Advanced biofuel production: Engineering metabolic pathways for butanol and propane biosynthesis.

Greenhouse gases emitted from energy production and transportation are dramatically changing the climate of Planet Earth. As a consequence, global warming is affecting the living conditions of numerous plant and animal species, including ours. Thus the development of sustainable and renewable liquid fuels is an essential global challenge in order to combat the climate change. In the past decades many technologies have been developed as alternatives to currently used petroleum fuels, such as bioethanol and biodiesel. However, even with gradually increasing production, the market penetration of these first generation biofuels is still relatively small compared to fossil fuels. Researchers have long ago realized that there is a need for advanced biofuels with improved physical and chemical properties compared to bioethanol and with biomass raw materials not competing with food production. Several target molecules have been identified as potential fuel candidates, such as alkanes, fatty acids, long carbon‐chain alcohols and isoprenoids. The current study focuses on the biosynthesis of butanol and propane as possible biofuels. The scope of this research was to investigate novel heterologous metabolic pathways and to identify bottlenecks for alcohol and alkane generation using Escherichia coli as a model host microorganism. The first theme of the work studied the pathways generating butyraldehyde, the common denominator for butanol and propane biosynthesis. Two ways of generating butyraldehyde were described, one via the bacterial fatty acid elongation machinery and the other via partial overexpression of the acetone‐butanol‐ethanol fermentation pathway found in Clostridium acetobutylicum. The second theme of the experimental work studied the reduction of butyraldehyde to butanol catalysed by various bacterial aldehyde‐reductase enzymes, whereas the final part of the work investigated the in vivo kinetics of the cyanobacterial aldehyde deformylating oxygenase (ADO) for the generation of hydrocarbons. The results showed that the novel butanol pathway, based on fatty acid biosynthesis consisting of an acyl‐ACP thioesterase and a carboxylic acid reductase, is tolerant to oxygen, thus being an efficient alternative to the previous Clostridial pathways. It was also shown that butanol can be produced from acetyl‐CoA using acetoacetyl CoA synthase (NphT7) or acetyl‐CoA acetyltransferase (AtoB) enzymes. The study also demonstrated, for the first time, that bacterial biosynthesis of propane is possible. The efficiency of the system is clearly limited by the poor kinetic properties of the ADO enzyme, and for proper function in vivo, the catalytic machinery requires a coupled electron relay system.

Germination, initial growth and cotyledon protein content of bean cultivars under salinity stress

Excess salts in the root zone inhibit water uptake by plants, affect nutrient uptake and may result in toxicities due to individual salts in the soil solution. Excess exchangeable sodium in the soil may destroy the soil structure to a point where water penetration and root aeration become impossible. Sodium is also toxic to many plants. Beans (Phaseolus vulgaris L.) are consumed as protein source in northeastern Brazil, although little is known about common bean cultivar tolerance to salinity. The germination of bean cultivars under salt stress was studied. The cultivars 'Carioca' and 'Mulatinho' were submitted to germination test in a germinator at 25ºC, at the Seed Analysis Laboratory of the Brazilian Agricultural Research Corporation unit in the Semi- Arid region (Embrapa Semi Árido), Petrolina, Pernambuco State. These seeds were germinated on "germitest" papers imbibed in distilled water or in 10, 50, 100 e 200 mol.m-3sodium chloride (NaCl) solutions. At the first and second counts of the germination test, normal seedlings were counted, measured, weighed and dried, supplying data for vigor, total germination, fresh matter weight and dry matter weight and seedlings length. Total protein was quantified in cotyledons at 3, 6 and 9 days after sowing. The results indicated that the NaCl content influenced seed germination and concentrations above 50 mol.m-3 decreased germination and seedling growth.

Square Root Finding In Graphs

Abstract: Root and root finding are concepts familiar to most branches of mathematics. In graph theory, H is a square root of G and G is the square of H if two vertices x,y have an edge in G if and only if x,y are of distance at most two in H. Graph square is a basic operation with a number of results about its properties in the literature. We study the characterization and recognition problems of graph powers. There are algorithmic and computational approaches to answer the decision problem of whether a given graph is a certain power of any graph. There are polynomial time algorithms to solve this problem for square of graphs with girth at least six while the NP-completeness is proven for square of graphs with girth at most four. The girth-parameterized problem of root fining has been open in the case of square of graphs with girth five. We settle the conjecture that recognition of square of graphs with girth 5 is NP-complete. This result is providing the complete dichotomy theorem for square root finding problem.

Effet de l’irrigation par l’eau usée sur la biomasse aérienne et souterraine d’une culture intensive de saules en courtes rotations

Le but de cette étude est de comprendre l’effet d’une irrigation par les eaux usées et /ou de la fertilisation par les engrais chimiques sur la productivité aérienne et souterraine d’une plantation de saule Salix miyabeana SX67 en CICR dans un contexte de filtre végétal. Nous avons d’une part évalué l’impact de diverses doses d’eau usées et/ou de la fertilisation minérale sur les rendements en biomasse ligneuse d’une culture de saules au cours d’un cycle de croissance de deux ans. D’autre part et pour la même période nous avons comparé le développement racinaire (biomasse, morphologie et distribution dans le sol) suite aux divers traitements. Les résultats ont montré qu’au terme de deux ans de croissance, les traitements par les eaux usées aussi bien que celle par les engrais a permis l’augmentation des rendements de la biomasse aérienne de notre culture de saules avec un effet plus prononcé suite au traitements des eaux usées qu’à celui du fertilisant chimique. Nous avons mesuré des productivités en biomasse aussi élevées que 39,4 Mg ha-1 et 54,7 Mg ha-1 et ce pour les parcelles qui ont reçu la plus grande quantité d'eaux usées, respectivement pour les saules non fertilisé et fertilisé (D3-NF et D3-F). La majeure partie du système racinaire était en superficie avec 92-96% des racines (racine fine et racine grosse) concentrées dans les premiers 40 cm de sol et nous avons trouvé que la biomasse des racines fines était comprise entre 1,01 et 1,99 Mg ha-1. Généralement la fertilisation chimique n’a pas eu d’effet sur les rendements en biomasse des racines totales et/ou fines. Bien que l’irrigation par les eaux usées ait entraîné une réduction statistiquement significative de la biomasse racinaire, néanmoins cette réduction n'était pas linéaire (avec une réduction de la biomasse de D0 à D1, une augmentation de D1 à D2 pour réduire de nouveau de D2 à D3). Cette tendance porte à penser qu'au-delà d'une certaine quantité d'eau et de nutriments (suite à l’irrigation par les eaux usées), le développement du système racinaire des saules est affecté négativement, et bien que la biomasse aérienne soit restée élevée sous le traitement D3, nous pensons que le développement de la plante a été quelque peu déséquilibré. Aucun changement significatif n'a été constaté dans les traits morphologiques liés à l'irrigation par les eaux usées.

