Objective
A. BACKGROUND
Denitrification, one of the main branches of the global nitrogen cycle, is an energy-yielding process in which microorganisms utilise nitrate as terminal respiratory electron acceptor under oxygen limited conditions. The overall reaction sequence employs the following intermediates in a pathway where nitrate is reduced via nitrite to gaseous products:
Nitrate r Nitrite r Nitric oxide r Nitrous oxide r Dinitrogen
Denitrification is also called nitrate respiration or dissimilatory nitrate reduction, where these terms stress different physiological roles of the process. For many years it was believed to be performed exclusively by eubacteria. However, there are indications that some fungi (e.g. the pathogenic species Fusarium oxysporum) and archaea are also able to denitrify. Bacteria of many different systematic groups can perform denitrification. However, some microorganisms can reduce nitrate only to nitrite and others only to nitrous oxide. Several bacteria utilize nitrite or nitrous oxide but not nitrate. Overall, approximately 50 % of all known bacteria can perform denitrification (or at least some of its partial reactions). The occurrence of denitrification in organisms of totally unrelated affiliations suggests that denitrification has been distributed evolutionary by lateral gene transfer.
The single steps of the denitrification process are catalysed by specific reductases. The first reaction, the conversion of nitrate to nitrite, is catalysed by a Mo-containing nitrate reductase. All nitrate reductases have a molybdopterin-cofactor and contain FeS centres, and, in addition, some possess cytochrome b or c. Several forms of nitrate reductases exist (membrane-bound, periplasmic and also an assimilatory enzyme with similar characteristics to the dissimilatory enzymes) making the study of this step interesting but also complex, particularly since some organisms contain more than one type of enzyme.
The next step in the denitrification pathway is the reduction of nitrite to nitric oxide catalysed by nitrite reductase. Bacteria employ two different forms of nitrite reductase, containing either cytochrome cd1- or Cu in their prosthetic group. Organisms possess either of the two enzymes. The cytochrome cd1 containing nitrite reductase appears to be more widespread among bacteria, whereas the Cu-enzyme apparently is more conserved evolutionary. The product of the reaction, NO, in addition to being toxic and highly reactive, is an important signal molecule for plant or animal life. Intact denitrifying organisms evolve at best minute amounts of this gas, which is effectively utilized by the cytochrome b and c containing nitric oxide reductase. The conversion of NO to N2O catalysed by this enzyme involves the formation of the dinitrogen double (triple) bond, which is a biochemically extremely interesting reaction that is poorly understood currently. Finally, nitrous oxide is reduced to the dinitrogen molecule by nitrous oxide reductase which contains Cu-atoms in a novel tetra-nuclear cluster at the active site.
By the action of denitrifying microorganisms, the global dinitrogen content in the atmosphere is largely in balance due to the formation of the dinitrogen gas from terrestrial nitrate. Nitrate is the main N-source for the growth of plants in agriculture but can simultaneously by used also by microorganisms in soils. Denitrification is generally regarded as an anaerobic process, but there are indications that it may take place also in well-aerated soils. The conditions favouring denitrification in soils have not yet been elucidated in much detail. It is, however, clear that any use of nitrate by bacteria means a loss of N for the growth of plants. Thus, denitrification has severe impact on agriculture, and the COST Action will particularly put emphasis on these agricultural aspects. In addition, products of denitrification (nitrate respiration) have manifold other, mainly adverse effects on soils but also on the atmosphere and on waters. The major concern is thus agriculture, but it does not make sense to leave out environmental impacts of denitrification in this COST Action. Such impacts can be summarized as follows:
In contrast to ammonia, which is tightly bound in soil, nitrate is easily washed out and transported to the groundwater where it (and its reduction product nitrite) adversely affects water quality. On the other hand, denitrifying bacteria, together with nitrifying microbes, play a crucial role in the two-step process of biological sewage treatment. Denitrification also contributes in a beneficial way to the removal of nitrogen from polluted, eutrophic lakes. In addition, nitrogenous oxides released from soils and waters have several impacts on the atmosphere. Nitrous oxide is next to CO2 and CH4 in its importance as a potent greenhouse gas. Nitric acid and its chemical oxidation product NO2 are major constituents of acid rain, and NO and also N2O interact with ozone in complex reactions and are major causes of the destruction of the protective ozone layer in the stratosphere.
