氮源，因此使用硫酸铵和酵母膏作为复合氮源。 本论文采用了BP神经网络结合遗传算法的方法来优化红发夫酵母的发酵培养基，得到红发夫酵母发酵培养基的最佳配比为：蔗糖45.10 g/L、硫酸铵3.00 g/L、硫酸镁0.80 g/L、磷酸二氢钾1.40 g/L、酵母膏3.00 g/L、氯化钙0.50 g/L，使用优化后的培养基发酵类胡萝卜素产量达到8.20 mg/L，干重达到9.47 g/L，类胡萝卜素的产量比起始培养基提高了95.90%，干重提高了89.40%。 从代谢途径出发对红发夫酵母合成虾青素调控调控，选择谷氨酸、乙醇、VB1作为添加剂，通过正交试验设计得出三者添加水平分别为0.2 g/L，0.1% (V/V)，10 mg/L时，类胡萝卜素产量提高了25.73%，达到了10.31mg/L。 通过上述优化培养，本论文中红发夫酵母的虾青素产量从1.33 mg/L提高到9.12 mg/L，产量提高了6.86倍；总类胡萝卜素产量从4.23 mg/L提高到10.31 mg/L，产量提高了2.44倍；细胞干重从5.00 g/L提高到11.35 g/L，提高了2.27倍，总体提高效果显著。 红发夫酵母属于中低温菌，本论文采用紫外复合诱变的方式，通过高温筛选，得到一株能在35 ℃下能生长的突变株，但所产类胡萝卜素中虾青素所占比例很小，可能是诱变改变了红发夫酵母的代谢途径，阻断了虾青素的合成。 Phaffia rhodozyma is a heterobasidiomyceteous yeast that was originally isolated from the slime fluxes of brich tree wounds in mountain regions of northern Japan and southern Alaska. Phaffia rhodozyma produces astaxanthin as its principal carotenoid pigment, which has potential applications in acquaculture, food and pharmaceutical industry. This paper researched ways to break cell, analysis of astaxanthin, characteristics of growth, culture parameters and the effects of components of medium on growth and astaxanthin formation , optimization of culture medium, control of astaxanthin synthesis and mutagenesis of Phaffia rhodozyma. It is necessary to disrupt the yeast cell for extracting astaxanthin considering the yeast accumulating carotenoids in cell. Dimethyisulphoxide was the most effective solvent for breaking the yeast cell; acetone and chloroform were effective to extract carotenoids out of the disrupted cell. The optimum pH for growth and carotenoids synthesis is 5.5, the optimum medium volume is 30 mL (in 250 mL flask), the optimum culture time of inoculum is 36 h, the optimum inoculum concentration is 10%. The research on culture medium showed: sucrose is the best one of 6 carbon sources for growth and astaxanthin synthesis. Peptone is the best nitrogen source for growth and astaxanthin synthesis. Uniform Design was used for trial design of the formula medium components, then back-propagation neural network was established to modeling the relationships between the carotenoid yield and the concentration of medium components. Genetic algorithm (GA) was used for global optimization of the model. The optimum combination of the medium was obtained: sucrose 45.10 g/L, ammonium sulfate 3.00 g/L, magnesium sulfate 0.80 g/L, potassium dihydrogen phosphate 1.40 g/L, yeast extract 3.00 g/L, calcium chloride 0.50 g/L. The yield of carotenoid reached 8.20 mg/L, which was 95.90% higher than that of the original medium. Glu, VB1 and ethanol were selected as fermentation addictives, after Orthogonal Test, the carotenoid contents increased by 25.73% when adding 0.16 g/L Glu, VB1 10 mg/L and ethanol 0.1% (V/V). After the above optimization, the astaxanthin content increased 6.86 folds, which is 9.12 mg/L. The carotenoids content increased 2.44 folds, which is 10.31 mg/L. The biomass increased 2.27 folds, which is 11.35 g/L. Phaffia rhodozyma grows in the mild temperature range of 0 to 27 ℃, in this work, a thermotolerant mutant was selected through UV-irradiation. It can grows at 35 ℃, and showed increased carotenoid content. The optimal growth temperature for this mutant is 30 ℃. But the mutant can only produce carotenoids with little astaxanthin accumulation.

Substrate induction and statistical optimization for the production of chitosanase from Microbacterium sp OU01

The chitosanase production was markedly enhanced by substrate induction, statistical optimization of medium composition and culture conditions by Microbacteritan sp. OU01 in shake-flask. A significant influence of (NH4)(2)SO4, MgSO4 center dot 7H(2)O and initial pH on chitosanase production was noted with Plackett-Burman design. It was then revealed with the method of steepest ascent and response surface methodology (RSM) that 19.0 g/L (NH4)(2)SO4, 1.3 g/L MgSO4 and an initial pH of 2.0 were optimum for the production of chitosanase; colloidal chitosan appeared to be the best inducer for chitosanase production by Microbacterium sp. OU01. This optimization strategy led to the enhancement of chitosanase from 3.6 U/mL to 118 U/mL. (c) 2006 Elsevier Ltd. All rights reserved.