Séquestration du carbone atmosphérique dans la biomasse racinaire de plantations de saules

Le cycle du carbone (C) est, depuis la révolution industrielle, déstabilisé par l’introduction dans l’atmosphère de C autrefois fossilisé. Certaines mesures de mitigation prometteuses impliquent la séquestration accrue du CO2 atmosphérique dans les sols via le développement du réseau racinaire des arbres. Ce projet de recherche visait à : 1) quantifier la biomasse racinaire ligneuse produite annuellement par unité de surface par le Salix miyabeana cultivé en régie intensive à courtes rotations, 2) doser la concentration en C et en N des racines de saule en fonction de leur profondeur et de leur diamètre et 3) déterminer l’influence des propriétés pédoclimatiques du milieu sur la séquestration du carbone organique (Corg) par les racines. Pour y arriver, six souches de saules ont été excavées à partir de huit sites (n=48) et neuf carottes de recolonisation ont été implantées à cinq sites (n=45) pour évaluer la productivité racinaire fine. Les échantillons séchés ont été pesés pour quantifier la biomasse racinaire produite, et ont été analysés pour le C et le N. La productivité en biomasse racinaire ligneuse du saule en plantation pour tout le réseau d'échantillonnage varie de 0,7 – 1,8 Mg/ha/an. La proportion de C dans la biomasse racinaire s’étend de 31,3% à 50,4% et sa variance dans les tissus est expliquée par le diamètre racinaire et par les conditions environnementales des sites de provenance. Les conditions climatiques constituent la principale influence sur la production de biomasse racinaire. La variance de la biomasse racinaire est significativement contrôlée (p :0,004) par la quantité de précipitation de l’été et de l’année qui contrôlent ensemble 83,4 % du r2 ajusté. La précipitation de l’été est inversement liée à la productivité racinaire puisque les protéines expansines des racines sont stimulées par les carences hydriques du sol. La production de racines fines des plantations (1,2 à 2,4 Mg/ha/an) est, elle, plus fortement contrôlée par les conditions pédologiques du site qui expliquent 36,5% de la variance de productivité des racines fines contre 37,5% de la variance expliquée par les facteurs pédoclimatiques. Le P et le N du sol ont des rôles prépondérants sur la production de racines fines. Une disponibilité en P accrue dans le sol stimule la biomasse racinaire fine alors qu’une quantité supérieure de N dans le sol limite la croissance racinaire tout en favorisant la croissance des parties aériennes de la plante. Ce projet a permis d’améliorer notre compréhension des conditions pédologiques et climatiques qui engendrent, au Québec méridional, une productivité et une séquestration en Corg accrue dans le réseau racinaire du saule.

Spatial and Temporal Variation of Microbial Community Structure In Surficial Sediments of Cochin Estuary

This thesis entitled spatial and temporal variarion of microbial community structure in surficial sediments of cochin estuary.In the estuarine and coastal systems, organic matter (OM) is derived not only from autochthonous primary production, but also from allochthonous (terrestrial) organic matter (OM) delivered by river discharge and runoff. A significant portion of the OM sinks through the water column and is ultimately stored in carbon pool in the sediments.Analysis of spatial and temporal variation in benthic microbial community of a tropical estuary was conducted for the first time using non selective measures that affirms that PLFA approach is a sensitive and reliable method in determining microbial community structures of surficial sediments of estuary.The close relationship between the concentrations of the microbial fatty acids and total biomass indicates that bacteria could account for the largest proportion of the biomass in the sediments.This is first study that has documented the changes in microbial community composition linkage to biotic and abiotic variables in benthic estuarine ecosystem. This contemporaneous community will be the backdrop for understanding the response of autochthonous community to increasing anthropogenic stress.

Die mikrobielle Besiedlung von Wurzeloberfläche und Rhizosphäre in ihrer Bedeutung für Stoffumsätze in Böden

