Trading nitrogen for carbon: Nitrogen and carbon translocation in a plant/fungal (Metarhizium spp.) symbiosis

While nitrogen is critical for all plants, they are unable to utilize organically bound nitrogen in soils. Therefore, the majority of plants obtain useable nitrogen through nitrogen fixing bacteria and the microbial decomposition of organic matter. In the majority of cases, symbiotic microorganisms directly furnish plant roots with inorganic forms of nitrogen. More than 80% of all land plants form intimate symbiotic relationships with root colonizing fungi. These common plant/fungal interactions have been defined largely through nutrient exchange, where the plant receives limiting soil nutrients, such as nitrogen, in exchange for plant derived carbon. Fungal endophytes are common plant colonizers. A number of these fungal species have a dual life cycle, meaning that they are not solely plant colonizers, but also saprophytes, insect pathogens, or plant pathogens. By using 15N labeled, Metarhizium infected, wax moth larvae (Galleria mellonella) in soil microcosms, I demonstrated that the common endophytic, insect pathogenic fungi Metarhizium spp. are able to infect living soil borne insects, and subsequently colonize plant roots and furnish ts plant host with useable, insect-derived nitrogen. In addition, I showed that another ecologically important, endophytic, insect pathogenic fungi, Beauveria bassiana, is able to transfer insect-derived nitrogen to its plant host. I demonstrated that these relationships between various plant species and endophytic, insect pathogenic fungi help to improve overall plant health. By using 13C-labeled CO2, added to airtight plant growth chambers, coupled with nuclear magnetic resosnance spectroscopy, I was able to track the movement of carbon from the atmosphere, into the plant, and finally into the root colonized fungal biomass. This indicates that Metarhizium exists in a symbiotic partnership with plants, where insect nitrogen is exchanged for plant carbon. Overall these studies provide the first evidence of nutrient exchange between an insect pathogenic fungus and plants, a relationship that has potentially useful implications on plant primary production, soil health, and overall ecosystem stability.

Molecular biodiversity of microbial communities in polluted soils and their role in soil phytoremediation

Les métaux lourds (ML) s’accumulent de plus en plus dans les sols à l’échelle mondiale, d’une part à cause des engrais minéraux et divers produits chimiques utilisés en agriculture intensive, et d’autre part à cause des activités industrielles. Toutes ces activités génèrent des déchets toxiques qui s’accumulent dans l’environnement. Les ML ne sont pas biodégradables et leur accumulation cause donc des problèmes de toxicité des sols et affecte la biodiversité des microorganismes qui y vivent. La fertilisation en azote (N) est une pratique courante en agriculture à grande échelle qui permet d’augmenter la fertilité des sols et la productivité des cultures. Cependant, son utilisation à long terme cause plusieurs effets néfastes pour l'environnement. Par exemple, elle augmente la quantité des ML dans les sols, les nappes phréatiques et les plantes. En outre, ces effets néfastes réduisent et changent considérablement la biodiversité des écosystèmes terrestres. La structure des communautés des champignons mycorhiziens à arbuscules (CMA) a été étudiée dans des sols contaminés par des ML issus de la fertilisation à long terme en N. Le rôle des différentes espèces de CMA dans l'absorption et la séquestration des ML a été aussi investigué. Dans une première expérience, la structure des communautés de CMA a été analysée à partir d’échantillons de sols de sites contaminés par des ML et de sites témoins non-contaminés. Nous avons constaté que la diversité des CMA indigènes a été plus faible dans les sols et les racines des plantes récoltées à partir de sites contaminés par rapport aux sites noncontaminés. Nous avons également constaté que la structure de la communauté d'AMF a été modifiée par la présence des ML dans les sols. Certains ribotypes des CMA ont été plus souvent associés aux sites contaminés, alors que d’autres ribotypes ont été associés aux sites non-contaminés. Cependant, certains ribotypes ont été observés aussi bien dans les sols pollués que non-pollués. Dans une deuxième expérience, les effets de la fertilisation organique et minérale (N) sur les différentes structures des communautés des CMA ont été étudiés. La variation de la structure de la communauté de CMA colonisant les racines a été analysée en fonction du type de fertilisation. Certains ribotypes de CMA étaient associés à la fertilisation organique et d'autres à la fertilisation minérale. En revanche, la fertilisation minérale a réduit le nombre de ribotypes de CMA alors que la fertilisation organique l’a augmenté. Dans cette expérience, j’ai démontré que le changement de structure des communautés de CMA colonisant des racines a eu un effet significatif sur la productivité des plantes. Dans une troisième expérience, le rôle de deux espèces de CMA (Glomus irregulare et G. mosseae) dans l'absorption du cadmium (Cd) par des plants de tournesol cultivés dans des sols amendés avec trois niveaux différents de Cd a été évalué. J’ai démontré que les deux espèces de CMA affectent différemment l’absorption ou la séquestration de ce ML par les plants de tournesol. Cette expérience a permis de mieux comprendre le rôle potentiel des CMA dans l'absorption des ML selon la concentration de cadmium dans le sol et les espèces de CMA. Mes recherches de doctorat démontrent donc que la fertilisation en N affecte la structure des communautés des CMA dans les racines et le sol. Le changement de structure de la communauté de CMA colonisant les racines affecte de manière significative la productivité des plantes. J’ai aussi démontré que, sous nos conditions expériemntales, l’espèce de CMA G. irregulare a été observée dans tous les sites (pollués et non-pollués), tandis que le G. mosseae n’a été observé en abondance que dans les sites contaminés. Par conséquent, j’ai étudié le rôle de ces deux espèces (G. irregulare et G. mosseae) dans l'absorption du Cd par le tournesol cultivé dans des sols amendés avec trois différents niveaux de Cd en serre. Les résultats indiquent que les espèces de CMA ont un potentiel différent pour atténuer la toxicité des ML dans les plantes hôtes, selon le niveau de concentration en Cd. En conclusion, mes travaux suggèrent que le G. irregulare est une espèce potentiellement importante pour la phytoextration du Cd, alors que le G. mosseae pourrait être une espèce appropriée pour phytostabilisation du Cd et du Zn.

