Denitrification, Dissimilatory Nitrate Reduction, and Methanogenesis in the Gut of Earthworms (Oligochaeta): Assessment of Greenhouse Gases and Genetic Markers
- The earthworm gut is an anoxic microzone in aerated soils, shows a high water content and high amounts of nitrite and organic carbon. These conditions are in marked contrast to those in the pre-ingested material (i.e., substrate) and ideal for microorganisms as those capable of fermentations, for denitrifiers, and dissimilatory nitrate reducers (DNR). Thus, substrate-derived denitrifiers in the earthworm gut (Lumbricidae) emit the greenhouse gas N2O as well as N2. One large species (Megascolecidae) emitted no N2O, leading to the hypothesis that large earthworms cannot emit nitrogenous gases, i.e., N2O and N2. There was no emission of the greenhouse gas CH4 reported for earthworm species.
The current study analyzed ten earthworm species of different families, sizes, and feeding guilds (i.e., burrow and feeding habits) from Brazil for the emission of nitrogenous gases and CH4. Taxa affiliated with these emissions, i.e., denitrifiers, DNR, and methanogens were analyzed with cloning and pyrosequencing of marker genes, from gut contents and substrates of earthworm species from Brazil (Amynthas gracilis, Glossoscolex paulistus, Eudrilus eugeniae), Germany (Aporrectodea caliginosa, Lumbricus terrestris, Lumbricus rubellus), and New Zealand (Octochaetus multiporus) on gene and partly on transcript levels. Potential denitrifiers and methanogens were isolated and enriched from earthworm gut contents, respectively. Sequences of narG (encoding for a nitrate reductase; targets denitrifiers and DNR), nirK, nirS (both encoding for a nitrite reductase; target denitrifiers), nosZ (encoding for a N2O reductase; targets denitrifiers), and mcrA/mrtA (encoding for the methyl-CoM reductase and its isoenzyme; target methanogenic Archaea) were analyzed. For nirK and nirS, cutoff values were calculated to define species-level affiliations according to their sequence similarities.
Perionyx excavatus, A. gracilis (both Megascolecidae), Pontoscolex corethrurus, Rhinodrilus alatus (both Glossoscolecidae), Dichogaster annae, Dichogaster sp. (both Acanthodrilidae), and E. eugeniae (Eudrilidae) emitted nitrogenous gases; G. paulistus, Glossoscolex sp. (both Glossoscolecidae), and Eisenia andrei (Lumbricidae) did not. Earthworm substrates emitted smaller amounts of nitrogenous gases, predominantly N2. When provided with nitrite, G. paulistus emitted nitrogenous gases but total emissions and the ratio of N2O to N2 were higher for A. gracilis. It was shown that earthworms of all families, sizes, and feeding guilds can emit nitrogenous gases, and that the earthworm substrate, size, and feeding guild were influencing but not determinative factors taken alone.
In earthworms gut contents, denitrifiers were predominantly affiliated with Bradyrhizobiaceae (Rhizobiales), indicating that here, these taxa might be responsible for the emission of nitrogenous gases. Active DNR were predominantly affiliated with Mycobacterium (Actinomycetales), and it is anticipated that these Bacteria compete with denitrifiers for nitrate. Gene analyses and isolation approaches demonstrated that (i) both denitrifiers and DNR in the earthworm gut were derived from ingested material and (ii) diversity in the gut was influenced by the earthworm feeding guild. Analyses of genes and transcripts from earthworms from Germany demonstrated that there was a selective activation of substrate-derived denitrifiers and DNR in the gut.
E. eugeniae emitted the highest amounts of CH4, P. corethrurus and R. alatus emitted less, all other tested species no CH4. One substrate emitted minor amounts of CH4, all others did not emit CH4. Certain substrates appeared to influence the emission of CH4 by earthworms but the substrate taken alone was not a determinative factor. The capacity to emit CH4 by E. eugeniae was not significantly affected by supplemental H2/CO2 and was at least partly retained when maintained on diverse alternative substrates.
Analysis of mcrA/mrtA revealed that selectively activated hydrogenotrophic and acetoclastic methanogens of the Methanosarcinaceae and Methanobacteriacea were the source of the CH4 emitted by E. eugeniae. These methanogens were assumed to be substrate-derived although a symbiotic affiliation with the earthworm cannot be excluded. Certain earthworms emitted both CH4 and nitrogenous gases, suggesting that methanogenesis and denitrification can be concomitant processes in the earthworm gut.
This study demonstrated that (i) earthworms from all families, sizes, and feeding guilds can emit N2O and N2, (ii) substrate-derived and selectively activated denitrifiers within the Rhizobiales are the main source of N2O and N2 whereas Actinomycetales are the main active DNR, (iii) the earthworm feeding guild affects the selective activation of ingested denitrifiers and DNR, (iv) certain earthworms emit CH4, and Methanosarcinaceae and Methanobacteriaceae appear to be the main source of this CH4, and (v) certain earthworms can concomitantly emit N2O, N2, and CH4.