Thus the impacts of products of denitrification on agriculture as well as on waters and the atmosphere are complex, but are of extreme relevance for human welfare. In particular, the increasing N2O content of the atmosphere is giving cause for serious environmental concern. However, many aspects of the denitrification processes are poorly understood at present. This COST Action will significantly contribute to increase the knowledge in the field, particularly to understand complex interactions of the biological N-cycle by bringing together scientists from different countries and different areas of competence. There are no series of International Congresses on denitrification in contrast to other fields of the N-cycle (e.g. dinitrogen fixation, nitrate assimilation). Therefore this COST Action is necessary to concentrate European activities and to stimulate the progress in the field. It should be stressed that such goals will not be achieved without the instalment of this COST Action.
B. OBJECTIVES AND BENEFITS
The main objective of the Action is to better understand factors governing the loss of N-fertilisers in agriculture due to the microbial activities. Factors resulting in the production of deleterious substances produced by biological denitrification will also be elucidated. This will be achieved by
(a)increasing the knowledge in the molecular biology and biochemistry of denitrification, particularly studying the expression of genes coding for structural and regulatory proteins,
(b)understanding factors which direct the levels of denitrification to take place in natural environments (in particular in soils),
(c)determining the extent of deleterious nitrogenous compounds formed as side products in denitrification.
In this first phase of experiments, it is not attempted to reduce or enhance the soil microbial population. At the current state of knowledge, it is appropriate to assess the diversity in the populations of denitrifying bacteria in soils and other habitats by the use of modern molecular techniques as well as of standard activity measurements. Any manipulation of the population of denitrifying bacteria in soils on a larger scale with impacts on the environment seems to be unrealistic and out of scope. It is, however, suitable to assess alterations in the microbial life after N-fertilization of soils in pot experiments or in small size field trials. Clearly, this COST Action will be centred around studies with soils, but it does not at all make sense to exclude experiments in the aqueous habitats, as problems are closely related and as many interlinks exist between soils and waters. As molecular biological techniques like PCR are nowadays essential tools for studying microbial populations (not only in denitrification but everywhere), data obtained by the use of such techniques will be disseminated to the public. Examples of potential exploitations of molecular ecological results are numerous and might also be expected for denitrification.
This COST Action will focus on four objectives which are interlinked and which will be implemented through working groups (WG). The benefits of the Action to the scientific community will include stimulation of active communication involved at all levels of research in the field of denitrification, particularly within Europe. This will promote and enhance a multidisciplinary approval to the study of the complex reactions in denitrification, both in the laboratory and under field conditions. The benefits to the public is that factors will be identified which govern the loss of N-fertilizers in soils (loss for the growth of crops). In addition, the formation of deleterious nitrogenous compounds affecting mankind in manifold ways will be studied. This might lead to computer-based predictions of the impacts of N-fertilizations of soils on the environments (climate, waters etc) in the long run. It is believed that more than 50% of the deleterious NOx compounds (NO, NO2, N2O) are not produced by a result of human activities (industry, cars etc.) but are formed biologically by both denitrification and nitrification on a global scale. Little is known about the distribution of denitrifying bacteria in soils and aqueous habitats and the factors governing their activities in natural environments. In recent years, molecular techniques have been developed which allow this important issue to be addressed in field conditions. It is expected that the increase in the knowledge in this subject will be enormous in the next ten years and the dissemination of the results to the public for potential exploitation will be one of the major goals of the COST Action. For these reasons, the instalment of the COST Action is timely and adequate at the present date.
C. SCIENTIFIC PROGRAMME
To achieve such goals, the scientific program will focus on four working groups:
WG 1: Molecular biology and biochemistry of denitrification
This WG will concentrate on studying the genes that code for all steps (enzymes) of denitrification with modern molecular biological techniques. This includes both structural and regulatory genes. The environmental factors (dioxygen concentration, availability of carbon sources) which direct the expression of these genes (both at the transcriptional and translational levels) will be elucidated. At the protein level, studies will even reach X-ray analyses. Several proteins involved in denitrification have been crystallised (cd1 and Cu-containing nitrite reductases, N2O reductase, NO reductase), and it is expected that further ones will be analysed by X-ray crystallography in the near future. In denitrification, several enzymes might be in the centre of the studies:
(a)the different forms of nitrate reductases, several organisms can express three or even more isoenzyme complexes.