Improving the microbial production of biofuels through metabolic engineering

Les défis conjoints du changement climatique d'origine anthropique et la diminution des réserves de combustibles fossiles sont le moteur de recherche intense pour des sources d'énergie alternatives. Une avenue attrayante est d'utiliser un processus biologique pour produire un biocarburant. Parmi les différentes options en matière de biocarburants, le bio-hydrogène gazeux est un futur vecteur énergétique attrayant en raison de son efficacité potentiellement plus élevé de conversion de puissance utilisable, il est faible en génération inexistante de polluants et de haute densité d'énergie. Cependant, les faibles rendements et taux de production ont été les principaux obstacles à l'application pratique des technologies de bio-hydrogène. Des recherches intensives sur bio-hydrogène sont en cours, et dans les dernières années, plusieurs nouvelles approches ont été proposées et étudiées pour dépasser ces inconvénients. À cette fin, l'objectif principal de cette thèse était d'améliorer le rendement en hydrogène moléculaire avec un accent particulier sur l'ingénierie métabolique et l’utilisation de bioprocédés à variables indépendantes. Une de nos hypothèses était que la production d’hydrogène pourrait être améliorée et rendue plus économiquement viable par ingénierie métabolique de souches d’Escherichia coli producteurs d’hydrogène en utilisant le glucose ainsi que diverses autres sources de carbone, y compris les pentoses. Les effets du pH, de la température et de sources de carbone ont été étudiés. La production maximale d'hydrogène a été obtenue à partir de glucose, à un pH initial de 6.5 et une température de 35°C. Les études de cinétiques de croissance ont montré que la μmax était 0.0495 h-1 avec un Ks de 0.0274 g L-1 lorsque le glucose est la seule source de carbone en milieu minimal M9. .Parmi les nombreux sucres et les dérivés de sucres testés, les rendements les plus élevés d'hydrogène sont avec du fructose, sorbitol et D-glucose; 1.27, 1.46 et 1.51 mol H2 mol-1 de substrat, respectivement. En outre, pour obtenir les interactions entre les variables importantes et pour atteindre une production maximale d'hydrogène, un design 3K factoriel complet Box-Behnken et la méthodologie de réponse de surface (RSM) ont été employées pour la conception expérimentale et l'analyse de la souche d'Escherichia coli DJT135. Le rendement en hydrogène molaire maximale de 1.69 mol H2 mol-1 de glucose a été obtenu dans les conditions optimales de 75 mM de glucose, à 35°C et un pH de 6.5. Ainsi, la RSM avec un design Box-Behken était un outil statistique utile pour atteindre des rendements plus élevés d'hydrogène molaires par des organismes modifiés génétiquement. Ensuite, l'expression hétérologue de l’hydrogénases soluble [Ni-Fe] de Ralstonia eutropha H16 (l'hydrogénase SH) a tenté de démontrer que la mise en place d'une voie capable de dériver l'hydrogène à partir de NADH pourrait surpasser le rendement stoechiométrique en hydrogène.. L’expression a été démontrée par des tests in vitro de l'activité enzymatique. Par ailleurs, l'expression de SH a restaurée la croissance en anaérobie de souches mutantes pour adhE, normalement inhibées en raison de l'incapacité de réoxyder le NADH. La mesure de la production d'hydrogène in vivo a montré que plusieurs souches modifiées métaboliquement sont capables d'utiliser l'hydrogénase SH pour dériver deux moles d’hydrogène par mole de glucose consommé, proche du maximum théorique. Une autre stratégie a montré que le glycérol brut pourrait être converti en hydrogène par photofermentation utilisant Rhodopseudomonas palustris par photofermentation. Les effets de la source d'azote et de différentes concentrations de glycérol brut sur ce processus ont été évalués. À 20 mM de glycérol, 4 mM glutamate, 6.1 mol hydrogène / mole de glycérol brut ont été obtenus dans des conditions optimales, un rendement de 87% de la théorie, et significativement plus élevés que ce qui a été réalisé auparavant. En prolongement de cette étude, l'optimisation des paramètres a également été utilisée. Dans des conditions optimales, une intensité lumineuse de 175 W/m2, 30 mM glycérol et 4.5 mM de glutamate, 6.69 mol hydrogène / mole de glycérol brut ont été obtenus, soit un rendement de 96% de la valeur théorique. La détermination de l'activité de la nitrogénase et ses niveaux d'expression ont montré qu'il y avait relativement peu de variation de la quantité de nitrogénase avec le changement des variables alors que l'activité de la nitrogénase variait considérablement, avec une activité maximale (228 nmol de C2H4/ml/min) au point central optimal. Dans la dernière section, la production d'hydrogène à partir du glucose via la photofermentation en une seule étape a été examinée avec la bactérie photosynthétique Rhodobacter capsulatus JP91 (hup-). La méthodologie de surface de réponse avec Box-Behnken a été utilisée pour optimiser les variables expérimentales de façon indépendante, soit la concentration de glucose, la concentration du glutamate et l'intensité lumineuse, ainsi que d'examiner leurs effets interactifs pour la maximisation du rendement en hydrogène moléculaire. Dans des conditions optimales, avec une intensité lumineuse de 175 W/m2, 35 mM de glucose, et 4.5 mM de glutamate,, un rendement maximal d'hydrogène de 5.5 (± 0.15) mol hydrogène /mol glucose, et un maximum d'activité de la nitrogénase de 246 (± 3.5) nmol C2H4/ml/min ont été obtenus. L'analyse densitométrique de l'expression de la protéine-Fe nitrogenase dans les différentes conditions a montré une variation significative de l'expression protéique avec un maximum au point central optimisé. Même dans des conditions optimales pour la production d'hydrogène, une fraction significative de la protéine Fe a été trouvée dans l'état ADP-ribosylée, suggérant que d'autres améliorations des rendements pourraient être possibles. À cette fin, un mutant amtB dérivé de Rhodobacter capsulatus JP91 (hup-) a été créé en utilisant le vecteur de suicide pSUP202. Les résultats expérimentaux préliminaires montrent que la souche nouvellement conçue métaboliquement, R. capsulatus DG9, produit 8.2 (± 0.06) mol hydrogène / mole de glucose dans des conditions optimales de cultures discontinues (intensité lumineuse, 175 W/m2, 35 mM de glucose et 4.5 mM glutamate). Le statut d'ADP-ribosylation de la nitrogénase-protéine Fe a été obtenu par Western Blot pour la souche R. capsulatus DG9. En bref, la production d'hydrogène est limitée par une barrière métabolique. La principale barrière métabolique est due au manque d'outils moléculaires possibles pour atteindre ou dépasser le rendement stochiométrique en bio-hydrogène depuis les dernières décennies en utilisant les microbes. À cette fin, une nouvelle approche d’ingénierie métabolique semble très prometteuse pour surmonter cette contrainte vers l'industrialisation et s'assurer de la faisabilité de la technologie de la production d'hydrogène. Dans la présente étude, il a été démontré que l’ingénierie métabolique de bactéries anaérobiques facultatives (Escherichia coli) et de bactéries anaérobiques photosynthétiques (Rhodobacter capsulatus et Rhodopseudomonas palustris) peuvent produire de l'hydrogène en tant que produit majeur à travers le mode de fermentation par redirection métabolique vers la production d'énergie potentielle. D'autre part, la méthodologie de surface de réponse utilisée dans cette étude représente un outil potentiel pour optimiser la production d'hydrogène en générant des informations appropriées concernant la corrélation entre les variables et des producteurs de bio-de hydrogène modifiés par ingénierie métabolique. Ainsi, un outil d'optimisation des paramètres représente une nouvelle avenue pour faire un pont entre le laboratoire et la production d'hydrogène à l'échelle industrielle en fournissant un modèle mathématique potentiel pour intensifier la production de bio-hydrogène. Par conséquent, il a été clairement mis en évidence dans ce projet que l'effort combiné de l'ingénierie métabolique et la méthodologie de surface de réponse peut rendre la technologie de production de bio-hydrogène potentiellement possible vers sa commercialisation dans un avenir rapproché.

