Bacteria in the biosynthesis of animal nutrition components for crews of autonomous transport systems

. The International Space Station (ISS) is currently using a water electrolysis process to produce oxygen. The hydrogen produced as a by-product is removed overboard. Carbon dioxide from crew breathing, dissolved in the atmosphere of the station, are extracted and also removed outside the station, but NASA expects to use it and convert it by the physicochemical reaction Sabatier in methane and water. Water will be used for crew needs and electrolysis, and methane is also planned to be removed overboard. When these interrelated processes work, very important substances are lost: methane, carbon dioxide, and hydrogen. This situation can be remedied by including in the life support system (LSS) of the ATS closed biotechnological regeneration cycles operating in a waste-free mode and synergistically solving the problems of complex provision of the crew with oxygen, water, and, most importantly, food. The process of biological oxidation of methane by methanotrophs bacteria is observed in nature in the first link of the food chain of the world's ocean and has long been used in terrestrial conditions for the industrial production of animal biomass for food and feed purposes. The same can be said about the use of hydrogen bacteria, which use carbon dioxide as a carbon source, with hydrogen as their energy source. Tolerance of these cultures to common nutrient media and the same technological parameters of biosynthesis allows their joint cultivation to obtain animal ingredients for crew food. The proposed work is devoted to the study of alternative processes, integrated use of carbon dioxide, methane, and hydrogen in LSS ATS.


Introduction
Physico-chemical life support systems based on consumables stocks ensured the performance of animal space flights on rockets and artificial Earth satellites, human space flight programmes on spaceships such as Vostok, Voskhod, Soyuz, Mercury, Gemini, Apollo, Shuttle and the Skylab orbital station; they made it possible to implement space flight programmes for crews of the Salyut and Mir orbital space stations, and are now successfully operating on the ISS [1][2][3][4][5][6][7][8][9][10][11].
LSS ATS of forthcoming space missions must learn to reliably and qualitatively solve life support tasks without hope of delivery and replenishment.Such tasks primarily include: providing the crew with oxygen, removing carbon dioxide and harmful micro impurities from the station atmosphere, supplying the crew with the necessary amount of drinking water and water for sanitary and household needs, supplying the crew with the necessary amount of food of a given composition and caloric content, vitamins and mineral salts, ensuring microbiological safety, ensuring the transformation of physiological and household waste [7].The fulfilment of missions and safety of ATS crews depends on qualitative and reliable LSS operation.Therefore, the design of LSS ATS should use the most modern solutions, including first of all biotechnological solutions, as they are closest to nature-like Earth technologies.In recycled ecologies (RE) the circulation of biogenic elements is organised in such a way that substances used at a certain rate by some parts of these systems are regenerated at the same rate from the final products of their exchange to the initial state by other parts and then used again in the same biological cycles [2].The biosphere of the Earth is a prime example of natural RE.Artificial RE, especially within ATS, should have as little waste as possible.In RE, there is a circulation of mass transfer flows between synthesisers of substances and their destructors.In LSS aimed at growing crops, vegetables and microalgae biomass, the main work is based on photosynthesis -these phototrophic systems ensure the growth of synthesised substances.Destructors oxidise the substances received in the process of photosynthesis and products of their vital activity to carbon dioxide and water with obtaining mineral complexes, which in a closed cycle are again used by phototrophs.Man in this system is the main heterotrophic link, he forms the requirements for the work of all other links and sets the intensity of this cycle in order to meet his needs in oxygen, water and food, while exometabolites of human activity are also included in the cycle.Onboard LSS ATS based on RE thus become a serious competitor to the physicochemical systems currently used in LSS on the ISS, with the help of which it is impossible to create a complete cycle, because the ways of physicochemical synthesis of complete food ingredients are not known.At the same time, physicochemical methods can complement RE. "The Earth is the cradle of mankind, but one cannot live in the cradle forever!", said K. E. Tsiolkovsky in his time.Up to now, only the short-term arrival of astronauts to the Moon and missions of international automatic stations to Mars can be credited as a breakthrough asset of these aspirations of mankind.The main problem of long-term manned human missions is the reliable life support of the crews both in flight and on the surface of the space objects being explored.It is necessary not only to create comfortable conditions for human stay in an aggressive environment, to provide oxygen, water and food, but also to utilise the wastes of human activity, and all these tasks should be solved without loss of substance and energy, i.e., in interconnected closed regeneration cycles, which are impossible without inclusion of biological and biotechnological links in the LSS composition [3].
The principle industrial scheme of bacterial biomass production from methane using methane-oxidising bacteria in terrestrial conditions is presented in Fig. 1.Bacteria are grown in large containers -bioreactors filled with nutrient medium saturated with necessary salts, microelements, oxygen for bacteria respiration and methane -a source of carbon and energy for microorganisms.Gaseous methane is poorly soluble in water, so it is necessary to ensure maximum mass exchange in the entire volume of the bioreactor, for which purpose recycling, special nozzles and systems of gas pre-dissolution are used.The bacterial generation process occurs at a relatively high rate in a continuous mode, the biomass is isolated from the culture liquid, inactivated and dried by thermal treatment.The biomass is a protein and vitamin concentrate with a protein (crude protein) value of over 80%, containing a wide range of amino acids (Table 1), including essential amino acids, essential trace elements and B vitamins.