The regulation of digestive enzyme release in the two-spotted field cricket Gryllus bimaculatus (de Geer): effects of endogenous and environmental factors
- Insects are the most abundant animal species on earth with a huge economical and ecological impact. In spite of intensive research in the field of integrated pest management there are still a lot of questions concerning the adaptation mechanism of insects to their environment. As the digestive tract displays a putative target for effective pest management, this study worked on the effects of endogenous and environmental factors on digestive enzyme release in the omnivorous cricket, Gryllus bimaculatus.
The age-dependent enzyme release of carbohydrases, proteases and lipase correlates with the daily feeding rate of the crickets and peaked between days 2 to 4 in last instar larvae as well as in adult crickets. In contrast, the secretion of chitinase was affected by the moulting cycle of the insects reaching maximum activity at the day of moult. Therefore, chitinase plays only a minor role in food digestion. The cellulase activity in the midgut of G. bimaculatus resulted from an endogenous cellulase and was not caused by bacteria or eukaryotic endosymbionts in the digestive tract. The endoprotease trypsin was stored in the caecal tissue as an inactive precursor, and is secreted to the lumen by exocytosis. Following activation Gryllus-trypsin (~24 kDa) is protected from proteolytic degradation, but there is no endogenous protease inhibitor in the midgut.
Gene knockdown by RNA interference was used to analyse the endogenous regulation of digestive enzyme release by the neuropeptides allatostatin A and sulfakinin, which had already been shown to affect feeding in G. bimaculatus. Functional analysis of the AST-A gene was investigated for last instar larvae and adult crickets, whereby female crickets seemed to be more sensitive to this method. The gene suppression of AST-A resulted in a decreased synthesis of amylase, trypsin, aminopeptidase and lipase in the caecal tissue, but enzyme release varied between sexes and developmental stages. The knockdown of SK expression led to an increase of amylase and cellulase secretion in female crickets, and to a reduction of protease and lipase release in males.
As food plays a fundamental role in digestive enzyme release, both quality and quantity of nutrition are ample factors. There was always a higher digestive enzyme activity in fed crickets compared to starved ones. Furthermore, starvation resulted in a decrease of enzyme synthesis in the caecal tissue. In general, nutrients in the incubation medium led to a stimulation of digestive enzyme secretion, but in the case of cellulase the presence of both cellubiose in the incubation medium and cellulose in the diet caused a strong decline in cellulase release. Addition of the plant protease inhibitor SBTI to the diet caused a dose-dependent inhibition of protease activity in the caeca, whereby minor concentrations of SBTI were compensated by enzyme hyperproduction.
In addition to the food uptake, the daily light-dark cycle seems to affect digestive enzyme release. Crickets started to feed at the beginning of the scotophase, which led to an increase of protease and lipase secretion in larvae and adults. The secretion of carbohydrases was highest during the photophase. This means that enzyme release is not solely affected by the time of food uptake.
Temperature is one of the most important environmental factors, but seems to play only a minor role in the release of digestive enzymes. All tested enzymes showed a broad optimal temperature range (30°C-40°C), but there was no difference in the release of amylase or lipase after tissue incubation at 25°C or 35°C. In contrast, trypsin and aminopeptidase showed a higher secretion after incubation at 35°C compared to 25°C. Furthermore, insect rearing at 22°C and 32°C during various developmental stages resulted in a positive acclimation of trypsin secretion to rearing temperature.
Quantifying water use by temperate deciduous forests in South Korea: roles of species diversity, canopy structure, and complex terrain
- About seventy percent of South Korea is covered with forests, most of which are found in the mountain regions since mountains receive more rainfall and are difficult terrains not suitable for agriculture. Because mountains are important water sources for cities and human population downstream, performing water balance for forest catchments has become a research priority. The ongoing shift from coniferous to species-rich deciduous forests due to a changing government policy and the anticipated changes in future climate, associated with increasing amount of rainfall and temperature will also impact forest water use, calling for an urgent need to understand how forests, in their current status, use water. The knowledge is vital for predicting water requirements for the future forest. The warm-deciduous temperate forests found in South Korea, however, have a high diversity of tree species, have multi-layered canopies and are mostly located on rugged mountainous terrains, which make it difficult to quantify forest water use, a basic requirement for catchment water budgeting. The main objectives of this study were to: (1) identify the roles of species diversity in tree and forest water use, (2) examine the impact of canopy structure on forest transpiration, and (3) evaluate the influence of terrain on forest water use.
