Entsymaattisen hydrolyysin vaikutuksista mikrokiteisen selluloosan rakenteeseen

Cellulose can be used as a renewable raw material for energy production. The utilization requires degradation of cellulose into glucose, which can be done with the aid of enzymatic hydrolysis. In this thesis, various x-ray methods were used to characterize sub-micrometer changes in microcrystalline cellulose during enzymatic hydrolysis to clarify the process and factors slowering it. The methods included wide-angle x-ray scattering (WAXS), small-angle x-ray scattering (SAXS) and x-ray microtomography. In addition, the samples were studied with transmission electron microscopy (TEM). The studied samples were hydrolyzed by enzymes of the Trichoderma reesei species for 6, 24, and 75 hours, which corresponded to 31 %, 58 %, and 68 % degrees of hydrolysis, respectively. Freeze-dried hydrolysis residues were measured with WAXS, SAXS and microtomography, whereas some of them were re-wetted for the wet SAXS and TEM measurements. The microtomography measurements showed a clear decrease in particle size in scale of tens of micrometers. In all the TEM pictures similar cylindrical and partly ramified structures were observed, independent of the hydrolysis time. The SAXS results were ambiguous and partly imprecise, but showed a change in the structure of wet samples in scale of 10-30 nm. According to the WAXS results, the degrees of crystallinity and the crystal sizes remained the same. The gained results support the assuption, that the cellulosic particles are hydrolyzed mostly on their surface, since the enzymes are unable to penetrate into the nanopores of wet cellulose. The hydrolysis therefore proceeds quickly in easily accessible particles and leaves the unaccesible particles almost untouched. The structural changes observed in the SAXS measurements might correspond to slight loosening of the microfibril aggregates, which was seen only in the wet samples because of their different pore structure.

Computational Methods for Reconstruction and Analysis of Genome-Scale Metabolic Networks

Metabolism is the cellular subsystem responsible for generation of energy from nutrients and production of building blocks for larger macromolecules. Computational and statistical modeling of metabolism is vital to many disciplines including bioengineering, the study of diseases, drug target identification, and understanding the evolution of metabolism. In this thesis, we propose efficient computational methods for metabolic modeling. The techniques presented are targeted particularly at the analysis of large metabolic models encompassing the whole metabolism of one or several organisms. We concentrate on three major themes of metabolic modeling: metabolic pathway analysis, metabolic reconstruction and the study of evolution of metabolism. In the first part of this thesis, we study metabolic pathway analysis. We propose a novel modeling framework called gapless modeling to study biochemically viable metabolic networks and pathways. In addition, we investigate the utilization of atom-level information on metabolism to improve the quality of pathway analyses. We describe efficient algorithms for discovering both gapless and atom-level metabolic pathways, and conduct experiments with large-scale metabolic networks. The presented gapless approach offers a compromise in terms of complexity and feasibility between the previous graph-theoretic and stoichiometric approaches to metabolic modeling. Gapless pathway analysis shows that microbial metabolic networks are not as robust to random damage as suggested by previous studies. Furthermore the amino acid biosynthesis pathways of the fungal species Trichoderma reesei discovered from atom-level data are shown to closely correspond to those of Saccharomyces cerevisiae. In the second part, we propose computational methods for metabolic reconstruction in the gapless modeling framework. We study the task of reconstructing a metabolic network that does not suffer from connectivity problems. Such problems often limit the usability of reconstructed models, and typically require a significant amount of manual postprocessing. We formulate gapless metabolic reconstruction as an optimization problem and propose an efficient divide-and-conquer strategy to solve it with real-world instances. We also describe computational techniques for solving problems stemming from ambiguities in metabolite naming. These techniques have been implemented in a web-based sofware ReMatch intended for reconstruction of models for 13C metabolic flux analysis. In the third part, we extend our scope from single to multiple metabolic networks and propose an algorithm for inferring gapless metabolic networks of ancestral species from phylogenetic data. Experimenting with 16 fungal species, we show that the method is able to generate results that are easily interpretable and that provide hypotheses about the evolution of metabolism.