Carbon/Nitrogen Ratio Optimization and Periphyton Development on the Production and Sustainability of Penaeus Monodon(Fabricius)in Extensive Culture System

Controlling the inorganic nitrogen by manipulating carbon / nitrogen ratio is a method gaining importance in aquaculture systems. Nitrogen control is induced by feeding bacteria with carbohydrates and through the subsequent uptake of nitrogen from the water for the synthesis of microbial proteins. The relationship between addition of carbohydrates, reduction of ammonium and the production of microbial protein depends on the microbial conversion coefficient. The carbon / nitrogen ratio in the microbial biomass is related to the carbon contents of the added material. The addition of carbonaceous substrate was found to reduce inorganic nitrogen in shrimp culture ponds and the resultant microbial proteins are taken up by shrimps. Thus, part of the feed protein is replaced and feeding costs are reduced in culture systems.The use of various locally available substrates for periphyton based aquaculture practices increases production and profitability .However, these techniques for extensive shrimp farming have not so far been evaluated. Moreover, an evaluation of artificial substrates together with carbohydrate source based farming system in reducing inorganic nitrogen production in culture systems has not yet been carried-out. Furthermore, variations in water and soil quality, periphyton production and shrimp production of the whole system have also not been determined so-far.This thesis starts with a general introduction , a brief review of the most relevant literature, results of various experiments and concludes with a summary (Chapter — 9). The chapters are organised conforming to the objectives of the present study. The major objectives of this thesis are, to improve the sustainability of shrimp farming by carbohydrate addition and periphyton substrate based shrimp production and to improve the nutrient utilisation in aquaculture systems.

Spatial and Temporal Variation of Microbial Population in the Grassland Soils of Tropical Montane Forest: Influence of Soil Physico-Chemical Characteristics and Nutrients

Present study is focused on the spatiotemporal variation of the microbial population (bacteria, fungus and actinomycetes) in the grassland soils of tropical montane forest and its relation with important soil physico-chemical characteristics and nutrients. Different physico-chemical properties of the soil such as temperature, moisture content, organic carbon, available nitrogen, available phosphorous and available potassium have been studied. Results of the present study revealed that both microbial load and soil characteristics showed spatiotemporal variation. Microbial population of the grassland soils were characterized by high load of bacteria followed by fungus and actinomycetes. Microbial load was high during pre monsoon season, followed by post monsoon and monsoon. The microbial load varied with important soil physico-chemical properties and nutrients. Organic carbon content, available nitrogen and available phosphorous were positively correlated with bacterial load and the correlation is significant at 0.05 and 0.01 levels respectively. Available nitrogen and available phosphorous were positively correlated with fungus at 0.05 level significance. Moisture content was negatively correlated with actinomycetes at 0.01 level of significance. Organic carbon negatively correlated with actinomycetes load at 0.05 level of significance

Salinity-induced changes in the microbial use of sugarcane filter cake added to soil