Im Vordergrund der Arbeit stand die Erfassung der mikrobiellen Biomasse bzw. Residualmasse an der Wurzeloberfläche, im Rhizosphärenboden und im umgebenden Boden. Durch den Vergleich von verschiedenen Methoden zur Erfassung der mikrobiellen Biomasse wurden die Gehalte von pilzlichem und bakteriellem Kohlenstoff an der Rhizoplane und in der Rhizosphäre quantifiziert. Dabei wurde die Fumigations-Extraktions-Methode zur Erfassung der mikrobiellen Biomasse eingesetzt. Ergosterol diente als Indikator für die pilzliche Biomasse und die Aminozucker Glucosamin und Muraminsäure sollten Aufschluss geben über die bakterielle und pilzliche Biomasse bzw. Residualmasse in den drei Probenfraktionen. Dazu wurden Umrechnungsfaktoren erstellt, die zur Berechnung des bakteriellen und pilzlichen Kohlenstoffs aus den Gehalten von Muraminsäure und Pilz-Glucosamin dienten. Die Bestimmung von Aminozuckern wurde insoweit modifiziert, dass sowohl in Boden- als auch in Wurzelhydrolysaten die Messung von Glucosamin, Galactosamin, Muraminsäure und Mannosamin gleichzeitig als automatisiertes Standardverfahren mit Hilfe der HPLC erfolgen konnte. Es wurden drei Gefäßversuche durchgeführt: Im ersten Versuch wurde der Einfluss der Pflanzenart auf die mikrobielle Besiedlung der Wurzeloberflächen untersucht. Dabei wurden Wurzeln und Rhizosphärenboden von 15 verschiedenen Pflanzenarten miteinander verglichen. Im zweiten Versuch stand der Einfluss der mikrobiellen Biomasse eines Bodens auf die mikrobielle Besiedlung von Wurzeloberflächen im Vordergrund. Deutsches Weidelgras (Lolium perenne L.) wurde auf sieben verschiedenen Böden angezogen. Bei den Böden handelte es sich um sechs Oberböden, die sich hinsichtlich des Bodentyps und der Bewirtschaftungsform voneinander unterschieden, und einen Unterboden. Im dritten Versuch wurde die mikrobielle Besiedlung von Wurzeln nach teilweiser und vollständiger Entfernung der oberirdischen Biomasse beobachtet. Welsches Weidelgras (Lolium multiflorum Lam.) wurde 24 Tage nach der Aussaat beschnitten. Anschließend wurde über einen Versuchszeitraum von acht Tagen die mikrobielle Besiedlung an den Wurzeln und in den Bodenfraktionen bestimmt. Es bestätigte sich, dass der Einfluss der einzelnen Pflanzenart von entscheidender Bedeutung für die mikrobielle Besiedlung von Wurzeln ist. Bei fast allen Pflanzen wurde die mikrobielle Biomasse an den Wurzeln von Pilzen dominiert. Das Verhältnis von pilzlichem zu bakteriellem Kohlenstoff an den Wurzeln der 15 Pflanzenarten lag im Mittel bei 2,6. Bei der Betrachtung verschiedener Böden zeigte sich, dass die mikrobielle Besiedlung in tieferen Bodenschichten signifikant niedriger ist als in den Oberböden. Dabei war der Pilzanteil an der mikrobiellen Biomasse im Unterboden deutlich erhöht. Der Vergleich der Oberböden untereinander ergab, dass sowohl der Bodentyp als auch die Bewirtschaftungsform einen signifikanten Einfluss auf mikrobielle Besiedlung ausüben. Durch die teilweise oder vollständige Entfernung der oberirdischen Biomasse wurde eine Veränderung der mikrobiellen Besiedlung an den Wurzeln beobachtet. Das Verhältnis von pilzlichem zu bakteriellem Kohlenstoff sank in dem Versuchszeitraum von 2,5 auf 1,4. Dabei war die Förderung der Pilze in der Variante mit teilweise entfernter oberirdischer Biomasse relativ größer als in der Variante mit vollständig entfernter oberirdischer Biomasse. Entgegen der weit verbreiteten Annahme, dass bei den wurzelbesiedelnden Mikroorganismen die Bakterien gegenüber den Pilzen dominieren, zeigten die Ergebnisse ein gegensätzliches Bild. In allen drei Versuchen ergab sich gleichermaßen, dass sowohl im Boden als auch an den Wurzeln die Pilze gegenüber den Bakterien dominieren.

Studies on arbuscular mycorrhiza (AM) in the Alentejo (Portugal) using pea mutants resistant to AM fungi as a control tool for field conditions

The utilization and management of arbuscular mycorrhiza (AM) symbiosis may improve production and sustainability of the cropping system. For this purpose, native AM fungi (AMF) were sought and tested for their efficiency to increase plant growth by enhanced P uptake and by alleviation of drought stress. Pot experiments with safflower (Carthamus tinctorius) and pea (Pisum sativum) in five soils (mostly sandy loamy Luvisols) and field experiments with peas were carried out during three years at four different sites. Host plants were grown in heated soils inoculated with AMF or the respective heat sterilized inoculum. In the case of peas, mutants resistant to AMF colonization were used as non-mycorrhizal controls. The mycorrhizal impact on yields and its components, transpiration, and P and N uptake was studied in several experiments, partly under varying P and N levels and water supply. Screening of native AMF by most probable number bioassays was not very meaningful. Soil monoliths were placed in the open to simulate field conditions. Inoculation with a native AMF mix improved grain yield, shoot and leaf growth variables as compared to control. Exposed to drought, higher soil water depletion of mycorrhizal plants resulted in a haying-off effect. The growth response to this inoculum could not be significantly reproduced in a subsequent open air pot experiment at two levels of irrigation and P fertilization, however, safflower grew better at higher P and water supply by multiples. The water use efficiency concerning biomass was improved by the AMF inoculum in the two experiments. Transpiration rates were not significantly affected by AM but as a tendency were higher in non-mycorrhizal safflower. A fundamental methodological problem in mycorrhiza field research is providing an appropriate (negative) control for the experimental factor arbuscular mycorrhiza. Soil sterilization or fungicide treatment have undesirable side effects in field and greenhouse settings. Furthermore, artificial rooting, temperature and light conditions in pot experiments may interfere with the interpretation of mycorrhiza effects. Therefore, the myc- pea mutant P2 was tested as a non-mycorrhizal control in a bioassay to evaluate AMF under field conditions in comparison to the symbiotic isogenetic wild type of var. FRISSON as a new integrative approach. However, mutant P2 is also of nod- phenotype and therefore unable to fix N2. A 3-factorial experiment was carried out in a climate chamber at high NPK fertilization to examine the two isolines under non-symbiotic and symbiotic conditions. P2 achieved the same (or higher) biomass as wild type both under good and poor water supply. However, inoculation with the AMF Glomus manihot did not improve plant growth. Differences of grain and straw yields in field trials were large (up to 80 per cent) between those isogenetic pea lines mainly due to higher P uptake under P and water limited conditions. The lacking N2 fixation in mutants was compensated for by high mineral N supply as indicated by the high N status of the pea mutant plants. This finding was corroborated by the results of a major field experiment at three sites with two levels of N fertilization. The higher N rate did not affect grain or straw yields of the non-fixing mutants. Very efficient AMF were detected in a Ferric Luvisol on pasture land as revealed by yield levels of the evaluation crop and by functional vital staining of highly colonized roots. Generally, levels of grain yield were low, at between 40 and 980 kg ha-1. An additional pot trial was carried out to elucidate the strong mycorrhizal effect in the Ferric Luvisol. A triplication of the plant equivalent field P fertilization was necessary to compensate for the mycorrhizal benefit which was with five times higher grain yield very similar to that found in the field experiment. However, the yield differences between the two isolines were not always plausible as the evaluation variable because they were also found in (small) field test trials with apparently sufficient P and N supply and in a soil of almost no AMF potential. This similarly occurred for pea lines of var. SPARKLE and its non-fixing mycorrhizal (E135) and non-symbiotic (R25) isomutants, which were tested in order to exclude experimentally undesirable benefits by N2 fixation. In contrast to var. FRISSON, SPARKLE was not a suitable variety for Mediterranean field conditions. This raises suspicion putative genetic defects other than symbiotic ones may be effective under field conditions, which would conflict with the concept of an appropriate control. It was concluded that AMF resistant plants may help to overcome fundamental problems of present research on arbuscular mycorrhiza, but may create new ones.