Identification of the fungal catabolic D-galacturonate pathway

Pectin is a natural polymer consisting mainly of D-galacturonic acid monomers. Microorganisms living on decaying plant material can use D-galacturonic acid for growth. Although bacterial pathways for D-galacturonate catabolism had been described previously, no eukaryotic pathway for D-galacturonate catabolism was known at the beginning of this work. The aim of this work was to identify such a pathway. In this thesis the pathway for D-galacturonate catabolism was identified in the filamentous fungus Trichoderma reesei. The pathway consisted of four enzymes: NADPH-dependent D-galacturonate reductase (GAR1), L-galactonate dehydratase (LGD1), L-threo-3-deoxy-hexulosonate aldolase (LGA1) and NADPH-dependent glyceraldehyde reductase (GLD1). In this pathway D-galacturonate was converted to pyruvate and glycerol via L-galactonate, L-threo-3-deoxy-hexulosonate and L-glyceraldehyde. The enzyme activities of GAR1, LGD1 and LGA1 were present in crude mycelial extract only when T. reesei was grown on D-galacturonate. The activity of GLD1 was equally present on all the tested carbon sources. The corresponding genes were identified either by purifying and sequencing the enzyme or by expressing genes with homology to other similar enzymes in a heterologous host and testing the activities. The new genes that were identified were expressed in Saccharomyces cerevisiae and resulted in active enzymes. The GAR1, LGA1 and GLD1 were also produced in S. cerevisiae as active enzymes with a polyhistidine-tag, and purified and characterised. GAR1 and LGA1 catalysed reversible reactions, whereas only the forward reactions were observed for LGD1 and GLD1. When gar1, lgd1 or lga1 was deleted in T. reesei the deletion strain was unable to grow with D-galacturonate as the only carbon source, demonstrating that all the corresponding enzymes were essential for D-galacturonate catabolism and that no alternative D-galacturonate pathway exists in T. reesei. A challenge for biotechnology is to convert cheap raw materials to useful and more valuable products. Filamentous fungi are especially useful for the conversion of pectin, since they are efficient producers of pectinases. Identification of the fungal D-galacturonate pathway is of fundamental importance for the utilisation of pectin and its conversion to useful products.

Characterization of hydrophobin proteins at interfaces and in solutions using x rays

Hydrophobins are a group of particularly surface active proteins. The surface activity is demonstrated in the ready adsorption of hydrophobins to hydrophobic/hydrophilic interfaces such as the air/water interface. Adsorbed hydrophobins self-assemble into ordered films, lower the surface tension of water, and stabilize air bubbles and foams. Hydrophobin proteins originate from filamentous fungi. In the fungi the adsorbed hydrophobin films enable the growth of fungal aerial structures, form protective coatings and mediate the attachment of fungi to solid surfaces. This thesis focuses on hydrophobins HFBI, HFBII, and HFBIII from a rot fungus Trichoderma reesei. The self-assembled hydrophobin films were studied both at the air/water interface and on a solid substrate. In particular, using grazing-incidence x-ray diffraction and reflectivity, it was possible to characterize the hydrophobin films directly at the air/water interface. The in situ experiments yielded information on the arrangement of the protein molecules in the films. All the T. reesei hydrophobins were shown to self-assemble into highly crystalline, hexagonally ordered rafts. The thicknesses of these two-dimensional protein crystals were below 30 Å. Similar films were also obtained on silicon substrates. The adsorption of the proteins is likely to be driven by the hydrophobic effect, but the self-assembly into ordered films involves also specific protein-protein interactions. The protein-protein interactions lead to differences in the arrangement of the molecules in the HFBI, HFBII, and HFBIII protein films, as seen in the grazing-incidence x-ray diffraction data. The protein-protein interactions were further probed in solution using small-angle x-ray scattering. Both HFBI and HFBII were shown to form mainly tetramers in aqueous solution. By modifying the solution conditions and thereby the interactions, it was shown that the association was due to the hydrophobic effect. The stable tetrameric assemblies could tolerate heating and changes in pH. The stability of the structure facilitates the persistence of these secreted proteins in the soil.

Enzymatic cross-linking of β-casein and its impact on digestibility and allergenicity