Five laboratory incubation experiments were carried out to assess the salinity-induced changes in the microbial use of sugarcane filter cake added to soil. The first laboratory experiment was carried out to prove the hypothesis that the lower content of fungal biomass in a saline soil reduces the decomposition of a complex organic substrate in comparison to a non-saline soil under acidic conditions. Three different rates (0.5, 1.0, and 2.0%) of sugarcane filter cake were added to both soils and incubated for 63 days at 30°C. In the saline control soil without amendment, cumulative CO2 production was 70% greater than in the corresponding non-saline control soil, but the formation of inorganic N did not differ between these two soils. However, nitrification was inhibited in the saline soil. The increase in cumulative CO2 production by adding filter cake was similar in both soils, corresponding to 29% of the filter cake C at all three addition rates. Also the increases in microbial biomass C and biomass N were linearly related to the amount of filter cake added, but this increase was slightly higher for both properties in the saline soil. In contrast to microbial biomass, the absolute increase in ergosterol content in the saline soil was on average only half that in the non-saline soil and it showed also strong temporal changes during the incubation: A strong initial increase after adding the filter cake was followed by a rapid decline. The addition of filter cake led to immobilisation of inorganic N in both soils. This immobilisation was not expected, because the total C-to-total N ratio of the filter cake was below 13 and the organic C-to-organic N ratio in the 0.5 M K2SO4 extract of this material was even lower at 9.2. The immobilisation was considerably higher in the saline soil than in the non-saline soil. The N immobilisation capacity of sugarcane filter cake should be considered when this material is applied to arable sites at high rations. The second incubation experiment was carried out to examine the N immobilizing effect of sugarcane filter cake (C/N ratio of 12.4) and to investigate whether mixing it with compost (C/N ratio of 10.5) has any synergistic effects on C and N mineralization after incorporation into the soil. Approximately 19% of the compost C added and 37% of the filter cake C were evolved as CO2, assuming that the amendments had no effects on the decomposition of soil organic C. However, only 28% of the added filter cake was lost according to the total C and d13C values. Filter cake and compost contained initially significant concentrations of inorganic N, which was nearly completely immobilized between day 7 and 14 of the incubation in most cases. After day 14, N re-mineralization occurred at an average rate of 0.73 µg N g-1 soil d-1 in most amendment treatments, paralleling the N mineralization rate of the non-amended control without significant difference. No significant net N mineralization from the amendment N occurred in any of the amendment treatments in comparison to the control. The addition of compost and filter cake resulted in a linear increase in microbial biomass C with increasing amounts of C added. This increase was not affected by differences in substrate quality, especially the three times larger content of K2SO4 extractable organic C in the sugarcane filter cake. In most amendment treatments, microbial biomass C and biomass N increased until the end of the incubation. No synergistic effects could be observed in the mixture treatments of compost and sugarcane filter cake. The third 42-day incubation experiment was conducted to answer the questions whether the decomposition of sugarcane filter cake also result in immobilization of nitrogen in a saline alkaline soil and whether the mixing of sugarcane filter cake with glucose (adjusted to a C/N ratio of 12.5 with (NH4)2SO4) change its decomposition. The relative percentage CO2 evolved increased from 35% of the added C in the pure 0.5% filter cake treatment to 41% in the 0.5% filter cake +0.25% glucose treatment to 48% in the 0.5% filter cake +0.5% glucose treatment. The three different amendment treatments led to immediate increases in microbial biomass C and biomass N within 6 h that persisted only in the pure filter cake treatment until the end of the incubation. The fungal cell-membrane component ergosterol showed initially an over-proportionate increase in relation to microbial biomass C that fully disappeared at the end of the incubation. The cellulase activity showed a 5-fold increase after filter cake addition, which was not further increased by the additional glucose amendment. The cellulase activity showed an exponential decline to values around 4% of the initial value in all treatments. The amount of inorganic N immobilized from day 0 to day 14 increased with increasing amount of C added in comparison to the control treatment. Since day 14, the immobilized N was re-mineralized at rates between 1.31 and 1.51 µg N g-1 soil d-1 in the amendment treatments and was thus more than doubled in comparison with the control treatment. This means that the re-mineralization rate is independent from the actual size of the microbial residues pool and also independent from the size of the soil microbial biomass. Other unknown soil properties seem to form a soil-specific gate for the release of inorganic N. The fourth incubation experiment was carried out with the objective of assessing the effects of salt additions containing different anions (Cl-, SO42-, HCO3-) on the microbial use of sugarcane filter cake and dhancha leaves amended to inoculated sterile quartz sand. In the subsequent fifth experiment, the objective was to assess the effects of inoculum and temperature on the decomposition of sugar cane filter cake. In the fourth experiment, sugarcane filter cake led to significantly lower respiration rates, lower contents of extractable C and N, and lower contents of microbial biomass C and N than dhancha leaves, but to a higher respiratory quotient RQ and to a higher content of the fungal biomarker ergosterol. The RQ was significantly increased after salt addition, when comparing the average of all salinity treatments with the control. Differences in anion composition had no clear effects on the RQ values. In experiment 2, the rise in temperature from 20 to 40°C increased the CO2 production rate by a factor of 1.6, the O2 consumption rate by a factor of 1.9 and the ergosterol content by 60%. In contrast, the contents of microbial biomass N decreased by 60% and the RQ by 13%. The effects of the inoculation with a saline soil were in most cases negative and did not indicate a better adaptation of these organisms to salinity. The general effects of anion composition on microbial biomass and activity indices were small and inconsistent. Only the fraction of 0.5 M K2SO4 extractable C and N in non-fumigated soil was consistently increased in the 1.2 M NaHCO3 treatment of both experiments. In contrast to the small salinity effects, the quality of the substrate has overwhelming effects on microbial biomass and activity indices, especially on the fungal part of the microbial community.