Hydrogen bacteria, biosynthesis and biomass characteristics
Hydrogen bacteria use carbon dioxide as a carbon source, are able to combine with water electrolysis to supply the ATS crew with oxygen, purify water from human exometabolites, and most importantly, they are able to synthesise high-protein biomass [5].
The most studied microorganism is the bacterium Alcaligenes eutrophus.This microorganism is a typical representative of hydrogen bacteria, aerobe, grows on fully mineralised media, the gas phase of which includes CO2, hydrogen and oxygen.This culture tolerates high concentrations of hydrogen and oxygen and has a high growth rate.Crude protein levels in the biomass exceed 70% and true protein levels exceed 60%, for comparison, beef has 21-25% true protein.
Analyses of regeneration methods have shown that LSS with hydrogen bacteria are effective for flights lasting more than a year if 20% of the human diet is made up of biomass components of these bacteria [10].
Therefore, various aspects of the physiology of hydrogen bacteria important for their use in LSS have been investigated: genetic stability, utilisation of human exometabolites, effects of long-term storage conditions and sensitivity to ionising radiation [4] and [9].The optimal method of culturing hydrogen bacteria in LSS ATS conditions is continuous, in chemostat mode.Hydrogen bacteria in LSS can also act as disposers of crew exometabolites and other wastes, as well as participate in the process of water regeneration.
Hydrogen bacteria are autotrophs and have been shown to be able to assimilate urea nitrogen, which makes up 95% of urine.With a complete balance of gas and water metabolism, LSS on hydrogen bacteria can fulfil human needs for most trace elements, proteins, essential acids and some vitamins.A complete nutritional balance can be achieved by combining hydrogen bacteria LSS with components of vegetarian LSS.
Biotechnologies based on the use of the potential of living systems can solve key problems of life support: production of food, minerals and energy resources, creation of new materials, means of diagnostics and treatment, disposal of toxic waste and many others.Academician S. N. Vinogradsky's discovery of chemoautotrophy as a new way of life gained practical interest -the use of hydrogen-oxidising bacteria as a regenerative link in space closed-loop life-support systems [6].
Active research on hydrogen-oxidising bacteria was initiated by Academician G.A. Zavarzin at the S.N.Vinogradsky Institute of Microbiology.S.N.Vinogradsky Institute of Microbiology of the Russian Academy of Sciences (Moscow), where representatives of this interesting microbiological group were isolated, systematised and described [5].
In a relatively short period of time, it was possible to develop a series of laboratory facilities for mass cultivation of bacteria, to obtain and study a stable flow-through culture.Extensive experimental material on the kinetics and physiology of bacterial growth was accumulated, and changes in their metabolism under the influence of different factors were studied [1].

Fig.3. Fermenter with a volume of 3 m³ as the main element of pilot production of hydrogen bacteria biomass
In the 1980s, pilot production of a new preparation -hydrogen bacterial biomass (HBB) was created.A series of zootechnical, veterinary, medical-biological and toxicological experiments on farm animals and fur-bearing animals showed that the product as a source of protein has a high nutritional value.