Changes in amino acid enantiomers and microbial performance in soils from a subtropical mountain oasis in Oman abandoned for different periods

An important feature of maintaining the agricultural stability in millennia-old mountain oases of northern Oman is the temporary abandonment of terraces. To analyse the effects of a fallow period on soil microbial performance, i.e. microbial activity and microbial biomass, samples of eight terrace soils abandoned for different periods were collected in situ, assigned to four fallow age classes and incubated for 30 days in the laboratory after rewetting. The younger fallow age classes of 1 and 5 years were based on the records of the farmers’ recollections, the two older fallow age classes of 10–20 and 25–60 years according to the increase in the D -to- L ratio of valine and leucine enantiomers. The increase in these two ratios was in agreement with that of the D -to- L ratio of lysine. The strongest relationship was observed between the increase in the D -to- L ratio of lysine and the decrease in soil microbial biomass C. However, the most stringent coherence between the increase in fallow age and soil properties was revealed by the decreases in cumulative respiration and net N mineralisation rates with decreasing availability of substrate to soil microorganisms. During the 30-day incubation following rewetting, relative changes in microbial activity (respiration and net N mineralisation) and microbial biomass (C and N)indices were similar in the eight terrace soils on a fallow age-class-specific level, indicating that the same basic processes occurred in all of the sandy terrace soils investigated.

Effects of fertilizer type and rate on partitioning of soil organic matter into pools of different stability

Type and rate of fertilizers influence the level of soil organic carbon (Corg) and total nitrogen (Nt) markedly, but the effect on C and N partitioning into different pools is open to question. The objectives of the present work were to: (i) quantify the impact of fertilizer type and rate on labile, intermediate and passive C and N pools by using a combination of biological, chemical and mathematical methods; (ii) explain previously reported differences in the soil organic matter (SOM) levels between soils receiving farmyard manure with or without biodynamic preparations by using Corg time series and information on SOM partitioning; and (iii) quantify the long-term and short-term dynamics of SOM in density fractions and microbial biomass as affected by fertilizer type and rate and determine the incorporation of crop residues into labile SOM fractions. Samples were taken from a sandy Cambisol from the long-term fertilization trial in Darmstadt, Germany, founded in 1980. The nine treatments (four field replicates) were: straw incorporation plus application of mineral fertilizer (MSI) and application of rotted farmyard manure with (DYN) or without (FYM) addition of biodynamic preparations, each at high (140 – 150 kg N ha-1 year-1; MSIH, DYNH, FYMH), medium (100 kg N ha-1 year-1; MSIM, DYNM, FYMM) and low (50 – 60 kg N ha-1 year-1; MSIL, DYNL, FYML) rates. The main findings were: (i) The stocks of Corg (t ha-1) were affected by fertilizer type and rate and increased in the order MSIL (23.6), MSIM (23.7), MSIH (24.2) < FYML (25.3) < FYMM (28.1), FYMH (28.1). Stocks of Nt were affected in the same way (C/N ratio: 11). Storage of C and N in the modelled labile pools (turnover times: 462 and 153 days for C and N, respectively) were not influenced by the type of fertilizer (FYM and MSI) but depended significantly (p ≤ 0.05) on the application rate and ranged from 1.8 to 3.2 t C ha 1 (7 – 13% of Corg) and from 90 to 140 kg N ha-1 (4-5% of Nt). In the calculated intermediate pool (C/N ratio 7), stocks of C were markedly higher in FYM treatments (15-18 t ha-1) compared to MSI treatments (12-14 t ha-1). This showed that differences in SOM stocks in the sandy Cambisol induced by fertilizer rate may be short-lived in case of changing management, but differences induced by fertilizer type may persist for decades. (ii) Crop yields, estimated C inputs (1.5 t ha-1 year-1) with crop residue, microbial bio¬mass C (Cmic, 118 – 150 mg kg-1), microbial biomass N (17 – 20 mg kg-1) and labile C and N pools did not differ significantly between FYM and DYN treatments. However, labile C increased linearly with application rate (R2 = 0.53) from 7 to 11% of Corg. This also applied for labile N (3.5 to 4.9% of Nt). The higher contents of Corg in DYN treatments existed since 1982, when the first sampling was conducted for all individual treatments. Contents of Corg between DYN and FYM treatments con-verged slightly since then. Furthermore, at least 30% of the difference in Corg was located in the passive pool where a treatment effect could be excluded. Therefore, the reported differences in Corg contents existed most likely since the beginning of the experiment and, as a single factor of biodynamic agriculture, application of bio-dynamic preparations had no effect on SOM stocks. (iii) Stocks of SOM, light fraction organic C (LFOC, ρ ≤ 2.0 g cm-3), light fraction organic N and Cmic decreased in the order FYMH > FYML > MSIH, MSIL for all sampling dates in 2008 (March, May, September, December). However, statistical significance of treatment effects differed between the dates, probably due to dif-ferences in the spatial variation throughout the year. The high proportion of LFOC on total Corg stocks (45 – 55%) highlighted the importance of selective preservation of OM as a stabilization mechanism in this sandy Cambisol. The apparent turnover time of LFOC was between 21 and 32 years, which agreed very well with studies with substantially longer vegetation change compared to our study. Overall, both approaches; (I) the combination of incubation, chemical fractionation and simple modelling and (II) the density fractionation; provided complementary information on the partitioning of SOM into pools of different stability. The density fractionation showed that differences in Corg stocks between FYM and MSI treatments were mainly located in the light fraction, i.e. induced by higher recalcitrance of the organic input in the FYM treatments. Moreover, the use of the combination of biological, chemical and mathematical methods indicated that effects of fertilizer rate on total Corg and Nt stocks may be short-lived, but that the effect of fertilizer type may persist for longer time spans in the sandy Cambisol.

