BACKGROUND

Micro-organisms are sustaining the circulation in Nature. This means degrading and re-shaping of organic material. Microbes carry out this essential task by using chemical energy that is stored into organic material. This release of energy takes place either aerobically with the presence of oxygen or anaerobically in an environment that contains no oxygen. Some of the microbes are also capable to carry out photosynthesis.

The decomposition of organic materia is after all just a fraction of the universal task of the microbes in the circulation of the materia. They also carry out reactions related with the circulation of elements such as nitrogen, sulphur, phosphorus. The microbial activities have thus a fundamental and wide purpose in plant's nutrition including also these elements.

Organic substances are different types of hydrocarbons. Thus organic chemistry is chemistry of carbon compounds. Hydrogen for its part is an extremely reactive substance and a reducing agent that reacts quickly once set free, in most cases with oxygen forming then water. Carbon in turn usually becomes oxidized into carbon dioxide. The disassembling reactions or biochemical paths or single reactions carried out by microbes are led by metabolic or genetic regulation systems.

Nitrogen is one of the most important substances for the functioning of the organic molecules. Most of the nitrogen on Earth is in gas phase, it is bound into biosphere i.a. as a component of proteins and nucleic acids. In the air 78% of the nitrogen is normally molecular nitrogen. Ammonium ions can be released as an ammonium vapor from liquid phase and thus a major part of the nitrogen in the ground can evaporate out of the reach of the plants.

When fertilization in fields, gardens or forest soil without industrially processed minerals is pursued, the significance of microbes is underlined. So called humus, that Is biogenic, microbe-containing soil, is needed by the plants. It has to contain enough nitrogen, phosphorus, potassium and other nutrients and micronutrients. Advantageously the humus should also contain degraded or cracking organic compounds (macromolecules).

The relatively low concentration of many nutrients sets limits when using organic fertilizers to replace mineral fertilizers. In other words it is hard to get the same levels of the nutrient concentrations that are common amongst the inorganic mineral fertilizers, for nitrogen, for example, from natural raw materials without chemical additions. For example with nitrogen this is problematic. Most of the nitrogen in manure, for example, is ammonium nitrogen that is easily evaporated out of the reach of the plants from the soil. Many microbes have the ability to bind nitrogen of the atmosphere. This usually happens in totally anaerobic circumstances. Microbes' nitrogen bonding can be symbiotic (for example the Rhizoblum-bacterta in the tubercles of many plants like legumes). The nitrogen bonging of microbes can be symbiotic. Non-symbiotic, autonomic nitrogen fixation can be carried out by many facultative, anaerobic bacteria from the genus Klebsiella, for example, and several of the strictly anaerobic (obligate) bacteria such as Clostridium

pasteurlanum.

Many different bacterial groups participate in the nitrogen cycle in soil. Correspondingly, many of the soil bacteria participate in nitrification, i.e. the oxidization of nitrogen compounds first from ammonium form to nitrite (nltritation) and then to nitrate (nitratation). As result of bacterial action called denitrification, nitrate may be reduced to nitrite and this may evaporate as nitrogen or the oxides of nitrogen. Nitrite may also transform into nitrous acid which consequently can add acidity to soil. Nitrogen gas of the atmosphere can bind back to soil through nitrogen binding bacteria.

The transformation of nitrogen in soil into nitrate form, which is from the aspect of the plants existence easily utilized and rather stable, is result of the right conditions and the many-sided interaction of the members of the microbial community. Based on current knowledge, no single microbe has been known to alone perform the complex reactions of taking this nitrogen into use in a way that is significant in relation to plant nutrition.

Nevertheless, it has been known that not only the amounts of nutrients such as nitrogen, but also their microbiological processing in soil can affect the so-called fertilization effect

Although microbial strains often are available within soil, artificial soil or organic fertilizers, or as an addition to them, to improve soil quality and microbial growth result, the deficiency due to nitrogen evaporation remains uncorrected in plant nutrition. Also from the nitrogen that is contained in mineral fertilizers, for reasons mentioned above, significant portions disappear into the atmosphere. This decreases the fertilizing effect and impact.