Co-culture of methanotrophic and hydrogen bacteria
In hydrogen biosynthesis of protein, the same hardware solutions as in methanotrophic biosynthesis can be used, but taking into account the explosiveness of hydrogen.In this case, nutrient media and technological parameters of biosynthesis are the same, which gives the right to their joint cultivation with obtaining mixed biomass suitable as animal protein concentrate for food preparation.
To implement the proposed method in complex with pre-fermentation and finishing technologies and apparatuses, Fig. 4 shows the Principal Scheme of the whole technological installation.The scheme shows how hydrogen, methane and air after passing through the filters (12), enter the system of feed medium cooling and pre-dissolution of gases (10).By means of a pressure pump (11), the feed medium is fed into the fermenter to the inlet of the mixing aerating head.In case it is impossible to exclude the outlet of fermentation gases, the scheme shows a filtration system of gas separation (13), with the subsequent direction of hydrogen and methane -for mixing, and carbon dioxide -for the synthesis of biomass of blue-green algae Spirulina (SBGA).The biomass is separated from the culture liquid (CL) on a centrifuge (19), further dried (21) and pelleted (22).The fugate obtained after biomass separation is normalised (3 and 9) and used in the recycling system.The system of nutrient medium formation consists of water treatment unit (3), dosing containers (2), mixers (4), consumable containers (5), sterilisers (6 and 7), and the apparatus for final formation of nutrient medium (8).
Bold arrows show two protein target products: bacterial mixed protein and SBGA protein.Abbreviations: b/m -biomass; n/m -nutrient medium; c/l -culture liquid; inoculum, inoculum mixed culture; fugate, liquid phase after culture fluid separation; SBGA, Spirulina blue-green algae.

Description of the plant operation
Nutrient medium, consisting of salts of microelements, from consumable containers-dosers (2) are fed into mixers (4) and further -into consumable containers (5), where it is mixed with water from the steriliser (3).Then the nutrient medium is sent to the steriliser (6) and then to the apparatus for final formation of nutrient medium (8), where it is mixed with a solution of nutrient factors coming from the mixer-steriliser of nutrient nutrition (7), where an aqueous solution based on sterile water, salts and solutions of P, N, K and Mg is prepared.The nutrient medium thus obtained is fed to the system for cooling the nutrient medium and pre-dissolving gases in it (10).Filtered hydrogen, methane and air are also sent here.Moreover, the gases obtained by separation at the gas separation filter (13) are also sent here.At the same time, the processes are adjusted so that the entire volume of gases is fully utilised in the fermenter.The device ( 10) is also a refrigerator, which contributes to improved dissolution of the gases.The cooled nutrient medium, enriched with gases, is fed under pressure to the bottom of the fermenter (1) by means of a pressure pump (11).The inoculum, in the form of a mixed culture, is fed into the fermenter once, after the nutrient medium has been partially fed.Excess carbon dioxide, after separation on the filter (13) is fed to the photobioreactor (15) for cultivation of blue-green algae, the biomass of which is separated on the  9) is analysed and sent to the water treatment unit (3) for normalisation for further use in the recycling water supply system.Two-stage saturation with methane, hydrogen and air, first -of cooled nutrient medium at the pre-fermentation stage, then of culture liquid in the process of biosynthesis, allows to fully provide the microorganisms of mixed culture with carbon and energy sources.
The proposed method of biosynthesis of protein compounds is based on the use of mixed culture of production microorganisms.As a result of biosynthesis, carbon dioxide is released, the high concentration of which in CL, inhibits the cellular metabolism of production cultures.To solve this problem, a production strain of hydrogen bacteria is introduced into the mixed culture, which utilises hydrogen and carbon dioxide dissolved in the CL as energy and carbon sources, respectively.To utilise the exometabolites accumulated during biosynthesis, a heterotrophic bacterial strain is included in the mixed culture.Due to the thus designed mixed culture, the proposed method solves many problems associated with excessive foaming, inhibition by a large concentration of carbon dioxide, supernormative oxidation, and the process itself is maximally balanced, does not create stress for microorganisms, maximally waste-free and environmentally safe.When operating without the use of hydrogen bacteria, carbon dioxide is removed from the fermenter and used in photo-biosynthesis of SBGA biomass.
In the LSS ATS, this biosynthesis can be realised in a continuous mode under chemostat conditions, which is a fully stirred biomass suspension into which nutrient medium is fed at a constant rate and from which culture is withdrawn at the same rate, while the total volume of culture liquid remains constant [8].
Chemostat culturing theory assumes that culture growth is limited by a single component of the medium and allows predicting the relationship between growth rate, population density and the concentration of the limiting substrate in the medium.The dilution rate (D) at steadystate, when the culture grows at a constant rate under constant conditions, is equal to the specific growth rate: Depending on the ratio of dilution rate (D) and specific growth rate (μ), the following options are possible (Fig. 5).At the beginning of growth, no fresh medium is added and biomass growth proceeds as in periodic culture.After feeding fresh medium at a rate of D is possible: 1) D > μmax biomass is washed out of the fermenter and the concentration of the growth-limiting substrate tends to increase to a level of S0.In the first case, the specific biomass growth rate will be lower than μmax and will be determined by the medium flow rate D. 2) If D < μmax biomass concentration increases and stabilises at a certain level, the concentration of the limiting component of the medium decreases to the threshold concentration level and limits the growth rate.This steady state is self-regulating.
The growth rate of microorganisms in the stationary state in the fermenter we calculate by the formula: here μmax is the maximum possible growth rate; KS is Mono's substrate constant, numerically equal to the concentration of the limiting substrate, which, while limiting growth, slows it down by half.In some cases, the culture fluid contains microbial metabolic products that, accumulating in the medium, inhibit the entire reaction chain leading from the assimilated substrate to the final products.This fact confirms the necessity to use heterotrophic culture strains in the association.
According to the studies of N.D. Jerusalemsky [6], the dependence of the specific growth rate on the concentration of the product-inhibitor can be represented in the form of the following equation: here P defines concentration of the growth-inhibiting product; KP -inhibition constant, numerically equal to the concentration of the product at which the growth rate reaches half of the maximum.
This equation in form corresponds to the equation of noncompetitive inhibition of enzymatic reactions.Sometimes, wishing to take into account the simultaneous action of substrate and inhibitor, the dependence of the specific growth rate μ on S and P is written down in a combined expression: In conditions of continuous chemostat cultivation with growth limitation by a certain component of the medium, the complex of acting factors is different from that of periodic cultivation.On the one hand, the microbial population undergoes constant action of the selecting factor, which is the flow rate of the medium, and on the other hand, the culture is under constant deficiency of one of the nutritional elements and, of course, is subjected to long-term influence of the constant level of metabolic products in the medium.
The method of continuous chemostat cultivation significantly expands the possibilities of protein biosynthesis in LSS, simplifies the process of microbial growth control, provides time-reproducible results, provides the possibility of biosynthesis control in automatic mode, and significantly increases the reliability of the system.These advantages, along with the use of highly productive resistant bacterial strains, predetermine the prospects of this method of continuous cultivation in the biosynthesis of animal nutrition components for crews of autonomous transport systems using closed regenerative biotechnological cycles.