Application of biogas slurries from energy crops to arable soils and their impact on carbon and nitrogen dynamics

The use of renewable primary products as co-substrate or single substrate for biogas production has increased consistently over the last few years. Maize silage is the preferential energy crop used for fermentation due to its high methane (CH4) yield per hectare. Equally, the by-product, namely biogas slurry (BS), is used with increasing frequency as organic fertilizer to return nutrients to the soil and to maintain or increase the organic matter stocks and soil fertility. Studies concerning the application of energy crop-derived BS on the carbon (C) and nitrogen (N) mineralization dynamics are scarce. Thus, this thesis focused on the following objectives: I) The determination of the effects caused by rainfall patterns on the C and N dynamics from two contrasting organic fertilizers, namely BS from maize silage and composted cattle manure (CM), by monitoring emissions of nitrous oxide (N2O), carbon dioxide (CO2) and CH4 as well as leaching losses of C and N. II) The investigation of the impact of differences in soil moisture content after the application of BS and temperature on gaseous emissions (CO2, N2O and CH4) and leaching of C and N compounds. III) A comparison of BS properties obtained from biogas plants with different substrate inputs and operating parameters and their effect on C and N dynamics after application to differently textured soils with varying application rates and water contents. For the objectives I) and II) two experiments (experiment I and II) using undisturbed soil cores of a Haplic Luvisol were carried out. Objective III) was studied on a third experiment (experiment III) with disturbed soil samples. During experiment I three rainfall patterns were implemented including constant irrigation, continuous irrigation with periodic heavy rainfall events, and partial drying with rewetting periods. Biogas slurry and CM were applied at a rate of 100 kg N ha-1. During experiment II constant irrigation and an irrigation pattern with partial drying with rewetting periods were carried out at 13.5°C and 23.5°C. The application of BS took place either directly before a rewetting period or one week after the rewetting period stopped. Experiment III included two soils of different texture which were mixed with ten BS’s originating from ten different biogas plants. Treatments included low, medium and high BS-N application rates and water contents ranging from 50% to 100% of water holding capacity (WHC). Experiment I and II showed that after the application of BS cumulative N2O emissions were 4 times (162 mg N2O-N m-2) higher compared to the application of CM caused by a higher content of mineral N (Nmin) in the form of ammonium (NH4+) in the BS. The cumulative emissions of CO2, however, were on the same level for both fertilizers indicating similar amounts of readily available C after composting and fermentation of organic material. Leaching losses occurred predominantly in the mineral form of nitrate (NO3-) and were higher in BS amended soils (9 mg NO3--N m-2) compared to CM amended soils (5 mg NO3--N m-2). The rainfall pattern in experiment I and II merely affected the temporal production of C and N emissions resulting in reduced CO2 and enhanced N2O emissions during stronger irrigation events, but showed no effect on the cumulative emissions. Overall, a significant increase of CH4 consumption under inconstant irrigation was found. The time of fertilization had no effect on the overall C and N dynamics. Increasing temperature from 13.5°C to 23.5°C enhanced the CO2 and N2O emissions by a factor of 1.7 and 3.7, respectively. Due to the increased microbial activity with increasing temperature soil respiration was enhanced. This led to decreasing oxygen (O2) contents which in turn promoted denitrification in soil due to the extension of anaerobic microsites. Leaching losses of NO3- were also significantly affected by increasing temperature whereas the consumption of CH4 was not affected. The third experiment showed that the input materials of biogas plants affected the properties of the resulting BS. In particular the contents of DM and NH4+ were determined by the amount of added plant biomass and excrement-based biomass, respectively. Correlations between BS properties and CO2 or N2O emissions were not detected. Solely the ammonia (NH3) emissions showed a positive correlation with NH4+ content in BS as well as a negative correlation with the total C (Ct) content. The BS-N application rates affected the relative CO2 emissions (% of C supplied with BS) when applied to silty soil as well as the relative N2O emissions (% of N supplied with BS) when applied to sandy soil. The impacts on the C and N dynamics induced by BS application were exceeded by the differences induced by soil texture. Presumably, due to the higher clay content in silty soils, organic matter was stabilized by organo-mineral interactions and NH4+ was adsorbed at the cation exchange sites. Different water contents induced highest CO2 emissions and therefore optimal conditions for microbial activity at 75% of WHC in both soils. Cumulative nitrification was also highest at 75% and 50% of WHC whereas the relative N2O emissions increased with water content and showed higher N2O losses in sandy soils. In summary it can be stated that the findings of the present thesis confirmed the high fertilizer value of BS’s, caused by high concentrations of NH4+ and labile organic compounds such as readily available carbon. These attributes of BS’s are to a great extent independent of the input materials of biogas plants. However, considerably gaseous and leaching losses of N may occur especially at high moisture contents. The emissions of N2O after field application corresponded with those of animal slurries.

Nitrogen and carbon dynamics in grassland soils and plants after application of digestate

A better understanding of effects after digestate application on plant community, soil microbial community as well as nutrient and carbon dynamics is crucial for a sustainable grassland management and the prevention of species and functional diversity loss. The specific research objectives of the thesis were: (i) to investigate effects after digestate application on grass species and soil microbial community, especially focussing on nitrogen dynamic in the plant-soil system and to examine the suitability of the digestate from the “integrated generation of solid fuel and biogas from biomass” (IFBB) system as fertilizer (Chapter 3). (ii) to investigate the relationship between plant community and functionality of soil microbial community of extensively managed meadows, taking into account temporal variations during the vegetation period and abiotic soil conditions (Chapter 4). (iii) to investigate the suitability of IFBB-concept implementation as grassland conservation measure for meadows and possible associated effects of IFBB digestate application on plant and soil microbial community as well as soil microbial substrate utilization and catabolic evenness (Chapter 5). Taken together the results indicate that the digestate generated during the IFBB process stands out from digestates of conventional whole crop digestion on the basis of higher nitrogen use efficiency and that it is useful for increasing harvestable biomass and the nitrogen content of the biomass, especially of L. perenne, which is a common species of intensively used grasslands. Further, a medium application rate of IFBB digestate (50% of nitrogen removed with harvested biomass, corresponding to 30 50 kg N ha-1 a-1) may be a possibility for conservation management of different meadows without changing the functional above- and belowground characteristic of the grasslands, thereby offering an ecologically worthwhile alternative to mulching. Overall, the soil microbial biomass and catabolic performance under planted soil was marginally affected by digestate application but rather by soil properties and partly by grassland species and legume occurrence. The investigated extensively managed meadows revealed a high soil catabolic evenness, which was resilient to medium IFBB application rate after a three-year period of application.