The microbial communities, their strains and mixed populations that act in soil and bioprocesses, perform as guided by their internal communication systems that relate to cell density and activity. These are for example quarum sensing and other sensing type of controlling mechanisms and signaling systems including signals forwarded by gases. The understanding of the involute effects in questions is not often at the level that it could be fully exploited in biotechnology. On the other hand, no single microbe or conditions have been found in which autonomic nitrogen binding would produce significant improvements to the result of nitrogen fertilizing from the plants' perspective.

Similarly, there is a lack of device solutions for various production tasks that could take into account the above- mentioned phenomena that relate to messaging between cells, whether they are within a microbial strain or inter strains. It is still not generally possible to utilize these phenomena in the controlling or adjustment of processes. These mechanisms within and between populations are, however, in an important role for example in the activity of intestinal microbes. In plant fertilization, organic fertilizers that contain protein, such as meat bone meal, have quite not been used commonly.

DESCRIPTION OF THE INVENTION

With a method according to this invention it is possible to increase the nitrogen content of an organic fertilizer, for example with meat bone meal, waste from the egg industry or fishing industry, without the nitrogen, when ending into soil, evaporating largely as ammonia. This happens with the help of microbe masses that have been prepared by fermentation and whose pH has by normal fermentation been made to decrease under the value of 4.5 or at least under the value of 5.0. Currently, for example with a device according to this invention (figures 1 and 2), nitrogen or nitrogen compounds that are evaporating in soil can be made to revert into liquid form that is usable for plants.

The nitrification process divides into two phases:

1. Nitritation

2. Nitratation

In relation to nitritation, the optimal pH is above 7.5 and the temperature above 30 'C. The oxidative bacteria in this phase are for example Nitrosomonas sp. When conditions are favorable, nitritation happens within a few days. In nitratation the optimal pH is 6.5 - 7.5 and the temperature 25 - 30 °C. A central bacteria species is Nitrobacter. The beginning of nitratation takes normally at least 2 - 3 weeks. Compared with nitritation, in this reaction even stronger oxidization is needed.

All in all, the realization of nitrification in an optimal way requires microbial strains that function in an optimal way. In a method and device according to this invention, if necessary, reactor interstices in which the nitrification process progresses (figure 2) can be separated between the nitritation and nitratation process with semipermeable membranes or filters. These can be exploited in the directing of metabolic, genetic or other internally by the strains or between the strains. Alternatively, the binding and transformation of nitrogen into a usable form for the plants.

In this control and adjustment also inoculation with a reverse loop, in which microbes of the later phase of the process are inoculated into its earlier phase, can advantageously be exploited. Similarly, an inoculation may be done from an earlier process phase "over" an intermediate phase or phases. In fertilizer production, biomasses, from which biochemical or other biorefining products have first been collected, can advantageously be exploited.

The microbes in these intermediate stages of the process flow can, if necessary be reinoculated from one intermediate stage to another, or also to nitritation or nitratation containers. Thus the microbial community, its succession and gradient, can be developed in this bioprocess based on the interaction of microbial strains. Denitrification, i.e. the reduction of nitrate back to nitrite and further into gaseous form as nitrogen gas or nitrogen oxides, can be prevented with sufficient oxidization.

In the production process, with a method and device according to this invention, also the internal quarum sensing type of control systems within the microbial communities and the signal effects between cells or populations, can be exploited. The metabolic action of microbes can be followed also by measuring evaporating or gaseous substances from the process.

Once it has been succeeded to develop, with the help of a method and device according to this invention, an optimal microbial community to effectuate the nitrification from a desired raw material, this microbiome can further be added into an organic fertilizer as a living component. In the production stage, new microbes may be added during the process, or they can be inoculated between process stages. The microbes in question can then when acting in the soil, prevent the evaporation of nitrogen and transform ammoniacal nitrogen to a very useful: nitrate form from the point of plants' very existence.