Impact of sward type, cutting date and conditioning temperature on mass and energy flows in the integrated generation of solid fuel and biogas from semi-natural grassland

Energy production from biomass and the conservation of ecologically valuable grassland habitats are two important issues of agriculture today. The combination of a bioenergy production, which minimises environmental impacts and competition with food production for land with a conversion of semi-natural grasslands through new utilization alternatives for the biomass, led to the development of the IFBB process. Its basic principle is the separation of biomass into a liquid fraction (press fluid, PF) for the production of electric and thermal energy after anaerobic digestion to biogas and a solid fraction (press cake, PC) for the production of thermal energy through combustion. This study was undertaken to explore mass and energy flows as well as quality aspects of energy carriers within the IFBB process and determine their dependency on biomass-related and technical parameters. Two experiments were conducted, in which biomass from semi-natural grassland was conserved as silage and subjected to a hydrothermal conditioning and a subsequent mechanical dehydration with a screw press. Methane yield of the PF and the untreated silage was determined in anaerobic digestion experiments in batch fermenters at 37°C with a fermentation time of 13-15 and 27-35 days for the PF and the silage, respectively. Concentrations of dry matter (DM), ash, crude protein (CP), crude fibre (CF), ether extract (EE), neutral detergent fibre (NDF), acid detergent fibre (ADF), acid detergent ligning (ADL) and elements (K, Mg, Ca, Cl, N, S, P, C, H, N) were determined in the untreated biomass and the PC. Higher heating value (HHV) and ash softening temperature (AST) were calculated based on elemental concentration. Chemical composition of the PF and mass flows of all plant compounds into the PF were calculated. In the first experiment, biomass from five different semi-natural grassland swards (Arrhenaterion I and II, Caricion fuscae, Filipendulion ulmariae, Polygono-Trisetion) was harvested at one late sampling (19 July or 31 August) and ensiled. Each silage was subjected to three different temperature treatments (5°C, 60°C, 80°C) during hydrothermal conditioning. Based on observed methane yields and HHV as energy output parameters as well as literature-based and observed energy input parameters, energy and green house gas (GHG) balances were calculated for IFBB and two reference conversion processes, whole-crop digestion of untreated silage (WCD) and combustion of hay (CH). In the second experiment, biomass from one single semi-natural grassland sward (Arrhenaterion) was harvested at eight consecutive dates (27/04, 02/05, 09/05, 16/05, 24/05, 31/05, 11/06, 21/06) and ensiled. Each silage was subjected to six different treatments (no hydrothermal conditioning and hydrothermal conditioning at 10°C, 30°C, 50°C, 70°C, 90°C). Energy balance was calculated for IFBB and WCD. Multiple regression models were developed to predict mass flows, concentrations of elements in the PC, concentration of organic compounds in the PF and energy conversion efficiency of the IFBB process from temperature of hydrothermal conditioning as well as NDF and DM concentration in the silage. Results showed a relative reduction of ash and all elements detrimental for combustion in the PC compared to the untreated biomass of 20-90%. Reduction was highest for K and Cl and lowest for N. HHV of PC and untreated biomass were in a comparable range (17.8-19.5 MJ kg-1 DM), but AST of PC was higher (1156-1254°C). Methane yields of PF were higher compared to those of WCD when the biomass was harvested late (end of May and later) and in a comparable range when the biomass was harvested early and ranged from 332 to 458 LN kg-1 VS. Regarding energy and GHG balances, IFBB, with a net energy yield of 11.9-14.1 MWh ha-1, a conversion efficiency of 0.43-0.51, and GHG mitigation of 3.6-4.4 t CO2eq ha-1, performed better than WCD, but worse than CH. WCD produces thermal and electric energy with low efficiency, CH produces only thermal energy with a low quality solid fuel with high efficiency, IFBB produces thermal and electric energy with a solid fuel of high quality with medium efficiency. Regression models were able to predict target parameters with high accuracy (R2=0.70-0.99). The influence of increasing temperature of hydrothermal conditioning was an increase of mass flows, a decrease of element concentrations in the PC and a differing effect on energy conversion efficiency. The influence of increasing NDF concentration of the silage was a differing effect on mass flows, a decrease of element concentrations in the PC and an increase of energy conversion efficiency. The influence of increasing DM concentration of the silage was a decrease of mass flows, an increase of element concentrations in the PC and an increase of energy conversion efficiency. Based on the models an optimised IFBB process would be obtained with a medium temperature of hydrothermal conditioning (50°C), high NDF concentrations in the silage and medium DM concentrations of the silage.