Site-specific studies were carried out in three different natural deciduous forests, namely, Gyebang (GB), Gwangneung (GN) and Haean (HA) forest sites, representing the general structure of S. Korean forests. GB site is known for its high species diversity, GN site is an old forest growth at climax, with clearly defined understory and overstory canopy layers while the HA site was located with in a catchment, with strong elevation changes within short horizontal distances, rising from 400 to 1,000 m a.s.l., and in different aspects. Four locations with varying elevations and aspects were chosen in the HA site. Tree water use (TWU) and canopy transpiration (EC) were estimated from sap flux density measured with thermal dissipation probes. Understory transpiration (EU) was measured using stem heat balance while ecosystem evapotranspiration (Eeco) was determined using eddy covariance technique. Air temperatures (Ta), precipitation, solar radiation, vapor pressure deficit (VPD), wind speed were measured from weather stations and soil water content was measured from frequency domain reflectometry (FDR) sensors at the respective study sites. Vegetation surveys, including diameter at breast height (DBH), tree density, species composition, sapwood area (AS), and leaf area index were performed in all the sites. Canopy conductance (GC) and stomatal sensitivity to VPD were assessed based on transpiration and microclimate measured at each site.
A functional allometric relationship was established between AS and DBH, and also between TWU and DBH for all the study sites; first for single species and then combining all the species either in a single site or in all the sites. Irrespective of tree species, AS and maximum TWU were significantly correlated with DBH in a power function for AS (R2 = 0.77, P <0.0001) and both in power (R2 = 0.63, P <0.0001) and sigmoid functions (R2 = 0.66, P <0.0001) for TWU, for the co-occurring species as well as across the sites, suggesting that DBH can be a good predictor of stand AS and maximum TWU, based on the established allometric functions.
Early bud break and development of the understory compared to the overstory canopy resulted in an earlier onset of forest transpiration, with EU contributing 22% and 14% between April and May to the total forest transpiration. This high contribution was favored by high radiation and VPD in the understory, since the overstory was still undeveloped and open. Despite diminishing VPD and light conditions in the understory between June and August, the understory continued to transpire a substantial amount of water, contributing 10% of the total transpiration. The seasonal patterns of both EO and EU were synchronized to canopy development, while VPD and radiation determined daily trends. EO and EU accounted for 80% of Eeco in spring but only 60% during the monsoon period due to lowered radiation input, VPD, and plant area index (PAI). Thus, Eeco is largely influenced by transpiration rate and its seasonal variation and also canopy structure.
Early saturation of EC at relatively low VPD and also a rapid decrease in GC with increasing VPD were observed in the forest stand located at the highest elevation studied (950 m) in the HA site, compared to the GN and the other forest stands in HA. These differences in transpiration rates and stomatal response can be explained by greater stomatal sensitivity to VPD of 0.83 found at the 950 m site compared to 0.63–0.66 in the other study sites. However, the main controlling factor of the change in stomatal sensitivity at the 950 m stand is uncertain. Although maximum daily EC were correlated with AS of the forest stands at different sites (R2 = 0.78, P <0.01), annual EC declined with increasing elevation, i.e., 176 >175 >110 >90 mm year−1 at 340 >450 >650 >950 m, respectively. Decline in total EC was due to the decline in annual Ta, daytime VPD, and length of growing season at higher elevations. The GB site, which was located at 960 m elevation, however, did not display a same response pattern as those observed at the 950 m site. It is likely because these sites were under different environmental conditions, i.e., GB site is exposed to higher Ta and higher humidity, and is sheltered (lower wind speeds). These observations emphasize the complexity associated with estimation of transpiration in rugged terrains, since general principles do not always apply and the spatial patterns of forest transpiration are complex.
Complexity arising from multiple tree species composition when estimating forest water use can be reduced by applying functional allometric relationship linking tree size and water use. Forest canopy structure and physical location should be taken into account since they influence the way forests use water resources by altering microclimate and plant physiology. Based on our findings, estimation of forest water use on rugged terrains require repeated measurements at relatively small spatial scales since the driving factors change rapidly over very narrow vertical distances.
RNA interference with allatoregulating neuropeptide genes affecting circadian rhythm, development, mating and reproduction of Spodoptera frugiperda (Lepidoptera: Noctuidae)
Intisar Taha Elhag Hassanien
- Juvenile hormones (JHs) have juvenoid functions but also act as a true gonadotropin in the Lepidoptera. JHs are released by the corpora allata (CA) into the hemolymph, which are under allatotropic and allatostatic neuropeptide control as well as under somatic and environmental stimulus.
Two types of allatostatins (Spofr-AS type Manse-AS and Spofr-AS type A or FGLamides) and two allatotropins (Spofr-AT 1 or Manse-AT and Spofr-AT 2) were identified and cloned from Spodoptera frugiperda. Their functions were already investigated in our laboratory. Most of these peptides are pleiotropic in function but their specific roles in the various developmental stages of S. frugiperda are still unclear.