(b)catalytic properties of the two different forms of nitrite reductase: the cytochrome cd1 and the Cu-containing enzymes: identification of factors which allow these enzymes to form NO as a reaction product.
(c)formation of di- (tri-) nitrogen bond in N2O from NO catalysed by the cytochrome bc containing NO-reductase.
(d)the regulatory proteins (signal molecules), in particular those that direct the expression of components of the nitrate respiratory chain.
WG 2: Ecology of denitrification
Approximately 50 % of the bacteria have the genetic capability for denitrification, which, however, occurs in systematically unrelated groups. The distribution of these bacteria in soils and aqueous habitats is virtually unknown. Likewise, factors directing the expression of denitrification genes in natural habitats have not yet been elucidated. Recent research suggests that the community composition of denitrifying bacteria may have a direct role in controlling the net emissions of intermediate products (mainly N2O) from soils. Furthermore, the regulation of the synthesis and activities of the different reductases involved appears to be of utmost importance for the high N2O emissions due to natural perturbations such as low temperature or drought. Participants of this WG aim at addressing these aspects both by classical techniques (gas release and uptake measurements) and by modern molecular approaches. Most of the bacterial species present in soils and water have yet to be cultured in the laboratory. However, gene probes have recently been developed (or are in the course to be developed, one goal of WG 1) which will allow to assess the potential occurrence of denitrification genes in any bacterium.
DNA (and presumably also mRNA in the near future) can be isolated from soils and aqueous habitats with which the relative percentage of denitrifying bacteria in a given environment can be determined by heterologous hybridisation using these probes. The DNA isolated from the different environmental samples can be used to amplify denitrification genes of bacteria by polymerase chain reaction (PCR) - based techniques. Separation of the PCR-products obtained by these techniques like denaturing gradient gel electrophoresis, followed by cloning and sequencing will give access to a whole regime of unknown denitrifying bacteria present in field samples, even to those which have proved as non-culturable to date.
Attempts to characterize the distribution of denitrifying bacteria in soils and aquafilms by in situ techniques using such gene probes have been progressed recently by the use of FISH, quantitative PCR and other techniques. Progress in methodological development in this field is currently rapid. Partners of this WG will actively take part in this field of modern molecular ecology, last not least with the help of this COST Action. Even though we begin to understand the enormous diversity of the community composition and its expression kinetics for the denitrification genes (reductases), it might still to be too premature to expect now useful data for the improvements of simulation models for N2O emissions to the atmosphere. One can, however, envisage that such knowledge may open up for improvements of the denitrification process in selected manipulated environments such as sewage treatment plants by deliberately controlling community composition and gene expression. The increase in the knowledge of molecular biology and biochemistry of denitrification (working group 1) is expected to be rapid, and this will undoubtedly have direct impact on the ecology of the subject (this working group) in diverse and obvious ways. Links to the following working groups are obvious, particularly in the applied aspects.