A bioaugmentation failure caused by phage infection and weak biofilm formation ability

Two biological aerated filters (BAF) were setup for ammonia removal treatment of the circulation water in a marine aquaculture. One of the BAFs was bioaugmented with a heterotrophic nitrifying bacterium, Lutimonas sp. H10, where the ammonia removal was not improved and the massive inoculation was even followed by a nitrification breakdown from day 9 to 18. The nitrification was remained stable in control BAF operated under the same conditions. Fluorescent in situ hybridization (FISH) with rRNA-targeted probes and cultivable method revealed that Lutimonas sp. H10 almost disappeared from the bioaugomented BAF within 3 d, and this was mainly due to the infection of a specific phage as revealed by flask experiment, plaque assay and transmission electron observation. Analyses of 16S rRNA gene libraries showed that bacterial groups from two reactors evolved differently and an overgrowth of protozoa was observed in the bioaugmented BAR Therefore, phage infection and poor biofilm forming ability of the inoculated strain are the main reasons for bioaugmentation failure. In addition, gazing by protozoa of the bacteria might be the reason for the nitrification breakdown in bioaugmented BAF during day 9-18.

Determination for the different combined-form inorganic carbon in marine sediments

Marine sediment is the important sources and sinks of carbon. The inorganic carbon(IC) in marine sediments plays an important role in carbon cycling. In order to understand IC function in carbon cycling, sequential extraction method based on IC combined chemical strength difference were established to get five phases: NaCl phase (step I), NH3 - H2O phase (step II), NaOH phase (step III), NH2OH . HCl phase (step IV) and HCl phase(step V). The best extraction conditions were obtained by a series of experiments. Extractants were added into plastic centrifuge tubes in Step I - M, the capped tube were placed on a shaker table to keep the solids suspended for two hours. The suspended solution was separated by centrifugal, the residues were washed with water. The two supernatant were combined and the CO, was finally determined by volumetric analysis. The residues were transferred into conical flask in step IV and V, and then the extractants were added. The produced CO2 was adsorbed by saturated Ba(OH)(2) solution, and determined by volumetric analysis. This method for IC has a good precision in the analysis sediment samples.

A simple and accurate method for determining accurately dissolved inorganic carbon in seawaters

Dissolved inorganic carbon (DIC) account for more than 95% of total carbon in seawater, so it is necessary to make reliable and precise measurements of DIC to study marine carbon cycling. In order to establish a simple and speed method, an airproof device of gas extraction-absorption was designed. Finally a simple method was developed for the determination of DIC in seawater through a large mount of experiments. The determination procedure is as follows: 100 similar to 150 mL seawater was put into conical flask, then add 10% H3PO4, the DIC in seawater sample was dissolved to form CO2 gas and carried by pure N-2, then the CO2 gas was absorbed by two grades 0.1 mol/L NaOH solution. Finally the absorbed solution was titrated by HCl standard solution of 0.01000 mol/L with the end points detected with the indicator phenolphthalein and bromocresol green-methyl red mixture. The precision and accuracy of the method were satisfied. This method was used to analyse seawater samples from Jiaozhou bay in June, 2003. The result shows that the average DIC in surface seawater is 2066 mumol/L, DIC in bottom seawater is 2075 mumol/L inside bay, but the average DIC in surface seawater is 1949 mumol/L, DIC in bottom seawater is 2147 mumol/L outside bay.

Engineering design of a bioprocess for the production of natural red colourants by submerged fermentation of the thermophilic fungus Penicillium purpurogenum GH2

The development of a new bioprocess requires several steps from initial concept to a practical and feasible application. Industrial applications of fungal pigments will depend on: (i) safety of consumption, (ii) stability of the pigments to the food processing conditions required by the products where they will be incorporated and (iii) high production yields so that production costs are reasonable. Of these requirements the first involves the highest research costs and the practical application of this type of processes may face several hurdles until final regulatory approval as a new food ingredient. Therefore, before going through expensive research to have them accepted as new products, the process potential should be assessed early on, and this brings forward pigment stability studies and process optimisation goals. Only ingredients that are usable in economically feasible conditions should progress to regulatory approval. This thesis covers these two aspects, stability and process optimisation, for a potential new ingredient; natural red colour, produced by microbial fermentation. The main goal was to design, optimise and scale-up the production process of red pigments by Penicillium purpurogenum GH2. The approach followed to reach this objective was first to establish that pigments produced by Penicillium purpurogenum GH2 are sufficiently stable under different processing conditions (thermal and non-thermal) that can be found in food and textile industries. Once defined that pigments were stable enough, the work progressed towards process optimisation, aiming for the highest productivity using submerged fermentation as production culture. Optimum production conditions defined at flask scale were used to scale up the pigment production process to a pilot reactor scale. Finally, the potential applications of the pigments were assessed. Based on this sequence of specific targets, the thesis was structured in six parts, containing a total of nine chapters. Engineering design of a bioprocess for the production of natural red colourants by submerged fermentation of the thermophilic fungus Penicillium purpurogenum GH2.