Protein modification via enzymatic cross-linking is an attractive way for altering food structure so as to create products with increased quality and nutritional value. These modifications are expected to affect not only the structure and physico-chemical properties of proteins but also their physiological characteristics, such as digestibility in the GI-tract and allergenicity. Protein cross-linking enzymes such as transglutaminases are currently commercially available, but also other types of cross-linking enzymes are being explored intensively. In this study, enzymatic cross-linking of β-casein, the most abundant bovine milk protein, was studied. Enzymatic cross-linking reactions were performed by fungal Trichoderma reesei tyrosinase (TrTyr) and the performance of the enzyme was compared to that of transglutaminase from Streptoverticillium mobaraense (Tgase). Enzymatic cross-linking reactions were followed by different analytical techniques, such as size exclusion chromatography -Ultra violet/Visible multi angle light scattering (SEC-UV/Vis-MALLS), phosphorus nuclear magnetic resonance spectroscopy (31P-NMR), atomic force (AFM) and matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (MALDI-TOF MS). The research results showed that in both cases cross-linking of β-casein resulted in the formation of high molecular mass (MM ca. 1 350 kg mol-1), disk-shaped nanoparticles when the highest enzyme dosage and longest incubation times were used. According to SEC-UV/Vis-MALLS data, commercial β-casein was cross-linked almost completely when TrTyr and Tgase were used as cross-linking enzymes. In the case of TrTyr, high degree of cross-linking was confirmed by 31P-NMR where it was shown that 91 % of the tyrosine side-chains were involved in the cross-linking. The impact of enzymatic cross-linking of β-casein on in vitro digestibility by pepsin was followed by various analytical techniques. The research results demonstrated that enzymatically cross-linked β-casein was stable under the acidic conditions present in the stomach. Furthermore, it was found that cross-linked β-casein was more resistant to pepsin digestion when compared to that of non modified β-casein. The effects of enzymatic cross-linking of β-casein on allergenicity were also studied by different biochemical test methods. On the basis of the research results, enzymatic cross-linking decreased allergenicity of native β-casein by 14 % when cross-linked by TrTyr and by 6 % after treatment by Tgase. It can be concluded that in addition to the basic understanding of the reaction mechanism of TrTyr on protein matrix, the research results obtained in this study can have high impact on various applications like food, cosmetic, medical, textile and packing sectors.

Discovery of oxidative enzymes for food engineering : Tyrosinase and sulfhydryl oxidase

Enzymes offer many advantages in industrial processes, such as high specificity, mild treatment conditions and low energy requirements. Therefore, the industry has exploited them in many sectors including food processing. Enzymes can modify food properties by acting on small molecules or on polymers such as carbohydrates or proteins. Crosslinking enzymes such as tyrosinases and sulfhydryl oxidases catalyse the formation of novel covalent bonds between specific residues in proteins and/or peptides, thus forming or modifying the protein network of food. In this study, novel secreted fungal proteins with sequence features typical of tyrosinases and sulfhydryl oxidases were iden-tified through a genome mining study. Representatives of both of these enzyme families were selected for heterologous produc-tion in the filamentous fungus Trichoderma reesei and biochemical characterisation. Firstly, a novel family of putative tyrosinases carrying a shorter sequence than the previously characterised tyrosinases was discovered. These proteins lacked the whole linker and C-terminal domain that possibly play a role in cofactor incorporation, folding or protein activity. One of these proteins, AoCO4 from Aspergillus oryzae, was produced in T. reesei with a production level of about 1.5 g/l. The enzyme AoCO4 was correctly folded and bound the copper cofactors with a type-3 copper centre. However, the enzyme had only a low level of activity with the phenolic substrates tested. Highest activity was obtained with 4-tert-butylcatechol. Since tyrosine was not a substrate for AoCO4, the enzyme was classified as catechol oxidase. Secondly, the genome analysis for secreted proteins with sequence features typical of flavin-dependent sulfhydryl oxidases pinpointed two previously uncharacterised proteins AoSOX1 and AoSOX2 from A. oryzae. These two novel sulfhydryl oxidases were produced in T. reesei with production levels of 70 and 180 mg/l, respectively, in shake flask cultivations. AoSOX1 and AoSOX2 were FAD-dependent enzymes with a dimeric tertiary structure and they both showed activity on small sulfhydryl compounds such as glutathione and dithiothreitol, and were drastically inhibited by zinc sulphate. AoSOX2 showed good stabil-ity to thermal and chemical denaturation, being superior to AoSOX1 in this respect. Thirdly, the suitability of AoSOX1 as a possible baking improver was elucidated. The effect of AoSOX1, alone and in combi-nation with the widely used improver ascorbic acid was tested on yeasted wheat dough, both fresh and frozen, and on fresh water-flour dough. In all cases, AoSOX1 had no effect on the fermentation properties of fresh yeasted dough. AoSOX1 nega-tively affected the fermentation properties of frozen doughs and accelerated the damaging effects of the frozen storage, i.e. giving a softer dough with poorer gas retention abilities than the control. In combination with ascorbic acid, AoSOX1 gave harder doughs. In accordance, rheological studies in yeast-free dough showed that the presence of only AoSOX1 resulted in weaker and more extensible dough whereas a dough with opposite properties was obtained if ascorbic acid was also used. Doughs containing ascorbic acid and increasing amounts of AoSOX1 were harder in a dose-dependent manner. Sulfhydryl oxidase AoSOX1 had an enhancing effect on the dough hardening mechanism of ascorbic acid. This was ascribed mainly to the produc-tion of hydrogen peroxide in the SOX reaction which is able to convert the ascorbic acid to the actual improver dehydroascorbic acid. In addition, AoSOX1 could possibly oxidise the free glutathione in the dough and thus prevent the loss of dough strength caused by the spontaneous reduction of the disulfide bonds constituting the dough protein network. Sulfhydryl oxidase AoSOX1 is therefore able to enhance the action of ascorbic acid in wheat dough and could potentially be applied in wheat dough baking.