My work mainly emphasizes on the AT 1 gene function on JH and ecdysteroid titers in the hemolymph of larvae and adults as well as on females’ and males’ reproductive tissues and on the daily egg rate of the females. Studies were carried out by knockdown of the AT 1 gene using RNA interference. My results show a significant role of this neuropeptide on the circadian rhythm of prepupal commitment and metamorphosis, but also on the profile of egg laying and the number of deposited eggs. In general, Spofr-AT 1 acts as a true allatotropin in larvae and adults, but its functions may be substituted, at least in part, by Spofr-AT 2.
Silencing of Spofr-AT 1 and AS type A genes in males supported either allatotropic or allatostatic actions on the accessory glands (AGs) of the males, but had no effect on the JH transferred to the female bursa copulatrix (BC) during mating, nor on the fertility of the females.
Greenhouse gas metabolizing prokaryotes in peatlands
- Peatlands in the northern hemisphere store substantial amounts of carbon and nitrogen, and are predicted to react sensitively to global warming. The main objective of the study was to study microbial processes involved in the fluxes of the greenhouse gases methane (CH4) and nitrous oxide (N2O) from peatlands. Five model peatlands with contrasting features (e.g., nitrate content, mean annual temperature, pH) were assessed.
A pH-neutral fen produced CH4 in situ as well as in unsupplemented anoxic microcosms. Supplemental N-Acetylglucosaminestimulated formation of fermentation products and CH4 and lead to increased accumulation of fermentation products when methanogenesis was inhibited by Bromoethanesulfonate. Supplemental H2/CO2 and formate greatly stimulated methanogenesis, while acetate and methanol stimulated methanogenesis to a much lesser extent. A high family-level diversity of bacterial 16S rRNA genes was detected. Within the detected families, genera known for syntrophic interactions with methanogens were found. Diversity of methanogens was lower than bacterial diversity, as only hydrogenotrophic Methanomicrobiales and Methanocellales were detected. Both process data and molecular data suggest that (i) hydrogenotrophic methanogenesis is the main process of CH4 formation in pH-neutral fen soil, and (ii) a high diversity of bacterial families are likely involved in diverse fermentations, providing substrates for methanogens.
Denitrification and N2O consumption potentials as well as denitrifier community composition were assessed in all five northern peatlands. In situ N2O emissions range from < 0.01 to 10 mg N2O*m-2*d-1,and were positively and negatively correlated with soil nitrate and ammonia contents, respectively. All soils produced and consumed N2O in anoxic microcosms without apparent delay. N2O production capacities and apparent affinities for nitrate were likewise positively correlated with soil nitrate content. Phylogenetic analyses of the nitrate reduction- and denitrification-associated genes narG, nirK/nirS, and nosZ indicated that the diversity of the denitifier community was highest in pH-neutral fen soil and positively correlated with pH. Detected narG affiliated mainly with Betaproteobacterial and Actinobacterial narG. . The number and the identity of observed operational taxonomic units (OTUs) of nirK, nirS, and nosZ in pH-neutral fen soil was clearly distinct from those of the more acidic soils, and indicated Alpha-, Beta-, and Gammaproteobacterial denitrifiers in all peatlands. Permafrost-affected soils mainly clustered together in the canonical correspondence analysis plots of the analyzed genes. pH was the most important factor determining community composition of nitrate reducers and denitrifiers.Significant influences of soil carbon content, precipitation, or temperature were also detected. The occurrence of certain OTUs of nirK and nirS was positively correlated with N2O emissions. The collective data indicate that (i) denitrification is an ongoing process in different types of pristine peatland soils, (ii) source and sink function of peatland denitrifiers for N2O are influenced by soil nitrate content and denitrifier community composition, and (iii) nitrate reducer and denitrifier community composition are affected by pH, temperature, precipitation, and soil carbon content.
Global warming is predicted to increase the frequency of extreme weather events, affecting on the water table level in peatlands and on the microbial communities involved in the production of CH4 and N2O. Thus, the influence of short-term water table manipulations including application of artificial drought conditions or excessive flooding was assessed in the acidicfen. Fermentative, methanogenic and denitrifying potentials in anoxic microcosm studies revealed that the potential activity of methanogens and denitrifiers was affected by changing water tables, whereas the potential activity of fermenters was largely unaffected. Changes in the copy numbers of mcrA, narG, and nosZ detected by quantitative PCR were rather small when compared to the observed changes in potential activity. Community composition of methanogens, nitrate and N2O-reducers was similar at all sampled timepoints of the manipulation experiments. The collective data indicate a stable microbial community in fen soil that is able to adapt its activity to the changing conditions quite rapidly.
Endocrine control of fat body composition and effects of the insect growth regulators methoprene and pyriproxyfen on the development and reproduction of the Argentinian cockroach, Blaptica dubia Serville (Blattaria: Blaberidae)
Ahmad Alhasan Alamer
- The present study analysis physiological and endocrine aspects of female adult life of the ovoviviparous Argentinian (Dubia) cockroach, Blaptica dubia.
Experiments were done during the first (days 5 to 25 after emergence) and second gonadotropic cycle (days 80 to 100 after emergence), and whilst the period of oothecal transport (gestation) and hatching of the nymphs (around day 70 of adult life for the first gonadotropic cycle).