Development and Integration of Biocontrol Products in Branched Broomrape (Phelipanche ramosa) management in Tomato

Parasitic weeds of the genera Striga, Orobanche, and Phelipanche pose a severe problem for agriculture because they are difficult to control and are highly destructive to several crops. The present work was carried out during the period October, 2009 to February, 2012 to evaluate the potential of arbuscular mycorrhizal fungi (AMF) to suppress P. ramosa on tomatoes and to investigate the effects of air-dried powder and aqueous extracts from Euphorbia hirta on germination and haustorium initiation in Phelipanche ramosa. The work was divided into three parts: a survey of the indigenous mycorrhizal flora in Sudan, second, laboratory and greenhouse experiments (conducted in Germany and Sudan) to construct a base for the third part, which was a field trial in Sudan. A survey was performed in 2009 in the White Nile state, Sudan to assess AMF spore densities and root colonization in nine fields planted with 13 different important agricultural crops. In addition, an attempt was made to study the relationship between soil physico-chemical properties and AMF spore density, colonization rate, species richness and other diversity indices. The mean percentage of AMF colonization was 34%, ranging from 19-50%. The spore densities (expressed as per 100 g dry soil) retrieved from the rhizosphere of different crops were relatively high, varying from 344 to 1222 with a mean of 798. There was no correlation between spore densities in soil and root colonization percentage. A total of 45 morphologically classifiable species representing ten genera of AMF were detected with no correlation between the number of species found in a soil sample and the spore density. The most abundant genus was Glomus (20 species). The AMF diversity expressed by the Shannon–Weaver index was highest in sorghum (H\= 2.27) and Jews mallow (H\= 2.13) and lowest in alfalfa (H\= 1.4). With respect to crop species, the genera Glomus and Entrophospora were encountered in almost all crops, except for Entrophospora in alfalfa. Kuklospora was found only in sugarcane and sorghum. The genus Ambispora was recovered only in mint and okra, while mint and onion were the only species on which no Acaulospora was found. The hierarchical cluster analysis based on the similarity among AMF communities with respect to crop species overall showed that species compositions were relatively similar with the highest dissimilarity of about 25% separating three of the mango samples and the four sorghum samples from all other samples. Laboratory experiments studied the influence of root and stem exudates of three tomato varieties infected by three different Glomus species on germination of P. ramosa. Root exudates were collected 21or 42 days after transplanting (DAT) and stem exudates 42 DAT and tested for their effects on germination of P. ramosa seeds in vitro. The tomato varieties studied did not have an effect on either mycorrhizal colonization or Phelipanche germination. Germination in response to exudates from 42 day old mycorrhizal plants was significantly reduced in comparison to non-mycorrhizal controls. Germination of P. ramosa in response to root exudates from 21 day old plants was consistently higher than for 42 day-old plants (F=121.6; P<.0001). Stem diffusates from non-mycorrhizal plants invariably elicited higher germination than diffusates from the corresponding mycorrhizal ones and differences were mostly statistically significant. A series of laboratory experiments was undertaken to investigate the effects of aqueous extracts from Euphorbia hirta on germination, radicle elongation, and haustorium initiation in P. ramosa. P. ramosa seeds conditioned in water and subsequently treated with diluted E. hirta extract (10-25% v/v) displayed considerable germination (47-62%). Increasing extract concentration to 50% or more reduced germination in response to the synthetic germination stimulants GR24 and Nijmegen-1 in a concentration dependent manner. P. ramosa germlings treated with diluted Euphorbia extract (10-75 % v/v) displayed haustorium initiation comparable to 2, 5-Dimethoxy-p-benzoquinon (DMBQ) at 20 µM. Euphorbia extract applied during conditioning reduced haustorium initiation in a concentration dependent manner. E. hirta extract or air-dried powder, applied to soil, induced considerable P. ramosa germination. Pot experiments were undertaken in a glasshouse at the University of Kassel, Germany, to investigate the effects of P. ramosa seed bank on tomato growth parameters. Different Phelipanche seed banks were established by mixing the parasite seeds (0 - 32 mg) with the potting medium in each pot. P. ramosa reduced all tomato growth parameters measured and the reduction progressively increased with seed bank. Root and total dry matter accumulation per tomato plant were most affected. P. ramosa emergence, number of tubercles, and tubercle dry weight increased with the seed bank and were, invariably, maximal with the highest seed bank. Another objective was to determine if different AM fungi differ in their effects on the colonization of tomatoes with P. ramosa and the performance of P. ramosa after colonization. Three AMF species viz. GIomus intraradices, Glomus mosseae and Glomus Sprint® were used in this study. For the infection, P. ramosa seeds (8 mg) were mixed with the top 5 cm soil in each pot. No mycorrhizal colonization was detected in un-inoculated control plants. P. ramosa infested, mycorrhiza inoculated tomato plants had significantly lower AMF colonization compared to plants not infested with P. ramosa. Inoculation with G. intraradices, G. mosseae and Glomus Sprint® reduced the number of emerged P. ramosa plants by 29.3, 45.3 and 62.7% and the number of tubercles by 22.2, 42 and 56.8%, respectively. Mycorrhizal root colonization was positively correlated with number of branches and total dry matter of tomatoes. Field experiments on tomato undertaken in 2010/12 were only partially successful because of insect infestations which resulted in the complete destruction of the second run of the experiment. The effects of the inoculation with AMF, the addition of 10 t ha-1 filter mud (FM), an organic residues from sugar processing and 36 or 72 kg N ha-1 on the infestation of tomatoes with P. ramosa were assessed. In un-inoculated control plants, AMF colonization ranged between 13.4 to 22.1% with no significant differences among FM and N treatments. Adding AMF or FM resulted in a significant increase of branching in the tomato plants with no additive effects. Dry weights were slightly increased through FM application when no N was applied and significantly at 36 kg N ha-1. There was no effect of FM on the time until the first Phelipanche emerged while AMF and N application interacted. Especially AMF inoculation resulted in a tendency to delayed P. ramosa emergence. The marketable yield was extremely low due to the strong fruit infestation with insects mainly whitefly Bemisia tabaci and tomato leaf miner (Tuta absoluta). Tomatoes inoculated with varied mycorrhiza species displayed different response to the insect infestation, as G. intraradices significantly reduced the infestation, while G. mosseae elicited higher insect infestation. The results of the present thesis indicate that there may be a potential of developing management strategies for P. ramosa targeting the pre-attachment stage namely germination and haustorial initiation using plant extracts. However, ways of practical use need to be developed. If such treatments can be combined with AMF inoculation also needs to be investigated. Overall, it will require a systematic approach to develop management tools that are easily applicable and affordable to Sudanese farmers. It is well-known that proper agronomical practices such as the design of an optimum crop rotation in cropping systems, reduced tillage, promotion of cover crops, the introduction of multi-microbial inoculants, and maintenance of proper phosphorus levels are advantageous if the mycorrhiza protection method is exploited against Phelipanche ramosa infestation. Without the knowledge about the biology of the parasitic weeds by the farmers and basic preventive measures such as hygiene and seed quality control no control strategy will be successful, however.