Efecto de cuatro substratos en el endurecimiento de vitroplantas de mora (Rubus glaucus benth) variedad risaralda, en el municipio de Las Sabanas, departamento de Madriz

El presente estudio se llevó a cabo en el municipio de Las Sabanas, Departamento de Madriz en el periodo de noviembre del 2004 a abril del 20 05. En el estudio se realizaron dos ensayos. En el primer ensayo se evaluó la sobrevivencia de vitroplantas de mora ( Rubus glaucus Benth.) cultivadas en cuatro su stratos o tratamientos. Los tratamientos utilizados en el ensayo 1 fueron: arena (tratamiento I), lombrihumus de cachaza (tratamiento II), arena + cascarilla de arroz (tratamiento III) y arena + cascarilla de arroz + suel o (tratamiento IV). En el ensayo 2 se utilizó arena como sustrato, que resulto ser el mejor trat amiento según los resultados del ensayo 1 y se evaluaron los tratamientos arena esterilizada + trichoderma + micorriza (tratamiento V), arena esterilizada + trichoderma (tratamiento VI), arena sin esterilizar + trichoderma + micorriza (tratamiento VII) y arena sin esterilizar + tr ichoderma (tratamiento VIII). Se estimó el porcentaje de sobrevivencia (%), la altura (cm) y el número de hojas. Se utilizó un diseño experimental completo al azar (DCA). Se realizó un análisis de varianza y separación de medias mediante la prueba de Tukey (  = 0.05). El porcentaje de sobrevivencia de los tratamientos V y VI fue 64.16 % y 60.00 %, respectivamente. En el caso de los tratamientos VII y VIII presentaron un porcentaje de sobreviv encia de 51.66 % y 52.50 % respectivamente. En el tratamiento VI se obtuvo la mayor altura promedio con 1.67 cm, seguido del tratamiento V con una altura de 1.49 cm. Los mayores porcentajes de hojas estuvieron dados por los tratamientos V con 2.02 hojas y el tratamiento VI con 2.01 hoj as. En los tratamientos III y IV se presentó la mayor incidencia por mal del talluelo en las vitroplantas, enfermedad que es causada por un complejo de hongos del suelo ( Fusarium sp; Rhizoctonia sp y Pythium sp,) que provocan la marchitez del tallo y pudrición de raíces. La utilización de substratos con combinaciones de arena esterilizada + trichod erma + micorriza, presentan los mejores resultados.

Efecto de tres distancias de siembra y tres alternativas de manejo de enfermedades en el cultivo orgánico de fresa (Fragaria spp) variedad festival, en Las Sabanas, Madriz