Body weight changes of adult females are closely related with the reproductive
processes of ovarian growth, ootheca formation, ootheca deposition, and hatching of the nymphs. The biochemical composition of the fat body was analyzed and revealed the storage lipids as main constituents, followed by glycogen, proteins and free carbohydrates. Changes in fat body chemical constituents as well as in fat body fresh weight correlate well with the reproductive processes.
Concentrations of juvenile hormone (JH) III and free ecdysteroids (ecdysone,
20-hydroxyecdysone) were measured in the haemolymph of adult females by
the use of HPLC-mass spectrometry. The JH III titer shows significant peaks
during vitellogenesis and ovarian growth, and towards the end of the period of
gestation. Changes in the concentration of free ecdysteroids in the
haemolymph were less clear.
Treatment of adult females with the juvenile hormone analogue methoprene during the first 20 days of adult life (topical application of 100 μg methoprene in acetone per day) retarded ovarian development and blocked the formation of the ootheca. The inhibition of the ootheca formation went in line with reduced titers of JH III and free ecdysteroids in the haemolymph of such treated females.
Methoprene treatment of females, which had normally developed during the first gonadotropic cycle, from day 30 to 70 of adult life resulted in a complete reabsorption of the ootheca. The JH III titer of such treated females was also
reduced, whereas free ecdysteroids were hardly affected.
Treatment of adult females with another JH mimic, pyriproxyfen (100 μg per
treatment) gave similar results than those for methoprene usage.
The results show that both JH analogues have a clear ovicidal activity resulting in “sterilization” of the females and can be used as “third generation
pesticides” in adult cockroach biocontrol.
Impact of extreme hydrological conditions on belowground carbon cycling and redox dynamics in peat soils from a northern temperate fen
Cristian Estop Aragonés
- Peatlands have an important role in the global carbon cycle and constitute the largest pool of carbon stored in terrestrial ecosystems due to their disproportionally high areal soil carbon density. This globally relevant carbon stock is the result of a process mostly initiated after the last glaciation period. A key factor for this long term carbon accumulation is the relative low decomposition of organic matter in these predominantly water logged ecosystems. Hydrological conditions play thus a fundamental role in peatlands and the feedback of carbon cycling in these ecosystems in response to climate change is under debate. Peatlands are important CO2 sinks but also constitute global sources of CH4. The atmospheric exchange and production rates of these greenhouse gases are strongly influenced by the hydrological regime. An increased frequency of extreme meteorological conditions resulting in drying and flooding events is predicted to occur in the future.
The major issue regarding the climate change debate at the global scale is how rapid these greenhouse gases are being released to the atmosphere. Despite the general consensus regarding the broad effects of drying and flooding on CO2 and CH4 exchange, belowground processes producing such greenhouse gases and their response to water table dynamics is underrepresented and usually simplified or overgeneralized. Temperature, moisture, oxygen content and nutrient content are among the major environmental controls for organic matter decomposition rates in peat soils. Another important and intrinsic control is peat quality or humification degree of organic matter. The interrelation and relevance of all these factors vary among sites and with hydrological condition in a temporal and spatial scale.
This work presents investigations focusing on belowground redox processes aiming to witness the dynamic interrelation of soil physical and chemical (soil gas and pore water chemistry) variables, and evaluates the relevance of some controls of organic matter decomposition during a wide range of hydrological conditions. Most of this work shows information under in situ conditions and complementary laboratory experiments were performed minding the in situ observations. The findings contribute to general knowledge by providing raw data in fen peats under fluctuating and contrasting water table conditions in a relatively high spatiotemporal resolved scale. Dryings led to increased air filled porosity, O2 intrusion, CO2 degassing, inhibition of methanogenesis and renewal of electron acceptors. The opposite trend occurred upon rewetting with pulses of iron and sulphate reduction and delayed methane production to a variable extent. Upon flooding, continued anaerobic conditions stimulated the accumulation of reduced products, methanogenic precursors (acetate and hydrogen) and CH4.
The general assumption that the water table directly controls the oxygen content in peat was relativized. This work shows that such relation is greatly influenced by peat physical properties, which partially control the changes in moisture. Based on these findings, the mineral content and the degree of compaction in organic soils can be implemented to more accurately represent the dynamics of aeration in peats upon water table changes. Another general assumption is that drying events, i.e. temporary decline of water table below mean position, lead to increased CO2 production and emission from peat soils to the atmosphere. Such statement was also relativized and must account for the depth distribution of respiration rates in relation to the mean water table of the peat deposit. Based on these findings, the high relative contribution of upper peat layers already exposed above the water table mask the effects of increased CO2 production in deeper peat upon water table drop. Additionally, the role of moisture was shown to be little for aerobic respiration. This work also evaluates the importance of drought severity by accounting for the post drought effects on methane production. More intense and prolonged drying events led to a greater regeneration of electron acceptors in peat soil, which broadly suppressed or limited methane production upon rewetting. This relation was not simple and several factors such as water table position, post drought water table fluctuations, temperature and organic matter content contributed to the recovery of methane production after drying. The provision of substrates by fermentation processes limited peat respiration during shallow water table and drying. In contrast, accumulation of acetate and hydrogen was observed during flooding indicating a decoupling of fermentation from terminal metabolism and favouring the co-occurrence of iron reduction, sulphate reduction and methanogenesis.