WG 3: Influence of agricultural activity on denitrification
As outlined above, denitrification has many implications for both earth and atmosphere. Partners of this WG will study the release of products of denitrification like NO, N2O and NO2 from soils to the atmosphere. They will determine the influence of the agricultural practice on the production of these detrimental gases. Factors to be mentioned in this context are the use of pesticides (herbicides, fungicides, insecticides and others), the application of manure and crop residues which all may influence the rate of these detrimental N-gases both on a local and global scale. Techniques to study denitrification activities in soils will comprise the conventional 15N isotope determination, gas chromatographic N2O-formation measurements (performed ñ C2H2 which specifically blocks N2O-reductase) and infrared spectrometry using sealed boxes in soil cores and others. Soil microbe plant interactions will be studied in the context of the present agricultural practices (nitrate fertilisation). This also includes studies on nitrate uptake (uptake carriers) and nitrate reduction (nitrate and nitrite reductase) as well on ammonium assimilation. Clearly, these topics are so broad that only a limited
range of experiments can be performed and the studies can be performed by no means comprehensively. The focus of this WG will be rather the influence of agricultural activity on denitrification than the impact of denitrification on agriculture. Depending on the type of soil, the climate and the crops cultivated an appropriate balance has to be found between the input of N-fertilizers and plant needs, to minimize loss of nitrate by denitrification and or leaching to the groundwater, to prevent potential environmental pollution and health problems. Agricultural practices that influence denitrification are of major concern for the environment In this respect, the activities and outcome of this WG will help to improve the agricultural practice and to implement a sustainable agriculture as well as a precise fertilization management. Although agricultural aspects will be in the centre of the interest of this WG, other applied aspects shall not be neglected. The use of N-fertilizers in soils (even in non excessive amounts) may results in a release of nitrate (and its product nitrite in anaerobic respiration) to the groundwaters, which is of major environmental concern. The newly discovered anammox process (see WG 4) is worth to be studied also in soils. More recent evidence suggests that NO is an important signal molecule not only in animals and in human life but also in plants. The extent by which NO is released by microorganisms in soils and waters and the role of denitrification in this NO formation is virtually unknown. The determination of the N2O (and NO and NO2)-fluxes from soils and water to the atmosphere, performed on a local scale, will provide valuable information to colleagues that develop mathematical simulation models of climate changes. It should, however, be stressed that this aspect of air chemistry can only be of marginal concern for this COST Action.
Partners within this WG will aim at studying all the aspects mentioned with special emphasis on applications, in particular in agriculture and in close collaboration with potential agricultural and industrial partners.
WG 4: Denitrification and related, mainly N-gas forming processes
Denitrification is part of the biological nitrogen cycle. While denitrification is generally regarded as an anaerobic process, it is not yet clear to which extent N-gases are formed also aerobically and how far denitrifying bacteria are involved in this process. Thus one major goal of this WG is to resolve to which extent detrimental N-gases are formed in reactions other than denitrification.
Of special interest might be the newly discovered anammox reaction where ammonia and nitrite are comproportionated mainly to dinitrogen gas. Bacteria performing this reaction may be ubiquitous in nature and were claimed to account for a substantial proportion of the bacterial populations of wastewater bioreactors. Studies on the competition between bacteria performing denitrification and nitrate ammonification on the availability of nitrate might reveal which bacteria are better suited for practical application (removal of N from waste water or eutrophic lakes). N2O and NO are seemingly also formed in the anammox reaction. The reaction may also take place in soils which remains to be elucidated.
Closely related to denitrification is nitrate ammonification which is performed by E. coli and other enteric bacteria. In this process nitrate is reduced via nitrite to ammonia. Thus the first step is shared by both denitrification and nitrate ammonification, whereas the dissimilation of nitrite is catalysed by a totally different enzyme.
Studies on other aspects of the N-cycle (nitrification, dinitrogen fixation, assimilatory nitrate reduction) will also be included, however, only when they are complementary and help to the understanding of the denitrification reactions. Nitrification is of particular interest, since the trace gases NO and N2O are also produced in this process, either in the conversion of ammonia to nitrite via hydroxylamine or in the oxidation of nitrite to nitrate, possibly with the involvement of enzymes also catalysing denitrification reactions. NO and N2O can also be produced, though in minute amounts, by plants and animals. NO is an important signal molecule in animal and human life, and it is not known how much denitrifying bacteria can contribute to the formation of NO. N2O is an alternative substrate to nitrogenase. The understanding of such reactions might be also of high interest for investigating related steps of denitrification.
This is also the case with dissimilatory sulfate reduction (sulfate respiration) which is performed by bacteria also under anaerobic conditions. Relatively few bacteria can perform both nitrate and sulfate reductions. The regulatory factors (signal molecules) which direct the expression of genes
may be similar in denitrification and sulfate reduction. Therefore, gene expression studies may be of mutual interest for the understanding of both processes. The same applies also to other anaerobic respirations (fumarate-, Fe3+-, and the artificial DMSO or TMSO reductions performed by some bacteria, with no obvious physiological function for these reductions). Such subjects should also be included, however, only when the studies performed and the results obtained bear significance to problems in denitrification.