Effects of grassland renovation on carbon concentrations and aggregate distribution in temperate grassland soils

The effects of continuous tillage on the distribution of soil organic matter (SOM) and aggregates have been well studied for arable soils. However, less is known about the effects of sporadic tillage on SOM and aggregate dynamics in grassland soils. The objectives of the present thesis were (I) to study the longer-term effects of sporadic tillage of grassland on organic carbon (Corg) stocks and the distribution of aggregates and SOM, (II) to investigate the combined effects of sporadic tillage and fertilization on carbon and nitrogen dynamics in grassland soils, and (III) to study the temporal dynamics of Corg stocks, aggregate distribution and microbial biomass in grassland soils. Soil samples were taken in three soil depths (0 – 10 cm; 10 – 25 cm; 25 – 40 cm) from a field trial with loamy sandy soils (Cambisols, Eutric Luvisols, Stagnosols, Anthrosols) north of Kiel, Germany. For Objective I we have sampled soil two and five years after one or two tillage operation(s). Treatments consisted of (i) permanent grassland, (ii) tillage of grassland followed by a re-establishment of grassland and (iii) tillage of grassland followed by a re-establishment of grassland with one season of winter wheat in between. The tillage in grassland led to a reduction in Corg stocks, large macroaggregates (>2000 µm) and SOM in the top 10 cm soil depth. These findings were still significant two years after tillage; however, five years after tillage no longer present. Regarding the soil profile (0 – 40 cm) no significant differences in the mentioned parameters between the tilled plots and the permanent grassland existed. A second tillage event and the insertion of one season of winter wheat did not lead to any further effects on Corg stocks as well as aggregate and SOM concentrations in comparison with a single tillage event in these grassland soils. Treatments adapted for Objective II included (i) long-term grassland and (ii) tillage of grassland followed by a re-establishment of grassland with one season of winter wheat in between. The plots were split and received either 240 kg N ha-1 year-1 in the form of cattle slurry or no cattle slurry application. The application of slurry within a period of four years had no effects on the Corg and total nitrogen stocks or the aggregate distribution, but led to a reduction of free and not physically protected SOM. However, the application of cattle slurry and the grassland renovation seems to change the plant species composition and therefore generalizations on the direct effects are not yet possible. For studying Objective III a further field trial was initiated in September 2010. Soil samples were taken six times within one year (from October 2010 to October 2011) (i) after the conversion from arable land into grassland, (ii) after the tillage of grassland followed by a re-establishment of grassland and (iii) in a permanent grassland. We found an increase in the microbial and fungal biomass after the conversion of arable land into grassland, but no effect on aggregate distribution and Corg stocks. A one-time tillage operation in grassland led to a reduction in large macroaggregates and Corg stocks in the top 10 cm soil depth with no effect on the sampled soil profile. However, we found large variations in the fungal biomass and aggregate distribution within one year in the permanent grassland, presumably caused by environmental factors. Overall, our results suggest that a single tillage operation in grassland soils markedly decreased the concentrations of Corg, larger aggregates and SOM. However, this does not result in long-lasting effects on the above mentioned parameters. The application of slurry cannot compensate the negative effects of a tillage event on aggregate concentrations or Corg stocks. However, while the Corg concentration is not subject to fluctuations within a year, there are large variations of the aggregate distribution even in a permanent grassland soil. Therefore conclusions of results from a single sampling time should be handled with care.

Anàlisi de la capacitat de regeneració en estadis inicials del desenvolupament en diverses espècies mediterrànies del gènere Quercus