Microbial response to restoration of tantalite mine soils in western Rwanda

Artisanal columbite-tantalite (coltan) mining has had negative effects on the rural economy in the great Lakes region of Africa through labor deficits, degradation and loss of farmland, food insecurity, high cost of living, and reduced traditional export crop production alongside secondary impacts that remotely affect the quality of air, water, soil, plants, animals, and human wellbeing. The situation is multifaceted and calls for a holistic approach for short and long-term mitigation of such negative effects. This study focuses on the effects of mine land restoration on soil microbiological quality in the Gatumba Mining District of western Rwanda. Some coltan mine wastelands were afforested with pine and eucalyptus trees while farmers directly cultivated others due to land scarcity. Farmyard manure (FYM) is the sole fertilizer applied on the wastelands although it is insufficient to achieve the desired crop yields. Despite this, several multi-purpose plants such as Tithonia diversifolia, Markhamia lutea, and Canavalia brasiliensis thrive in the area and could supplement FYM. The potential for these “new” amendments to improve soil microbial properties, particularly in the tantalite mine soils was investigated. The specific objectives of the study were to: (a) evaluate the effects of land use on soil microbial indices of the tantalite mine soils; (b) investigate the restorative effects of organic amendments on a Technosol; and (c) estimate the short-term N and P supply potential of the soil amendments in the soils. Fresh soils (0-20 cm) from an unmined native forest, two mine sites afforested with pine and eucalyptus forests (pine and eucalyptus Technosols), an arable land, and two cultivated Technosols (Kavumu and Kirengo Technosols) were analyzed for the physicochemical properties. Afterwards, a 28-day incubation (22oC) experiment was conducted followed by measurements of mineral N, soil microbial biomass C, N, P, and fungal ergosterol contents using standard methods. This was followed by a 12-week incubation study of the arable soil and the Kavumu Technosol amended with FYM, Canavalia and Tithonia biomass, and Markhamia leaf litter after which soil microbial properties were measured at 2, 8, and 12 weeks of incubation. Finally, two 4-week incubation experiments each were conducted in soils of the six sites to estimate (i) potential mineralizable N using a soil-sand mixture (1:1) amended with Canavalia and goat manure and (ii) P mineralization mixtures (1:1) of soil and anion exchange resins in bicarbonate form amended with Tithonia biomass and goat manure. In study one, afforestation increased soil organic carbon and total N contents in the pine and eucalyptus Technosols by 34-40% and 28-30%, respectively of that in the native forest soil. Consequently, the microbial biomass and activity followed a similar trend where the cultivated Technosols were inferior to the afforested ones. The microbial indices of the mine soils were constrained by soil acidity, dithionite-extractable Al, and low P availability. In study two, the amendments substantially increased C and N mineralization, microbial properties compared with non-amended soils. Canavalia biomass increased CO2 efflux by 340%, net N mineralization by 30-140%, and microbial biomass C and N by 240-600% and 240-380% (P < 0.01), respectively after four weeks of incubation compared with the non-amended soils. Tithonia biomass increased ergosterol content by roughly 240%. The Kavumu Technosol showed a high potential for quick restoration of its soil quality due to its major responses to the measured biological parameters. In study three, Canavalia biomass gave the highest mineralizable N (130 µg g-1 soil, P < 0.01) in the Kavumu Technosol and the lowest in the native forest soil (-20 µg g-1 soil). Conversely, the mineralizable N of goat manure was negative in all soils ranging from -2.5 µg N g-1 to -7.7 µg N g-1 soil except the native forest soil. However, the immobilization of goat manure N in the “cultivated soils” was 30-70% lower than in the “forest soils” signifying an imminent recovery of the amended soils from N immobilization. The mineralization of goat manure P was three-fold that of Tithonia, constituting 61-71% of total P applied. Phosphorus mineralization slightly decreased after four weeks of incubation due to sulfate competition as reflected in a negative correlation, which was steeper in the Tithonia treatment. In conclusion, each amendment used in this research played a unique role in C, N, and P mineralization and contributed substantially to microbial properties in the tantalite mine soils. Interestingly, the “N immobilizers” exhibited potentials for P release and soil organic carbon storage. Consequently, the combined use of the amendments in specific ratios, or co-composting prior to application is recommended to optimize nutrient release, microbial biomass dynamics and soil organic matter accrual. Transport of organic inputs seems more feasible for smallholder farmers who typically manage small field sizes. To reduce acidity in the soils, liming with wood ash was recommended to also improve P availability and enhance soil biological quality, even if it may only be possible on small areas. Further, afforestation with mixed-species of fast-growing eucalyptus and legume or indigenous tree species are suggested to restore tantalite mine wastelands. It is emphasized most of this research was conducted under controlled laboratory conditions, which exclude interaction with environmental variables. Also fine fractions of the amendments were used compared with the usual practice of applying a mixture of predominantly coarser fractions. Therefore, the biological dynamics reported in the studies here may not entirely reflect those of farmers’ field conditions.