El presente trabajo se realizó en el municipio “Las Sabanas”, ubicada en departamento de Somoto-Madríz, durante el periodo de julio del 2005-abril 2006, con el propósito de conocer el efecto de distancias de siembra y tratamiento orgánicos de enfermedades en el cultivo de fresa (fragaria spp). Se utilizó un diseño en Bloque Completo al Azar (BCA) con arreglos en parcelas divididas con tres réplicas, siendo el factor “A”: distancias de siembra (0.25, 0.30 y 0.35 m. entre plantas) y el factor “B”: Tratamientos de enfermedades(trichoderma harzianum, Caldo sulfocálcico y un testigo absoluto). A los efectos obtenidos se les realizó un análisis de varianza (ANDEVA), y separación de medias a través de la prueba de rangos múltiples de tukey (8=0.05). Los resultados del estudio determinaron que el factor densidad de siembra presentó efecto significativo sobre las variables de crecimiento tales como: longitud de foliolo, ancho del foliolo, longitud del pecíolo, número de hojas y número de coronas, destacándose un mayor efecto en la distancia de 0.25m para todas estas variables. Mientras que en los tratamientos para enfermedades hubo efecto significativo en todas las variables antes mencionadas a excepción de la longitud del pecíolo. El mayor efecto lo realizaron el caldo sulfocálcico y el trichodermaharzianum. En el caso de las variables de fruto el mayor efecto significativo lo realizaron las distancias de 0.25 y 0.30m. Con respecto a los grados brix y el espesor de fruto no presentaron significancia estadística. Aunque para los tratamientos el mejor efecto influenciado sobre los fruto fue el caldo sulfocálcico. En cuanto a el mejor rendimiento total obtenido durante los cinco meses de cosecha fue 7,444.44 kg ha-¹ con el tratamiento del Caldo sulfocálcico y la distancia de 0.25 m, seguido de 6550.00 kg ha-¹ con el tratamiento de Trichodermaharzianum y la distancia de 0.30 m.

Influencia de dos tipos de sustratos y tres tipos de desinfecciòn en el crecimiento y calidad de plantas de pinusoocarpa (Shiede) producidas en un vivero de San Fernando Nueva Segovia, Nicaragua

La finca forestal San Nicolás está ubicada en el municipio de San Fernando, departamento de Nueva Segovia, a 267 kilómetros de Managua. En dicha finca se realizó el presente estudio durante los meses de abril a julio del año dos mil trece, con el objetivo de evaluar la influencia de dos tipos de sustratos y tres tipos de desinfección en el crecimiento y calidad de plantas de plantas de Pinus oocarpa Schiede, producidas en vivero. En dicho estudio se trabajó con suelo de dos localidades diferentes Macuelizo y San Nicolás, suelo del bosque de pinares con el fin de garantizar las micorrizas. Ambos suelos se trabajaron como un solo ensayo experimental, pero sin hacer comparaciones. El diseño de campo utilizado fue de parcelas divididas con cuatro repeticion es, evaluando do sustratos como factor A y cuatro niveles de desinfección del sustrato como factor B. Los sustratos utilizados fueron mezcla de 70% suelo- 30% arena y 100% suelo; la desinfección consistió en: desinfección biológica con Trichoderma, desinfección química con Carbendazim, desinfección con cal, usada artesanalmente en Nicaragua para desinfección de suelo y el testigo al cual no se aplicó desinfección. Las variables evaluadas fueron: sobrevivencia, crecimiento en altura, diámetro, síntomas de enfermedades,peso húmedo de la parte aérea y radicular, peso seco de la parte aérea y radicular. Para evaluar la calidad de la planta se calcularon los índices: relación parte aérea y parte radical, índice de esbeltez, índice de calidad, índice de lignificación y un análisis estadístico. Los índices calculados muestran que los mejores resultados se obtuvieron del sustrato 70% suelo-30% arena en ambas localidades.

Evaluación de una plantación de Pinus oocarpa Schiede con plantas provenientes de diferentes condiciones de manejo en el vivero de la finca forestal San Nicolás, San Fernando, Nueva Segovia

La calidad de planta producida en vivero debe demostrarse en campo, considerando tanto la supervivencia como el desarrollo inicial en el ambiente en que fueron plantadas. Con la finalidad de evaluar el comportamiento de una plantación de Pinus oocarpa, con plantas que provenían de un ensayo experimental en el vivero de la finca forestal San Nicolás, San Fernando, Nueva Segovia, aplicando diferentes mezclas y 3 niveles de desinfección (cal, hongo del genero Trichoderma controlador de patógenos y testigo), se llevó a cabo un experimento en terrenos de la misma finca. Se utilizó un diseño de bloques completos al azar con 3 repeticiones por tratamiento. Se realizaron cinco mediciones, con una frecuencia mensual para las variables dasométricas (altura y diámetro basal). Se plantaron entre 6 y 8 plantas por tratamiento para un total de 264 individuos, la distancia utilizada entre plantas fue de 2.5 x 2.5 m. Se evaluaron las variables de sobrevivencia, diámetro basal, altura, así como la condición sanitaria de las plantas por tratamiento. Las plantas del tratamiento que obtuvieron mayor porcentaje de sobrevivencia corresponden al tratamiento M70DCAL con 100 %, por su parte las plantas del tratamiento M70DTRI alcanzaron el mayor incremento promedio mensual en altura con 6.67 cm, asi mismo las plantas del tratamiento que obtuvieron mayor incremento promedio mensual en diámetro basal corresponden al tratamiento M70D0 con 5.79 mm. Los principales daños causados por insectos y otras enfermedades fueron reportados principalmente por las plantas de los tratamientos N100DTRI y M100DTRI con 16.7 y 26. 67 % respectivamente. Al momento de comparar la calidad de planta obtenida en vivero y su posterior comportamiento en el sitio de plantación, las plantas de los tratamientos que se comportaron superior pertenecen a N100D0 y M70DTRI.