The peptidyl-prolyl isomerase Pin1 is required for maintenance of the spindle assembly checkpoint
- Chromosomes are replicated during S-phase and segregated during M-phase of the eukaryotic cell cycle. The two sister chromatids of each duplicated chromosome are topologically entrapped and, thus, paired by the ring-shaped protein complex cohesin. They are separated in anaphase of mitosis when cohesin is endoproteolytically cleaved by separase. Activation of this giant protease requires the degradation of its two inhibitors, securin and cyclin B1, which is mediated by the anaphase promoting complex or cyclosome (APC/C), a multisubunit ubiquitin ligase, in conjunction with its essential co-activator Cdc20.
The spindle assembly checkpoint (SAC) is a surveillance mechanism that monitors the chromosomes' interactions with the microtubules of the mitotic spindle apparatus. In response to even one erroneous attachment the affected kinetochore emits a "wait anaphase" signal, which is amplified and culminates in the quantitative sequestration of Cdc20 by the SAC components Mad2 and BubR1. The consequent inactivation of the APC/C causes a metaphase arrest and gives the cell time to correct the error. Given its great importance for chromosome segregation fidelity, it comes at no surprise that loss of the SAC causes cell death while its curtailing is associated with tumour formation.
Pin1 is a peptidyl-prolyl-isomerase with strong preference for phosphorylated Ser-Pro or Thr-Pro motives within its protein substrates. In the present thesis, evidence for the involvement of Pin1 in the maintenance of a robust SAC response is presented.
Antibodies against Pin1 were raised and used to establish the effective immunodepletion of Pin1 from extracts of Xenopus laevis eggs. While the SAC could readily be activated in mock-treated samples of this cell free system, securin was degraded despite the presence of unattached kinetochores when Pin1 had previously been removed. Proving the specificity of this effect, a SAC mediated arrest could be rescued by adding back recombinant Pin1 to depleted extracts. Similarly, addition of dominant negative but not of wild-type Pin1 to SAC-arrested extracts resulted in a checkpoint override.
Chemical inhibition of human Pin1 with two different molecules in two different cancer cells lines invariably forced the cells to exit mitosis in the absence of spindles. This resulted in the premature disappearance of securin, cyclin B1 and a mitosis-specific phosphorylation on Ser10 of histone H3. Thus, Pin1's role as a checkpoint component is conserved in mammals.
In search for the relevant target, Cdc20 was identified as a novel interaction partner of vertebrate Pin1. This association requires phosphorylation of Cdc20 on Ser-Pro/Thr-Pro sites and occurs only during mitosis. Importantly, the Pin1-Cdc20 interaction is direct and not bridged via another checkpoint component or a subunit of the core APC/C. The experimental data suggest that Pin1-dependent isomerization of Cdc20 might bias it to preferentially associate with Mad2 and BubR1 instead of APC/C.
Taken together, these findings contribute to a better understanding of the molecular mechanisms involved in SAC signalling and unravel a previously unappreciated role of Pin1 for genome integrity.
Mineral sequestration of CO2 by reaction with alkaline residues
- With the onset of industrialization within the last 150 years, a significant increase in the concentration of the greenhouse gas carbon dioxide (CO2) is recorded in the atmosphere. According to current scientific understanding the rising atmospheric CO2 levels can be linked with high probability to the observed phenomenon of global warming. Consequently, the reduction of anthropogenic greenhouse gas emissions has become a global challenge of environmental research and policy.
In this thesis, a novel approach to achieve a long-term mineral sequestration of CO2 was studied, using alkaline residue materials. This study examined for the first time a process that allows for rapid removal of CO2 from flue gas through reaction with lignite fly ashes in aqueous solution.
In this process the basicity of the residues is utilized for mineral trapping of CO2 by precipitating stable calcite. Lignite fly ashes are a cheap, inexpensive, highly reactive byproduct of coal combustion. Due to the exposure to heat, these waste streams generally contain high amounts of reactive Ca/Mg (hydr)oxides and thus offer a high alkalinity. The alkaline residues were therefore not considered as an environmental problem, but rather as useful reactants for technical CO2 neutralization in the context of combustion processes.
