TECHNICAL FIELD OF THE INVENTION

The present invention relates to an improved fermentation medium for the cultivation of methanotrophic organisms. In particular, the present invention relates to process for providing an improved fermentation medium.

BACKGROUND OF THE INVENTION

One of the main food resources for humans may come from meat from animals and fish, beans and peas, eggs, processed soy products, nuts, seeds etc. because of the high content of protein in the products, which has an important nutritional benefit effect on the human body and development.

Not only humans have an increasing demand for a protein rich diet, but also animals (pets or farmed animals) and fish are feed more protein rich diet in order to speed up growth and development and for the wellbeing of the animal.

However, with an increasing world population, reaching 9 billion by 2050, and an increasing demand for proteins in the animal farming and pet industry, there is strong evidence that agriculture will not be able to meet this demand and that there is serious risk of food shortage.

To meet this demand production of single cell protein (SCP) has shown to be a very interesting candidate as these SCP may be grown by fermentation of biomass through the growth of the microorganisms on hydrocarbon, nitrogen and other substrates. SCP production represents options of fail-safe mass food-production which can produce food reliably worldwide and even under harsh climate conditions.

SCP product may be used directly in food or feed products, e.g. as a liquid product or as a spray dried product. The SCP or the biomass may alternatively be further processed, e.g. by hydrolysis and/or separation, to provide special fractions, remove impurities or concentrating components, before use in a food or feed product.

For commercial production of e.g. SCP the fermentation process involves 3 fermentation stages:A batch fermentation; which is the initial propagation of the organisms where all materials except the organisms, required are decontaminated by autoclaving before, loaded to the reactor together with the organisms and the process starts. The organism used goes through all the growth phases (lag phase, exponential phase and steady state phase. Under this operation mode, conditions are continuously changed with time under unsteady-state system and require a lot work and involvement.A fed-batch fermentation; is a biotechnological operational process where one or more nutrients are feed to the bioreactor during cultivation and in which the product/s remain in the bioreactor until the end of the run. The fed-batch fermentation is following the batch fermentation and is provided to achieve very high cell concentrations of the organism, as batch fermentation would require inhibitory high concentrations of nutrients and would therefore not be possible. The fed-batch fermentation is preparing the cell culture for continuous fermentation.A continuous fermentation; is the production mode of the fermentation process where feeding the microorganism with sterile fermentation medium which is used for the cultivation of the organism and at the same time, removing part of the fermentation liquid comprising the cells with spent medium from the system. This makes a unique feature of continuous culture which is that a time-dependent steady-state that can be attained and which enables one to determine the relations between environmental conditions and microbial behaviour including both genetic and phenotypic expression.

For financial reasons, there is an interest and a drive in the industry to start the continuous and steady state fermentation as quickly as possible to save time and costs and provide the SCP product faster and profitable to the market.

Therefore, the traditional provided processes use a nitrate compound as the nitrogen source during the batch fermentation, which is a small-scale fermentation. When changing to fed-batch, which is a large-scale fermentation, where the biomass content is to be increased and the demand for nitrogen is significantly increased compared to the batch phase. Thus, in order to speed up the growth of the organism and to save costs on the nutrients, the nitrogen source is changed to an ammonium compound. During the continuous fermentation, the steady state fermentation, which is the large-scale production of the SCP where a constant of fermentation liquid comprising the biomass is withdrawn from the fermentation reactor together with addition of a constant volume fermentation medium. During this continuous fermentation, the steady state fermentation, the nitrogen source is changed to an ammonium compound.

The challenge of using ammonium as the nitrogen source is that there is a significant increased risk of poisoning the fermentation liquid with nitrite which may be formed when ammonia is oxidized under nitrification process which is the rate limiting step of nitrification. During nitrification, the microorganism is under extreme stress and nitrite may be formed. Since nitrite cannot be converted totally into nitrate the fermentation must be stopped and discarded and a new batch must be started. This process is called the route of death of the fermentation and is feared by all single cell protein produces.

Hence, an improved fermentation medium would be advantageous, and in particular it would be advantageous to provide a fermentation medium being more efficient, more reliable, capable of providing a faster growth, reducing the risk of failure, and without compromising the cost challenge in the industry.

SUMMARY OF THE INVENTION

Thus, an object of the present invention relates to an improved fermentation medium.

In particular, it is an object of the present invention to provide a new method of providing an improved fermentation medium that solves the above-mentioned problems of the prior art with cost, effectivity, speed of the fermentation, risk of failure and cost.

Thus, one aspect of the invention relates to a method for providing a fermentation medium for the cultivation of at least one methanotrophic organism, or for the cultivation of a combination of organisms comprising at least one methanotrophic organism, the method comprises the step of:(i) providing a main growth solution;(ii) optionally sterilizing the main growth solution provided in step (i);(iii) providing a trace metal solution;(iv) mixing the main growth solution (as provided in step (i) or (ii)) with the trace metal solution provided in step (iii) and providing the fermentation medium for the cultivation of at least one methanotrophic organism, or for the cultivation of a combination of organisms comprising at least one methanotrophic organism,

wherein the trace metal solution is provided in a two-step procedure and wherein the two-step procedure involves:(v) providing at least one precipitating trace metal compound;(vi) providing one or more trace metals,(vii) mixing the at least one precipitating trace metal compound (provided in step (v)) and the one or more trace metal (provided in step (vi)), providing the trace metal solution;wherein an acid is added to the precipitating trace metal compound, or to the one or more trace metal or to both the precipitating trace metal compound and to the one or more trace metal, providing an acidic dissolved composition, before the at least one precipitating trace metal compound (provided in step (v)) and the one or more trace metal (provided in step (vi)) are mixed, providing the trace metal solution.

Another aspect of the present invention relates to a fermentation medium obtainable by the method according to the present invention.

A further aspect of the present invention relates to a fermentation medium comprising the following constituents: water; one or more calcium compounds, in particular calcium chloride; an acidic dissolved precipitating trace metal compound; one or more trace metals; and one or more buffer solutions, wherein none of the constituent's precipitates.

Yet another aspect of the present invention relates to a fermentation reactor comprising a reactor tank, one or more fermentation medium inlet, a fermentation liquid outlet for withdrawing fermentation liquid from the fermentation reactor and an ion sensor or analyser.

Still another aspect of the present invention relates to a fermentation process for production of biomass by cultivating at least one methanotrophic organism, or a combination of organisms comprising at least one methanotrophic organism, the method comprises the step of:(a) adding at least one methanotrophic organism into a fermentation reactor;(b) supplying a fermentation medium according to the present invention to the fermentation reactor,(c) Allowing the at least one methanotrophic organism present in the fermentation medium to ferment, providing a fermentation liquid; and(d) recovering the fermentation liquid from the fermentation reactor providing the biomass.

An even further aspect of the present invention relates to a biomass product obtainable by the fermentation process according to the present invention.

A further aspect of the present invention relates to a protein product obtainable by the fermentation process according to the present invention.

A further aspect of the present invention relates to a food or feed product comprising the biomass product according to the present invention; or the protein product according to the present invention.

Yet an aspect of the present invention relates to the use of the feed product according to the present invention for preparing a fish feed or an animal feed, such as a pig feed, a cow feed, or a chicken feed.