Avaliação das condições ambientais e de trabalho em salas cirúrgicas e de necropsia - estudos de casos em hospitais públicos e IMLs da região metropolitana do Rio de Janeiro

A qualidade do ar é um importante indicador de saúde ambiental, sendo o seu monitoramento contínuo necessário. Apesar da relevância do tema, há muitos países em que os limites de exposição para agentes biológicos ainda não foram estabelecidos ou foram definidos de forma inadequada, podendo comprometer a qualidade ambiental. Os ambientes hospitalares, assim como as salas de necropsia podem apresentar problemas de contaminação do ar por agentes microbiológicos, necessitando de monitoramento contínuo a fim de evitar a ocorrência de doenças nos trabalhadores e na população em geral. Este estudo realizou a avaliação microbiológica do ar em hospitais públicos e IMLs da região metropolitana do Rio de Janeiro em salas cirúrgicas e de necropsia. A pesquisa exploratória e descritiva baseou-se em levantamento bibliográfico e investigação de campo, através de estudos de casos. Os dados foram obtidos por meio de entrevistas e observação direta nos locais de trabalho, onde foram realizadas as avaliações microbiológicas do ar. As variações em salas cirúrgicas para bactérias e fungos foram respectivamente de 14,99 ufc/m3 88,29 ufc/m3 e de 45,93 ufc/m3 - 742,09 ufc/m3. Já nas salas de necropsia os valores para bactérias e fungos variaram respectivamente de 18,96 ufc/m3 54,9 ufc/m3 e de 144,87 ufc/m3 - 1152,01 ufc/m3. Foram identificados tanto no ambiente cirúrgico como nas salas de necropsia a presença dos seguintes fungos: Aspergillus sp., Neurospora sp., Penicillium sp., Fusarium sp., Cladosporium sp., Curvularia sp., e Trichoderma sp. Já em relação às bactérias foram identificadas as presenças de Staphilococcus sp., Streptococcus sp. e Micrococcus sp. Foram traçadas recomendações para melhoria da qualidade ambiental e do ar. Os resultados indicaram que os valores são elevados quando comparados com as recomendações das normas internacionais. Foram encontrados valores inferiores aos sugeridos pela CP n. 109 da ANVISA. A presença de microrganismos patogênicos sugere adoção de medidas de controle ambiental. O estudo apontou a necessidade urgente do estabelecimento de valores de referência para ambientes hospitalares no Brasil a fim de garantir condições seguras que não venham a comprometer a saúde dos pacientes e profissionais de saúde envolvidos.

商陆抗真菌蛋白的基因克隆及其应用

商陆种子的7kD多肽被鉴定为一种抗真菌多肽，命名为PAFP（pokeweed antifungal protein）。它抑制Trichoderma viride, Fusatium及其它一些病原真菌的生长。本文构建了cDNA文库，而后从库中筛选和克隆PAFP基因。PAFP的编码序列－－201bp的DNA片段被扩增并插入pBluescript SK+载体。经酶切图谱分析和核苷酸顺序测定之后，这个片段与35S启动子连接并重组于双元载体pBin 19。此表达载体质粒转入农杆菌LBA 4404供转化植物之用。通过农杆菌介导的对西瓜的转化，所采用的基因还包括报告基因GUS和Bar，以及一种来自大麦的抗真菌蛋白的基因。以PCR扩增，GUS与NPT II活性检测，以及Southern杂交对转基因植物进行鉴定。