Carbonates are end-products of weathering processes at the earth surface and mineral carbonation is thus assessed to be a permanent and safe storage option of CO2. Compared to alternative forms of carbon storage (e.g. the injection into gas reservoirs) cost-intensive monitoring programs for safety reasons can be omitted. Also, the carbonation generally leads to heavy metal fixation in the residues, allowing for an environmentally less problematic disposal of the products or even their industrial re-use (e.g. road construction, cement industry). Due to the common high reactivity of alkaline fly ashes no pre-treatment (e.g. grinding, using chemical additives) is needed compared to the use of natural silicate minerals as feedstock material. For these reasons mineral carbonation of alkaline residues can be considered as a process with low costs and low energy consumption, thus making it an interesting CO2 reduction pathway from an economical point of view.
In Chapter 1, the mechanisms and rates of reactions between alkaline lignite fly ash and CO2 in aqueous suspensions were evaluated. Aqueous laboratory experiments showed that CO2 from flue gas can be bound directly as carbonate. Additionally, solutions with high dissolved inorganic carbon content are formed, which can be injected into aquifers for mineral CO2 sequestration. As the dissolution rates of the alkaline mineral phases are high, gas phase CO2 transfer into the aqueous phase is mostly the limiting factor for the overall carbonation process. CO2 dissolution is controlled by the solution pH, by the available surface area of the gas/water interface and by the gradient at that interface.
The maximum conversion of 5.2 moles of CO2 per kg fly ash (≈ 0.23 kg kg-1) obtained at 75 °C demonstrates the high potential of alkaline fly ashes to sequester CO2. This value accounts for a CO2 sequestration capacity of nearly 3.5 million t of CO2 in Germany alone based on the available lignite fly ash, which corresponds to 2 percents of the CO2 emissions from lignite power combustion (168 million t a-1 in 2009).
In Chapter 2, laboratory carbonation experiments are described, which were carried out with the individual mineral phases CaO and MgO in aqueous solution. The process showed parallels with the reactions observed during carbonation of lignite fly ashes, suggesting that Ca and Mg (hydr)oxides can be used as proxies to estimate alkaline waste reaction with CO2 in general.
The carbonation of CaO happens fast, occurs at high pH values > 12 and is controlled at the mineral surface by the dissolution of Ca(OH)2. As long as Ca(OH)2 is available CO2 uptake by the system is high and leads to the simultaneous precipitation of calcite (CaCO3). Under similar conditions MgO carbonation is a slower and much more complex process. In the presence of MgO an initial pH of ~ 10.8, indicating solubility equilibrium, was reached. Subsequently, TDIC concentrations and EC increased almost linearly. The pool of MgO based alkalinity can be made available for mineral trapping if the kinetic restrictions for precipitation of Mg-carbonate can be overcome, e. g. by running the processes at higher temperature (> 50 °C) and higher s/l-ratio. Corresponding to related work the precipitation of hydromagnesite (Mg5(CO3)4 (OH)2 ∙ 4H2O) is found for temperatures above 50 ° C already at a suspended amount of 4 g L-1. Precipitation of nesquehonite (MgCO3 ∙ 3H2O)) starts upon a suspended amount of MgO of more than 10 g L-1 at 25 ° C.
In Chapter 3 the setup and the results of a model are shown, which was used to simulate and evaluate the process of alkaline material carbonation over time. Experimentally derived specific dissolution rates for CaO/MgO and CO2 are used for the development of a kinetic geochemical model based on the freely available PHREEQC algorithm. The software offers the access to databases, which containing thermodynamic constants of all common dissolved species in natural and industrial processes.
Experimental assays conducted in an aqueous carbonation reactor (see Chapter 1 and 2) were used as reference to test the model and evaluate its robustness and sensitivity.
The reaction course of the experiments based on the use of the pure phases (CaO and MgO) was successfully reproduced by our simulations. The developed model may thus be used as a valuable tool for the optimization of technical scenarios/facilities for CO2 sequestration. In order to study different mineral sequestration scenarios for calcite precipitation, we used the simulation to test the variation of process parameters and the addition of chemical additives (CaCl2, CaSO4). Finally, the simulation of the carbonation of lignite fly ash was tested using our simplified model based on CaO, MgO, calcite, anhydrite as kinetic reactants. It was shown that advanced techniques to determine the exact mineralogy of combustion residues and the extension of the availability of thermodynamic data of specific mineral phases are necessary to improve geochemical modelling in future work.
In Chapter 4, the potential contribution of lignite fly ash to mineral CO2 trapping in a high anhydrite (CaSO4) containing aquifer were analyzed. The study examined the possibility of combining underground CO2 storage and geothermal heat/energy production from an anhydrite rich aquifer. In such a scenario Ca2+ for the precipitation of calcite could be provided from the dissolution of the calcium sulfate. The dissolution of anhydrite concurrently releases acid, being counterproductive with respect to the formation of carbonates. The possibility of pH buffering by the addition of alkaline lignite fly ash is therefore appraised to optimize the conditions of carbonate precipitation.