Facultative anaerobes or strict anaerobes, or for example cyanobacteria, that are capable of binding nitrogen, can also be included in the microbial strains of the fertilizer. They can return to the soil the nitrogen that evaporates from it. Thus they improve the usefulness and utilization of nitrogen added within the fertilizer. When microbes, cultured in a way according to this invention, were added as organic living fertilizers into the soil in plant cultivation, so that the total concentration of nitrogen was standardized, they increased to growth of the plant's green parts by up to 50%. Also microbes or strains that improve the resistance of cultivated plants, can be added to the product.

If the evaporation of ammoniacal nitrogen is intended to be prevented with the decrease of pH, this biomass pH decrease that takes place with the help of fermentation can decrease the pH below 4.5. If needed, the decrease of the pH can be stopped between 4.5 - 5.5. If it is desired to combine the pH decrease caused by carbohydrates to slightly decreased nitrification, the pH can be 5.5 - 6.5. In case the evaporation of ammoniacal nitrogen is slowed down or partially prevented with the help of pH adjustment, the optimal pH can be 6.5 - 7.5 or 6.5 - 7.8. In all conditions the objective is the prevention of the evaporation of nitrogen.

In addition to the solution models presented above, in our experimental activities we have exploited Clostridium pasteurianum bacterium, which is an strictly anaerobic spore-forming bacterium, to control the nitrogen balance of soil and organic fertilizer. Surprisingly, it has been possible to find an organism similar to C. pasteurianum strains that has multiple enzyme systems in its cells, which are suited to the improvement of plant growth results. This bacteria has been added to irrigation water as a pure culture, in which case a crop increase of 100% was gained for garden cress, ryegrass and napa cabbage that were used as test plants. In figure 3 the two right hand side Chinese cabbage pots have been irrigated with water containing C. pasteurianum culture.

An especially surprising matter is, that an autonomic (non-symbiotic) nitrogen binding bacterium similar to C. pasteurianum can, contrary to the existing perception in scientific literature, in a crucial way effect the growth of cultivated plants. To this, in our experimental activity, we have found an explanation that the bacterium in question has in its cells several active important enzyme systems:

nitrogenase

- rubredoxin

ferredoxin / flavodoxin

dehydrogenase

See also figure 4. Through the action of nitrogenase, molecular nitrogen from the atmosphere binds Into ammonium form. Rubredoxin binds it into cells and releases partially in nitrate form to soil and for the use of plants. The same happens to ammoniacal nitrogen that releases from protein degradation. In the reactions hydrogen is released and similarly as a result of the action of dehydrogenases, which balances the redox conditions. The said reactions can happen in an unaccepted way also when there is molecular oxygen in soil, which should normally inhibit the fixing of nitrogen by the nitrogenases. This is possible with the help of ferredoxins and in low iron ion concentration by flavodoxins. These enzymes can bind molecular oxygen to water molecules which supports the preservation of anaerobic conditions. Thus the enzyme systems that act together make possible the efficient binding of nitrogen in soil into a form that is useful to plants.

As high ammoniacal nitrogen concentration stimulates especially well the before mentioned enzyme systems, the use of bacterial broth, obtained from C. pasteurianum bacteria and similar microbes, together with a fertilizer or their inclusion into the fertilizer itself, is most productive from the plants' nitrogen economy point of view when the organic fertilizer component contains abundantly proteins or substances that derive from them.

When hydrogen gas is formed as result of action of C. pasteurianum bacteria or other similar strictly or facultatively anaerobic bacteria, which can be used in exploiting a method or device according to this invention, this gaseous hydrogen can be used in the production of energy or chemicals. In order to reach anaerobic conditions, either nitrogen or carbon dioxide from pressure bottles, gas generators or similar are led into the bioreactor. These gases that are led into the enrichment culture may bind or assimilate to the microbial culture in which they for their part affect the growth result and metabolism, for example in a way that promotes the binding of nitrogen.