稠油废水处理中优势微生物种群组成及变化规律

针对辽河油田锦采污水处理厂稠油废水，利用传统培养方法和PCR-DGGE诊断技术，对稠油废水处理过程中优势微生物种群组成和多样性进行全面系统的研究。结果表明，微生物对稠油废水生物处理的作用为细菌>真菌>放线菌。细菌数量、基因多样性指数与废水中TPH、CODCr均正相关，可以作为稠油废水水质评价的生物指标。 对影响稠油废水生物降解的主要因子进行优化表明，当30℃，pH值7.5，HRT为216h，添加N、P营养盐使N:P比为5.63:1时，CODCr去除率最高，去除后CODCr值满足污水综合排放一级标准（GB8978-1996）。利用GC-MS技术分析降解前后稠油废水中主要有机成分表明，微生物对饱和烃类化合物降解率最高，其次是低分子量芳香烃，而高分子量芳香烃、胶质和沥青质最低。 以稠油为唯一碳源，对筛选出的菌株进行摇瓶实验表明，各菌株对稠油均具有一定的降解能力，其中F0504除油能力最强，56d去除率可达63.3%；动力学方程拟合表明稠油生物降解过程符合一级动力学方程。降解后残油组分分析表明，B0505和F0501对烷烃、B0510、F0505和F0507对芳香烃、B0501和F0504对胶质、沥青质的去除率均较高，去除率都在30c%之间。 经鉴定，优势菌株B0501和B0505分别为液化金杆菌（Aureobaterium liquefaciens）和弗氏丙酸杆菌（Propionibacterium freuclenreichii），主要真菌有青霉（Penicillium）、曲霉（Aspergillus）、木霉（Trichoderma）和交链孢霉（Alternaria）。

CO2升高对土壤微生物群落影响的模拟研究

大气CO2浓度升高可以通过植物间接影响土壤生态系统。土壤生态系统的结构和功能改变将影响有机质矿化和营养物质循环，进而可能对CO2浓度升高产生正反馈或负反馈。微生物是土壤生态系统的主体，在对CO2浓度升高的反馈中起着至关重要的作用。本研究以开顶箱系统为平台，采用微生物分子生态学技术和现代酶学技术，通过对长期接受500 ppm CO2的红松幼树、长白赤松幼树和蒙古栎幼树非根际土壤连续两个生长季的测定，系统研究了高浓度CO2对温带森林土壤微生物群落的生物量和微生物活性的影响，检测了土壤微生物群落的结构和功能以及土壤化学性质变化，主要结论如下： （1）高浓度CO2处理提高了土壤有机碳含量。与对照组相比较，红松幼树土壤有机碳含量提高9.4%；长白赤松幼树土壤提高0.6%；蒙古栎幼树土壤提高1.3%。 （2）高浓度CO2处理使土壤磷酸酶（phosphatase）、几丁质酶（1,4-β-acetylglucosaminidase, 1,4-β-NAG）和多酚氧化酶（phenol oxidase）活性发生了显著变化，高浓度CO2使红松土壤 1,4-β-NAG活性提高7-25%，长白松土壤1,4-β-NAG平均活性降低14%，蒙古栎土壤1,4-β-NAG平均活性提高31%。 同时研究还发现，过氧化物酶（peroxidase）和多酚氧化酶(phenol oxidase)活性与微生物量碳和微生物量氮呈显著的正相关。相关分析还显示，土壤湿度与1,4-α-葡萄糖苷酶（1,4-α-glucosidase）活性、 微生物生物量碳和微生物生物量氮呈显著的正相关。 高浓度CO2在不同程度上改变了土壤转化酶活性和脱氢酶活性。高浓度CO2显著提高了红松和长白赤松土壤硝化酶活性；而显著降低反硝化酶活性。 （3）研究发现三种树土壤的真菌和细菌群落存在着季节性演替，并且高浓度CO2熏蒸处理使真菌群落结构发生了显著的变化，表现为一些种群优势度下降，另一些升高。虽然，细菌群落没有如真菌群落变化的明显，但研究中也发现高浓度CO2的确使个别细菌种群的优势度发生了显著改变。 亲缘关系与Calocybe carnea，Magmatodrilus obscurus密切的真菌是红松土壤优势种群，与Humicola fuscoatra关系相近的是长白松土壤的优势种群，并且此三种真菌的季节性变化不显著。研究发现高浓度CO2使红松土壤中亲缘关系与Pachyella clypeata，Cochlonema euryblastum，Lepiota cristata，Eimeriidae sp.， Trichoderma sp.相近的种群的丰富度显著提高，使蒙古栎土壤中亲缘关系与Serendipita vermifera，Calocybe carnea种群丰富度显著下降，使蒙古栎土壤中与Candida sp.，Magmatodrilus obscurus和Pachyella clypeata亲缘关系密切种群的丰富度显著提高。 （4）三种幼树叶的原位分解培养429天结果显示，红松和长白松凋落物的β-葡萄糖苷酶（1,4-β-glucosidase）和木糖苷酶（1,4-β-xylosidase）活性随着分解而逐渐增加，而这两种酶在蒙古栎凋落物分解过程中保持相对恒定；高浓度CO2显著影响叶凋落物分解磷酸酶（phosphatase），纤维二糖酶（cellobiohydrolase）, 几丁质酶(1,4-β-NAG)，多酚氧化酶（phenol oxidase）和过氧化物酶（peroxidase）的活性。研究发现，凋落物的生物化学性质变化能引起分解的微生物群落发生变化，进而引起分泌的胞外酶活性变化，科学印证了大气CO2浓度升高“通过影响凋落物质量进而影响分解叶凋落物的微生物群落的结构和功能”的猜测。 不同凋落物之间酶活性差异显著，真菌和细菌群落结构也显著不同。序列与Hyphodiscus hymeniophilus亲缘关系密切的真菌和亲缘关系与Verrucomicrobia bacterium密切的细菌是长白松凋落分解的最优势种群，序列与Lophium mytilinum亲缘关系密切的真菌是红松凋落分解的最优势种群。 另外，研究还发现，高浓度CO2使参与分解红松凋落物Beta proteobacterium OS-15A亲缘关系相近的细菌种群和与Azospirillum amazonense亲缘关系相近的种群丰富度显著降低；使与Luteibactor rhizovicina亲缘关系相近的种群和与Luteibactor rhizovicina亲缘关系相近的种群显著提高。高浓度CO2使定殖于长白松凋落物上Hyphodiscus hymeniophilus亲缘关系相近的种群和与Bionectria pityrodes亲缘关系相近的种群显著提高，而使与Neofabraea malicorticis亲缘关系相近的种群和与Hyphodiscus hymeniophilus亲缘关系相近的种群显著下降。