The performed laboratory experiments, as basis for thermodynamic simulations with PHREEC, confirmed that the buffering capacity derived from the fly ashes is essential for calcite precipitation in such a system. Already with an addition of 0.1 weight percent of fly ash per volume of the injection solution the amount of precipitated calcite was maximized. The dissolution of anhydrite is associated with a concurrent increase in pore space and can balance the porespace reduction by precipitation of carbonates and secondary silicates in the geothermal reservoir.
Impact of Oxygen and Pesticides on Microbial Cellulose Degradation in Aerated Agricultural Soils: A Microscaled Analysis of Processes and Prokaryotic Populations
- The polysaccharide cellulose is a major component of organic matter in terrestrial ecosystems and its mineralization drives carbon fluxes in soil. The degradation of plant-derived saccharides (e.g., cellulose, cellobiose, and glucose) is catalysed by a huge diversity of aerobic and anaerobic microorganisms (including Bacteria, fungi, and protists), but there is limited information about their phylogenetic identities and their in situ relevance in soil. Soil is a heterogeneous habitat in which oxic and anoxic microzones co-occur, and in which the distribution of O2 can change rapidly. Hence, the availability of O2 is an important factor that determines the activity of the saccharide-degrading community in microzones of aerated soil. Likewise, the accumulation of potential toxic pesticides in agricultural ecosystems might influence microbial activity. It is not resolved how active cellulolytic and saccharolytic taxa respond to rapid changes in the availabilities of O2. Furthermore, it is unclear if pesticides impact on the degradation of cellulose and cellulose-linked processes, and influence the activity of active saccharide-utilizing microorganisms. Hence, this study first identified cellulolytic and saccharolytic aerobic and anaerobic Prokaryotes that catalyzed the degradation of supplemented [13C]-labeled saccharides by 16S rRNA stable isotope probing. The metabolic response of major bacterial taxa to pesticides and fluctuating availabilities of O2 was further analyzed with taxon-specific qPCR assays. Eukaryotes that contributed to soil carbon flux were identified by targeting 18S rRNA genes by the collaborative group of Dr. A. Chatzinotas at the Helmholtz Centre (UFZ) in Leipzig. Cellulose, cellobiose, and glucose were mineralized to carbon dioxide under oxic conditions, whereas different fermentation products accumulated under anoxic conditions. Fermentations occurred concomitant with the apparent reduction of nitrate and ferric iron. The degradation of supplemented saccharides was stable under oxic and anoxic conditions. Archaea were no active constituents of the cellulose-degrading community in the investigated soil. [13C]-cellulose was mainly degraded by Kineosporiaceae (Actinobacteria), the cellulolytic taxon Cluster III Clostridiaceae (Firmicutes), and the new family-level taxon 'Cellu1-3' (Bacteroidetes) under anoxic conditions. Under oxic conditions, the new family-level taxa 'Sphingo1-4' (Bacteroidetes) and 'Deha1' (Chloroflexi), and Planctomycetaceae (Planctomycetes) were involved. Active community members in [13C]-cellobiose and [13C]-glucose treatments differed from those in [13C]-cellulose treatments, and were selectively activated by O2. Twenty-eight of the 48 labeled bacterial family-level taxa did not closely affiliate with cultured species. Labeled Eukaryotes belonged to the families Bodonidae, Eustigmataceae, Mallomonadaceae, Opistonectidae, unclassified Chrysophyceae, and unclassified Stramenopiles. These families inhabit autotrophic algae and bacteriovorus flagellates. It is likely that these active Eukaryotes were labeled by incorporation of [13C]-carbon derived from grazing on active cellulolytic and saccharolytic soil bacteria. Fungi were not labeled in stable isotope probing experiments. The pesticides Bentazon, MCPA and Nonylphenol impaired cellulose-linked microbial processes only at pesticide concentrations far above the recommended rate. The impairment was most pronounced under anoxic conditions. Planctomycetaceae and the new family-level taxon 'Sphingo1-4' were sensitive to pesticide addition under oxic conditions, whereas Cluster I Clostridiaceae and the new family-level taxon 'Cellu1-3' were reduced under anoxic conditions. Nevertheless, the impact of pesticides on the degradation of saccharides at in situ-relevant concentrations seems to be minimal. These collective findings suggest that (i) a large uncultured diversity of Bacteria was involved in the degradation of cellulose, (ii) O2 selectively activates different cellulolytic and saccharolytic taxa, (iii) Cluster III Clostridiaceae, and the new family-level taxa 'Sphingo1-4' and 'Cellu1-3' represent the major cellulolytic constituents of the microbial community in the investigated agricultural soil, whereas Cluster I Clostridiaceae, Intrasporangiaceae and Micrococcaceae are saccharolytic satellite organisms that utilize cellulose-derived carbon, and (iv) the degradation of plant-derived saccharides is a community function that is stabilized by the rapid response of active bacterial taxa and independent of fluctuating availabilities of O2 and of pesticide application.