A Possible Relationship Between Chitinozoa And Tintinnids

The biological affinity of the extinct microfossil order chitinozoa has been the source of much discussion in the fifty years since they were first discovered. Within this period these flask-shaped, organic-walled organisms have been variously attributed to rhizopods, flagellates, tintinnids, chrysomonads, metazoan eggs, dinoflagellates, and fungi. Most of these suggested relationships were made before it was recognised that chitinozoans were encapsulated and must therefore be resting cysts or eggs and not active individuals. There are no living organisms which combine all the characteristics of the chitinozoa. Of all the possibilities, a grouping of flask-shaped cysts which have been found in present-day marine plankton and sediment comes closest to characterising the morphology of chitinozoa. This grouping of flask-shaped cysts includes forms which have been found within tintinnid loricae. Another modern cyst type Pacillina arctica, which is believed to be a ciliate cyst, comes close to replicating the morphology of the chitinozoan genus Hoegisphaera. This paper discusses the structure of tintinnid, other flask-shaped cysts and Pacillina arctica in relation to chitinozoan morphology, drawing attention to similarities and differences. The occurrence and distribution of these cyst forms in present-day plankton is also described and interpreted.

Cell survival responses after exposure to modulated radiation fields.

In the present study survival responses were determined in cells with differing radiosensitivity, specifically primary fibroblast (AG0-1522B), human breast cancer (MDA-MB-231), human prostate cancer (DU-145) and human glioma (T98G) cells, after exposure to modulated radiation fields delivered by shielding 50% of the tissue culture flask. A significant decrease (P < 0.05) in cell survival was observed in the shielded area, outside the primary treatment field (out-of-field), that was lower than predicted when compared to uniform exposures fitted to the linear-quadratic model. Cellular radiosensitivity was demonstrated to be an important factor in the level of response for both the in- and out-of-field regions. These responses were shown to be dependent on secretion-mediated intercellular communication, because inhibition of cellular secreted factors between the in- and out-of-field regions abrogated the response. Out-of-field cell survival was shown to increase after pretreatment of cells with agents known to inhibit factors involved in mediating radiation-induced bystander signaling (aminoguanidine, DMSO or cPTIO). These data illustrate a significant decrease in survival out-of-field, dependent upon intercellular communication, in several cell lines with varying radiosensitivity after exposure to a modulated radiation field. This study provides further evidence for the importance of intercellular signaling in modulated exposures, where dose gradients are present, and may inform the refinement of established radiobiological models to facilitate the optimization of advanced radiotherapy treatment plans.

In-vitro investigation of out-of-field cell survival following the delivery of conformal, intensity-modulated radiation therapy (IMRT) and volumetric modulated arc therapy (VMAT) plans

The aim of this work is to determine the out-of-field survival of cells irradiated with either the primary field or scattered radiation in the presence and absence of intercellular communication following delivery of conformal, IMRT and VMAT treatment plans. Single beam, conformal, IMRT and VMAT plans were created to deliver 3 Gy to half the area of a T80 flask containing either DU-145 or AGO-1522 cells allowing intercellular communication between the in-and out-of-field cell populations. The same plans were delivered to a similar custom made phantom used to hold two T25 culture flasks, one flask in-field and one out-of-field to allow comparison of cell survival responses when intercellular communication is physically inhibited. Plans were created for the delivery of 8 Gy to the more radio-resistant DU-145 cells only in the presence and absence of intercellular communication. Cell survival was determined by clonogenic assay. In both cell lines, the out-of-field survival was not statistically different between delivery techniques for either cell line or dose. There was however, a statistically significant difference between survival out-of-field when intercellular communication was intact (single T80 culture flask) or inhibited (multiple T25 culture flasks) to in-field for all plans. No statistically significant difference was observed in-field with or without cellular communication to out-of-field for all plans. These data demonstrate out-of-field effects as important determinants of cell survival following exposure to modulated irradiation fields when cellular communication between differentially irradiated cell populations is present. This data is further evidence that refinement of existing radiobiological models to include indirect cell killing effects is required.