Shifts in rhizosphere microbial communities and enzyme activity of Poa alpina across an alpine chronosequence

This study quantifies the influence of Poa alpina on the soil microbial community in primary succession of alpine ecosystems, and whether these effects are controlled by the successional stage. Four successional sites representative of four stages of grassland development (initial, 4 years (non-vegetated); pioneer, 20 years; transition, 75 years; mature, 9500 years old) on the Rotmoos glacier foreland, Austria, were sampled. The size, composition and activity of the microbial community in the rhizosphere and bulk soil were characterized using the chloroform-fumigation extraction procedure, phospholipid fatty acid (PLFA) analysis and measurements of the enzymes beta-glucosidase, beta-xylosidase, N-acetyl-beta-glucosaminidase, leucine aminopeptidase, acid phosphatase and sulfatase. The interplay between the host plant and the successional stage was quantified using principal component (PCA) and multidimensional scaling analyses. Correlation analyses were applied to evaluate the relationship between soil factors (C-org, N-t, C/N ratio, pH, ammonium, phosphorus, potassium) and microbial properties in the bulk soil. In the pioneer stage microbial colonization of the rhizosphere of P. alpina was dependent on the reservoir of microbial species in the bulk soil. As a consequence, the rhizosphere and bulk soil were similar in microbial biomass (ninhydrin-reactive nitrogen (NHR-N)), community composition (PLFA), and enzyme activity. In the transition and mature grassland stage, more benign soil conditions stimulated microbial growth (NHR-N, total amount of PLFA, bacterial PLFA, Gram-positive bacteria, Gram-negative bacteria), and microbial diversity (Shannon index H) in the rhizosphere either directly or indirectly through enhanced carbon allocation. In the same period, the rhizosphere microflora shifted from a G(-) to a more G(+), and from a fungal to a more bacteria-dominated community. Rhizosphere beta-xylosidase, N-acetyl-beta-glucosaminidase, and sulfatase activity peaked in the mature grassland soil, whereas rhizosphere leucine aminopeptidase, beta-glucosidase, and phosphatase activity were highest in the transition stage, probably because of enhanced carbon and nutrient allocation into the rhizosphere due to better growth conditions. Soil organic matter appeared to be the most important driver of microbial colonization in the bulk soil. The decrease in soil pH and soil C/N ratio mediated the shifts in the soil microbial community composition (bacPLFA, bacPLFA/fungPLFA, G(-), G(+)/G(-)). The activities of beta-glucosidase, beta-xylosidase and phosphatase were related to soil ammonium and phosphorus, indicating that higher decomposition rates enhanced the nutrient availability in the bulk soil. We conclude that the major determinants of the microllora vary along the successional gradient: in the pioneer stage the rhizosphere microflora was primarily determined by the harsh soil environment; under more favourable environmental conditions, however, the host plant selected for a specific microbial community that was related to the dynamic interplay between soil properties and carbon supply. (C) 2004 Elsevier Ltd. All rights reserved.

Adaptation of the Integrated Nitrogen Model for Catchments (INCA) to seasonally snow-covered catchments

Testing of the Integrated Nitrogen model for Catchments (INCA) in a wide range of ecosystem types across Europe has shown that the model underestimates N transformation processes to a large extent in northern catchments of Finland and Norway in winter and spring. It is found, and generally assumed, that microbial activity in soils proceeds at low rates at northern latitudes during winter, even at sub-zero temperatures. The INCA model was modified to improve the simulation of N transformation rates in northern catchments, characterised by cold climates and extensive snow accumulation and insulation in winter, by introducing an empirical function to simulate soil temperatures below the seasonal snow pack, and a degree-day model to calculate the depth of the snow pack. The proposed snow-correction factor improved the simulation of soil temperatures at Finnish and Norwegian field sites in winter, although soil temperature was still underestimated during periods with a thin snow cover. Finally, a comparison between the modified INCA version (v. 1.7) and the former version (v. 1.6) was made at the Simojoki river basin in northern Finland and at Dalelva Brook in northern Norway. The new modules did not imply any significant changes in simulated NO3- concentration levels in the streams but improved the timing of simulated higher concentrations. The inclusion of a modified temperature response function and an empirical snow-correction factor improved the flexibility and applicability of the model for climate effect studies.

Shifts in rhizosphere microbial communities and enzyme activity of Poa alpina across an alpine chronosequence

This study quantifies the influence of Poa alpina on the soil microbial community in primary succession of alpine ecosystems, and whether these effects are controlled by the successional stage. Four successional sites representative of four stages of grassland development (initial, 4 years (non-vegetated); pioneer, 20 years; transition, 75 years; mature, 9500 years old) on the Rotmoos glacier foreland, Austria, were sampled. The size, composition and activity of the microbial community in the rhizosphere and bulk soil were characterized using the chloroform-fumigation extraction procedure, phospholipid fatty acid (PLFA) analysis and measurements of the enzymes beta-glucosidase, beta-xylosidase, N-acetyl-beta-glucosaminidase, leucine aminopeptidase, acid phosphatase and sulfatase. The interplay between the host plant and the successional stage was quantified using principal component (PCA) and multidimensional scaling analyses. Correlation analyses were applied to evaluate the relationship between soil factors (C-org, N-t, C/N ratio, pH, ammonium, phosphorus, potassium) and microbial properties in the bulk soil. In the pioneer stage microbial colonization of the rhizosphere of P. alpina was dependent on the reservoir of microbial species in the bulk soil. As a consequence, the rhizosphere and bulk soil were similar in microbial biomass (ninhydrin-reactive nitrogen (NHR-N)), community composition (PLFA), and enzyme activity. In the transition and mature grassland stage, more benign soil conditions stimulated microbial growth (NHR-N, total amount of PLFA, bacterial PLFA, Gram-positive bacteria, Gram-negative bacteria), and microbial diversity (Shannon index H) in the rhizosphere either directly or indirectly through enhanced carbon allocation. In the same period, the rhizosphere microflora shifted from a G(-) to a more G(+), and from a fungal to a more bacteria-dominated community. Rhizosphere beta-xylosidase, N-acetyl-beta-glucosaminidase, and sulfatase activity peaked in the mature grassland soil, whereas rhizosphere leucine aminopeptidase, beta-glucosidase, and phosphatase activity were highest in the transition stage, probably because of enhanced carbon and nutrient allocation into the rhizosphere due to better growth conditions. Soil organic matter appeared to be the most important driver of microbial colonization in the bulk soil. The decrease in soil pH and soil C/N ratio mediated the shifts in the soil microbial community composition (bacPLFA, bacPLFA/fungPLFA, G(-), G(+)/G(-)). The activities of beta-glucosidase, beta-xylosidase and phosphatase were related to soil ammonium and phosphorus, indicating that higher decomposition rates enhanced the nutrient availability in the bulk soil. We conclude that the major determinants of the microllora vary along the successional gradient: in the pioneer stage the rhizosphere microflora was primarily determined by the harsh soil environment; under more favourable environmental conditions, however, the host plant selected for a specific microbial community that was related to the dynamic interplay between soil properties and carbon supply. (C) 2004 Elsevier Ltd. All rights reserved.