Aquest treball es centra en l'estudi de la regeneració vegetativa en els estadis inicials del desenvolupament de Quercus coccifera, Q. humilis, Q. ilex i Q. suber, analitzant la capacitat de rebrotada després de l'eliminació de la biomassa aèria. S'ha realitzat una descripció, a nivell histològic, de l'ontogènesi del nus cotiledonar des de l'embrió fins a la plàntula de mig any d'edat de Q. coccifera, Q. humilis i Q. ilex. Així mateix, s'ha analitzat el contingut de midó i de nitrogen a la primavera, l'estiu i l'hivern en diferents parts de les plàntules de les quatre espècies, avaluant, alhora, l'efecte de la tala. D'altra banda, també s'ha estudiat la biometria de les glans, la capacitat de germinació i el creixement de les plàntules de les quatre espècies de Quercus. A partir dels resultats s'ha comprovat que les glans de Q. suber, en general, són més grans, tant en diàmetre com en longitud, i tenen un pes superior, mentre que les de Q. ilex són més petites i pesen menys. En les quatre espècies estudiades, el pes de la llavor està determinat per la longitud i el diàmetre, si bé, també hi ha un cert efecte de l'espècie. Amb les dades obtingudes de les tres variables de la gla s'ha calculat una funció discriminant a partir de la qual es pot determinar, amb un alt grau d'encert, a quina espècie pertany una determinada gla coneixent la mida i el pes. En relació a la capacitat de germinació, en Q. coccifera i Q. ilex la germinació depèn del pes de la gla, mentre que en Q. humilis i Q. suber és independent. Així mateix, també s'ha comprovat que el pes de la llavor afecta positivament en el creixement de les plàntules de tres i sis mesos d'edat. Possiblement un major pes de la gla implica un contingut de substàncies de reserva més elevat, i per tant representa un major subministrament de nutrients cap a la planta, repercutint així en el seu creixement inicial. En plàntules de nou mesos d'edat ja no s'ha trobat relació entre el pes de la gla i el creixement de la planta, la qual cosa es podria explicar pel fet que les substàncies de reserva de la llavor s'han esgotat. Pel que fa al creixement de la plàntula, les plàntules de Q. ilex han tingut la major taxa de creixement per quasi totes les variables estudiades. En aquesta espècie, al principi de l'experiment la grandària de les plàntules ha estat baixa, però al final han crescut més que les de les altres espècies. Les plàntules de Q. coccifera han presentat la menor taxa. En les plàntules de les quatre espècies s'ha constatat, també, que la biomassa del sistema radicular és superior a la de l'aeri. Les plàntules de Q. coccifera i Q. humilis han tingut una relació BA/BR (biomassa aèria/biomassa radicular) més baixa que les de Q. ilex i Q. suber. En espècies mediterrànies s'ha relacionat sovint una baixa relació BA/BR com una adaptació a la sequera. No obstant això, Q. humilis és una espècie que viu en zones més humides que la resta d'espècies estudiades. En referència a la regeneració vegetativa, amb aquest estudi es demostra que les plàntules de les quatre espècies tenen una elevada capacitat d'emissió de rebrots, quan s'elimina la biomassa aèria per sobre la zona d'inserció dels cotilèdons. Tanmateix, el grau de supervivència difereix segons l'espècie i la intervenció. Així, per exemple, les plàntules de Q. ilex han presentat una major mortalitat tant en la primera com en la segona tala, mentre que en Q. humilis i Q. suber la supervivència de les plàntules ha disminuït després de talar dues vegades. En el cas de Q. coccifera el grau de supervivència és bastant similar tant en la primera com en la segona intervenció. La tala successiva afecta negativament al vigor dels rebrots en Q. coccifera, Q. humilis i Q. ilex. Ara bé, en el cas de Q. suber s'ha trobat que els rebrots emesos després de talar dues vegades han estat més grans que els obtinguts després d'una sola tala. Després de tallar la biomassa aèria per sobre la zona d'inserció dels cotilèdons, els rebrots s'originen a partir de les gemmes del nus cotiledonar. L'estudi de l'ontogènesi del nus cotiledonar ens ha permès de constatar que el patró de desenvolupament d'aquest en Q. coccifera, Q. humilis i Q. ilex és similar, però difereix del descrit per a Q. suber. En les tres primeres espècies el nus cotiledonar pràcticament no s'allarga i només es desenvolupen gemmes cotiledonars just en l'axil·la del pecíol cotiledonar. En aquest sentit, cal ressaltar que en el cas de Q. coccifera i Q. ilex les gemmes es formen després de germinar la gla, mentre que en Q. humilis i Q. suber són ja presents en l'embrió. Tal i com també s'ha descrit en Q. suber, en l'estadi de plàntula, en Q. coccifera, Q. humilis i Q. ilex a l'axil·la de la gemma cotiledonar proliferen noves gemmes, de tal manera que es formen plaques de gemmes. Les anàlisis del contingut de midó han permès de determinar que el sistema radicular de les plàntules de Q. coccifera, Q. humilis, Q. ilex i Q. suber conté aproximadament un 90% del midó de tota la planta. Concretament de les diferents fraccions del sistema radicular (nus cotiledonar, 11 pimers centímetres de l'arrel i resta de l'arrel) la major concentració de midó es troba ens els 11 primers cm de l'arrel, que en el cas de Q. suber correspon al nus cotiledonar. Quant a les estacions, a l'estiu la concentració de midó de la part aèria i radicular disminueix en Q. coccifera, Q. humilis i Q. ilex, si bé es recuperen a l'hivern. En canvi en el cas de Q. suber els nivells més baixos de midó s'han obtingut a l'hivern. La tala provoca una disminució dels nivells de midó, ja que una part d'aquest és mobilitzat per a la síntesi dels nous brots. En les quatre espècies la major proporció de midó mobilitzat és en els primers 11 cm de l'arrel, és a dir, el nus cotiledonar en el cas de Q. suber. Per tant, amb aquest estudi es reafirma que el nus cotiledonar de Q. suber és un lignotúber. Tanmateix, Q. coccifera, Q. humilis i Q. ilex no presenten cap lignotúber o estructura especialitzada en la rebrotada, si bé el fet de tenir gemmes i substàncies de reserva els confereix igualment una elevada capacitat per rebrotar. En relació al nitrogen, en les quatre espècies el sistema radicular presenta aproximadament el 70% del nitrogen total de la planta. A l'estiu, la concentració de nitrogen del sistema radicular de Q. humilis i Q. suber és més baixa que a l'hivern, mentre que en Q. coccifera i Q. ilex els valors són bastant similars en ambdues estacions. La tala no provoca una davallada dels nivells de midó, això possiblement es degui a que les plàntules van rebre continuament un aport de nitrogen a través de l'aigua de reg.

The role of fungi and bacteria on the organic matter decomposition process in streams: interaction and relevance in biofilms

L'objectiu d'aquest estudi és el d'investigar sobre l'ús de matèria orgànica per part dels fongs i bacteris que colonitzen diferents substrats bentònics en rius Mediterranis i analitzar l'efecte dels factors ambientals i antròpics sobre l'estabilitat estructural i funcional de les comunitats del biofilm. La metodologia emprada en aquest estudi consisteix en: i) anàlisi de la biomassa bacteriana i fúngica, ii) anàlisi de la composició de les comunitats bentòniques (identificació d'hifomicets aquàtics i anàlisi del 16S rDNA bacterià), i iii) anàlisi de l'activitat enzimàtica extracel·lular relacionada amb el reciclatge de matèria orgànica en rius.