Conclusions
1.It is shown that along with higher plants, microalgae and biological objects, it is possible to produce food on board ATS on the basis of bacterial microorganisms producing balanced animal protein.
2. For biosynthesis of animal protein not only strains of methane-oxidising bacteria, but also strains of hydrogen-oxidising bacteria and associations of these cultures can be used.
3. The use of methane and hydrogen oxidising bacteria for the purposes of animal protein biosynthesis of crew food excludes losses of substances from physicochemical reactions in closed LSS regeneration cycles.4.This technology was the previously missing link that makes the LSS ATS biotechnological cycles gain a closed-loop regenerative character, giving hope for extended long-range missions without resupply.
5. The LSS ATS closed-loop biotechnological regeneration facility should be calculated based on mission duration and number of crew members.
6.It is shown that existing processes on Earth for the production of food protein from methane, carbon dioxide and hydrogen using methanotrophic and hydrogen-producing bacteria can be applied to produce food protein on board the ATS.
7. The safety of dietary protein should be confirmed by additional testing with the development of standards for their introduction into foodstuffs.
8. The possibility of application of continuous chemostat process of food protein biosynthesis for interplanetary flights, as well as in the conditions of lunar and planetary bases is proposed.

Table 1 .
Amino acid composition of methanotrophic protein (in g/100 g protein) , dried on the apparatus (17) and tableted if necessary, on the apparatus (18).The grown biomass of methanotrophic bacteria is separated from the fugate on a centrifuge (19) and fed to a dryer (21), where the biomass separated on a fugate filter (20) is also fed, after which the whole dried biomass is sent to a pelletiser (22) and packed.The chemical composition of the digestate (