Modelling the inorganic nitrogen behaviour in a small Mediterranean forested catchment, Fuirosos (Catalonia)

The aim of this work was to couple a nitrogen (N) sub-model to already existent hydrological lumped (LU4-N) and semi-distributed (LU4-R-N and SD4-R-N) conceptual models, to improve our understanding of the factors and processes controlling nitrogen cycling and losses in Mediterranean catchments. The N model adopted provides a simplified conceptualization of the soil nitrogen cycle considering mineralization, nitrification, immobilization, denitrification, plant uptake, and ammonium adsorption/desorption. It also includes nitrification and denitrification in the shallow perched aquifer. We included a soil moisture threshold for all the considered soil biological processes. The results suggested that all the nitrogen processes were highly influenced by the rain episodes and that soil microbial processes occurred in pulses stimulated by soil moisture increasing after rain. Our simulation highlighted the riparian zone as a possible source of nitrate, especially after the summer drought period, but it can also act as an important sink of nitrate due to denitrification, in particular during the wettest period of the year. The riparian zone was a key element to simulate the catchment nitrate behaviour. The lumped LU4-N model (which does not include the riparian zone) could not be validated, while both the semi-distributed LU4-R-N and SD4-R-N model (which include the riparian zone) gave satisfactory results for the calibration process and acceptable results for the temporal validation process.

Utilisation by ruminants of nitrogen compounds in silage-based diets

This paper considers the various complex changes that occur to nitrogen (N) containing compounds in forages through the processes of ensiling, rumen degradation and microbial synthesis, post-ruminal digestion and absorption and synthesis into milk protein. Particular emphasis is placed on reviewing recent data on the efficiency of utilisation of N-containing compounds in silages by rumen microbes, since low efficiency here is believed to be a major cause of large N losses to the environment on some silage-based diets. Data are reviewed which show that although rumen degradation of N compounds in silage is rapid and extensive, up to 10% of the soluble N can escape the rumen by being associated with the liquid phase. There is now firm evidence that the composition of the amino acids (AAs) absorbed is heavily dependent on the process of ensiling and that witting or use of certain silage additives conserve the initial amino acid profile of the forage. This provides an opportunity to manipulate the amino acid supply to better match demand thus potentially enhancing utilisation. This review confirms that utilisation of the N fractions in grass and legume silages in particular, is poor and the efficiency of microbial protein synthesis (EMPS) is consistently higher on maize silage-based diets. It is concluded that the way in which grass and legume silages in particular are produced and used in the future needs a radical rethink. New research needs to be aimed at enhancing the utilisation of N in the rumen through a better understanding of N/carbohydrate relationships and the ability of forages to supply degraded carbohydrate. Also more emphasis is needed on understanding of the potentially different role of the different N fractions that exist in silages. (C) 2004 Elsevier B.V. All rights reserved.

Nitrogen recycling through the gut and the nitrogen economy of ruminants: An asynchronous symbiosis

The extensive development of the ruminant forestomach sets apart their N economy from that of nonruminants in a number of respects. Extensive pregastric fermentation alters the profile of protein reaching the small intestine, largely through the transformation of nitrogenous compounds into microbial protein. This process is fueled primarily by carbohydrate fermentation and includes extensive recycling of N between the body and gut lumen pools. Nitrogen recycling occurs via blood and gut lumen exchanges of urea and NH3, as well as endogenous gut and secretory N entry into the gut lumen, and the subsequent digestion and absorption of microbial and endogenous protein. Factors controlling urea transfer to the gut from blood, including the contributions of urea transporters, remain equivocal. Ammonia produced by microbial degradation of urea and dietary and endogenous AA is utilized by microbial fermentation or absorbed and primarily converted to urea. Therefore, microbial growth and carbohydrate fermentation affect the extent of NH3 absorption and urea N recycling and excretion. The extensive recycling of N to the rumen represents an evolutionary advantage of the ruminant in terms of absorbable protein supply during periods of dietary protein deficiency, or asynchronous carbohydrate and protein supply, but incurs a cost of greater N intakes, especially in terms of excess N excretion. Efforts to improve the efficiency of N utilization in ruminants by synchronizing fermentable energy and N availability have generally met with limited success with regards to production responses. In contrast, imposing asynchrony through oscillating dietary protein concentration, or infrequent supplementation, surprisingly has not negatively affected production responses unless the frequency of supplementation is less than once every 3 d. In some cases, oscillation of dietary protein concentration has improved N retention compared with animals fed an equal amount of dietary protein on a daily basis. This may reflect benefits of Orn cycle adaptations and sustained recycling of urea to the gut. The microbial symbiosis of the ruminant is inherently adaptable to asynchronous N and energy supply. Recycling of urea to the gut buffers the effect of irregular dietary N supply such that intuitive benefits of rumen synchrony in terms of the efficiency of N utilization are typically not observed in practice.