D. ORGANISATIONAND TIMETABLE
The duration of the Action will be five years. A five-year duration is considered necessary as the Action is tackling rather complex biological processes. Multi-site experiments have to be carried out over a number of years, and similarly data processing and analysis. Co-ordination in this area of research has been weak in Europe, setting up the network and make it running therefore requires and justifies the five year period. The transfer of scientific results will need more than average co-ordination and communication efforts. That is why the maximum operating period of five years is sought.
A Management Committee (MC) will co-ordinate this COST Action. The MC will meet at least once a year to assess the progress of the Action. It also prepares the annual report to communicate its progress to all interested parties. The MC will organise Working Group meetings according to the need of the Action; whenever adequate, meetings of at least two working groups shall be organised together in order to stimulate exchange between the different groups, and attempts should be made for MC meetings to run concurrently with WG meetings. It is envisaged that all WGs meet at least once a year. The MC will also be responsible for ensuring that the results from experimental work are presented in an informal and interactive way. It is also planned that joint meetings with other COST Actions of related subjects (e. g. on hydrogenases) are organised at least twice during the course of this COST Action.
A final conference will be organised at the end of the Action. At this date at the latest, the progress in the field, with emphasis of the achievements of partners, will be summarised. This will take the form of either a joint book containing all relevant chapters on denitrification, or of writing a series of review articles to be published in FEMS Microbiology Reviews, Experientia or any other highly-ranked journal. A financial support by the Commission will be requested for this activity in due time.
Training of scientists working in the field will be an essential and high-profile element of this COST Action. Therefore, the MC will encourage Short Term Scientific Missions within laboratories participating in this COST Action. In addition, younger scientists will be asked to present their data orally at the WG meetings. It is stressed that this training program of younger scientists also from less-developed areas is a priority for the success of this COST Action.
Several of the experts interested in the Action have strong links with industry. Whenever feasible, colleagues from industry will be invited to participate in the meetings. It is the intention of this COST Action to facilitate the information flow from scientists from research institutes and industry.
Possible milestones would be a joint meeting with another COST Action, either in the domain of Agriculture or in the domain of the Environment. These interaction meetings could take place after two years and at the end of this COST Action.
E. ECONOMIC DIMENSION
Partners from 12 COST countries have actively participated in the preparation of the Action or otherwise indicated their interest: Belgium, Denmark, Finland, France, Germany, Hungary, Italy, Norway, Spain, Sweden, the Netherlands and the United Kingdom. Others will be contacted when the COST Action is installed.
On the basis of national estimates provided by the represenetitives of these countries, the economic dimension of the activities to be carried out under the Actions has been estimated, in 2001 prices, at roughly Euro 30 million.
This estimate is valid under the assumption that all the countries mentioned above but no other countries will participate in the Action. Any departure from this will change the total costs accordingly.
F. DISSEMINATION PLAN
The success of this COST Action will rely on establishing effective communication between the participating scientists and the wider scientific community. This will be achieved by
(a)distributing a brochure containing the addresses of all partner labs, with the names of the scientists currently working on denitrification, a summary of the current activities in the lab (approximately half page for each participant) and a list of the relevant publications of the last five years. The basis of this information has already been laid in the preparation for this Action (see addendum)
(b)Creation of a Web site specifically for this COST Action with information similar to that listed under a). The Web site will also be used as a kind of newsletter where all relevant information can easily be distributed to all participants.
(c)Regular workshops, partly also open to a wider scientific community.
(d)Short term scientific missions between participating laboratories leading to exchange of expertise.
(e)Designing a poster containing the essential aims of this COST Action. This poster will be displayed by the partners at relevant international meetings, also at fairs where agricultural and industrial partners meet.
(f)Writing joint publications, particularly at the end, either a book or a common review issue in a journal where the essential achievements obtained by the partners will be stressed. Participants will be expected to acknowledge the support by the COST Action at the end of their original publications.
(g)Summing up and widely distributing the results obtained annually. This brochure will contain abstracts of the oral presentations of the workshops, which took place during the year. Reports of the short term scientific missions will be included. This brochure will also contain all other relevant information about the COST Action. It is aimed at widely distributing this brochure, however, depending on the financial help by the Commission on this activity.