施肥和海洋微生物调控对桉树人工林土壤质量的影响

本文研究施化肥和海洋微生物制剂对桉树人工林土壤质量的影响。研究结果表明：施用不同的化肥对桉树人工林土壤质量影响具有明显差异；从海洋植物根际分离得到的微生物菌株制成的微生物制剂中的活性微生物菌株能够在桉树人工林土壤中定殖，对桉树生长具有一定促进作用。桉树凋落叶分解过程中是否释放化感物质是桉树人工林发展过程中人们普遍关注的问题之一，本论文也对该部分做了初步研究。 对施用长效尿素、芬兰复合肥、高峰复合肥三种不同化肥对桉树人工林土壤质量的的影响进行了初步研究，研究结果表明长效尿素在保障土壤氮素供应、促进土壤纤维素分离能力提高和增强土壤对磷元素吸收方面具有重要作用；芬兰复合肥在增强土壤呼吸作用和促进土壤酶活性提高方面优于长效尿素和高峰复合肥。 以两株海洋来源的枯草芽孢杆菌(Bacillus subtilis 3512, Bacillus subtilis 3728)和一株海洋木霉(Trichoderma TF4)为研究材料，在实验室条件下对其生防机理进行了研究，研究表明:两株枯草芽孢杆菌通过产生脂肽和蛋白酶对植物病原菌产生抑制作用；海洋木霉TF4则能够产生HCN，IAA类植物生长激素，同时还具有一定的解磷能力，具有很好的应用前途，采用传统分类学方法和分子系统学方法鉴定为棘孢木霉(T.asperellum)。这三株海洋菌株制成微生物菌剂，在原位条件和盆栽条件下考察了其对桉树生物量和土壤质量指标的影响，研究结果表明将三株海洋微生物混合后添加少量三叶草作辅剂，能有效改善桉树人工林土壤质量，并促进桉树树高和胸径的增加，具备进一步研究和开发成产品的价值。 用高效液相色谱(HPLC)对桉树凋落叶中毒性物质进行分离、纯化，以小麦、绿豆、大速生菜为指示植物，对分离到的物质进行毒性跟踪，分离到一个毒性较强的组分，经1H氢谱和NMR鉴定为3–β甲酰基–乌索酸。