DNA damage responses following exposure to modulated radiation fields

During the delivery of advanced radiotherapy treatment techniques modulated beams are utilised to increase dose conformity across the target volume. Recent investigations have highlighted differential cellular responses to modulated radiation fields particularly in areas outside the primary treatment field that cannot be accounted for by scattered dose alone. In the present study, we determined the DNA damage response within the normal human fibroblast AG0-1522B and the prostate cancer cell line DU-145 utilising the DNA damage assay. Cells plated in slide flasks were exposed to 1 Gy uniform or modulated radiation fields. Modulated fields were delivered by shielding 25%, 50% or 75% of the flask during irradiation. The average number of 53BP1 or ?H2AX foci was measured in 2 mm intervals across the slide area. Following 30 minutes after modulated radiation field exposure an increase in the average number of foci out-of-field was observed when compared to non-irradiated controls. In-field, a non-uniform response was observed with a significant decrease in the average number of foci compared to uniformly irradiated cells. Following 24 hrs after exposure there is evidence for two populations of responding cells to bystander signals in-and out-of-field. There was no significant difference in DNA damage response between 25%, 50% or 75% modulated fields. The response was dependent on cellular secreted intercellular signalling as physical inhibition of intercellular communication abrogated the observed response. Elevated residual DNA damage observed within out-of-field regions decreased following addition of an inducible nitric oxide synthase inhibitor (Aminoguanidine). These data show, for the first time, differential DNA damage responses in-and out-of-field following modulated radiation field delivery. This study provides further evidence for a role of intercellular communication in mediating cellular radiobiological response to modulated radiation fields and may inform the refinement of existing radiobiological models for the optimization of advanced radiotherapy treatment plans. © 2012 Trainor et al.

Production of bacterial biopolymers from industrial fat-containing wastes

Dissertation presented in partial fulfilment of the Requirements for the Degree of Master in Biotechnology

Production and characterization of Chitin-Glucan Complex by Komagataella pastoris

This thesis was focused on the production, extraction and characterization of chitin:β-glucan complex (CGC). In this process, glycerol byproduct from the biodiesel industry was used as carbon source. The selected CGC producing yeast was Komagataella pastoris (formerly known as Pichia pastoris), due the fact that to achieved high cell densities using as carbon source glycerol from the biodiesel industry. Firstly, a screening of K. pastoris strains was performed in shake flask assays, in order to select the strain of K. pastoris with better performance, in terms of growth, using glycerol as a carbon source. K. pastoris strain DSM 70877 achieved higher final cell densities (92-97 g/l), using pure glycerol (99%, w/v) and in glycerol from the biodiesel industry (86%, w/v), respectively, compared to DSM 70382 strain (74-82 g/l). Based on these shake flask assays results, the wild type DSM 70877 strain was selected to proceed for cultivation in a 2 l bioreactor, using glycerol byproduct (40 g/l), as sole carbon source. Biomass production by K. pastoris was performed under controlled temperature and pH (30.0 ºC and 5.0, respectively). More than 100 g/l biomass was obtained in less than 48 h. The yield of biomass on a glycerol basis was 0.55 g/g during the batch phase and 0.63 g/g during the fed-batch phase. In order to optimize the downstream process, by increasing extraction and purification efficiency of CGC from K. pastoris biomass, several assays were performed. It was found that extraction with 5 M NaOH at 65 ºC, during 2 hours, associated to neutralization with HCl, followed by successive washing steps with deionised water until conductivity of ≤20μS/cm, increased CGC purity. The obtained copolymer, CGCpure, had a chitin:glucan molar ratio of 25:75 mol% close to commercial CGC samples extracted from A. niger mycelium, kiOsmetine from Kitozyme (30:70 mol%). CGCpure was characterized by solid-state Nuclear Magnetic Resonance (NMR) spectroscopy and Differential Scanning Calorimetry (DCS), revealing a CGC with higher purity than a CGC commercial (kiOsmetine). In order to optimize CGC production, a set of batch cultivation experiments was performed to evaluate the effect of pH (3.5–6.5) and temperature (20–40 ºC) on the specific cell growth rate, CGC production and polymer composition. Statistical tools (response surface methodology and central composite design) were used. The CGC content in the biomass and the volumetric productivity (rp) were not significantly affected within the tested pH and temperature ranges. In contrast, the effect of pH and temperature on the CGC molar ratio was more pronounced. The highest chitin: β-glucan molar ratio (> 14:86) was obtained for the mid-range pH (4.5-5.8) and temperatures (26–33 ºC). The ability of K. pastoris to synthesize CGC with different molar ratios as a function of pH and temperature is a feature that can be exploited to obtain tailored polymer compositions.(...)