RU2811519C2 - Nutrient composition and improved methods of obtaining biomass and value added product - Google Patents

Abstract

FIELD: microorganisms cultivating.

SUBSTANCE: invention relates to a method of cultivating microorganisms. A method of producing biomass involves cultivating a microorganism selected from methanotrophs in a medium composition, adding a nutrient medium composition having a pH of less than 3 to the culture medium, and collecting the biomass. The composition of the nutrient medium with pH of less than 3 contains a microelement selected from the group consisting of MgSO₄.7H₂O, CaCl₂.2H₂O and their combinations; trace element selected from the group consisting of FeNa-EDTA, NaMoO₄.2H₂O, FeSO₄.7H₂O, ZnSO₄.7H₂O, H3BO₃, CoCl₂.6H₂O, Na₂-EDTA dihydrate, MnCl₂.4H₂O, NiCl₂.6H₂O, CuSO₄.5H₂O and combinations thereof; HNO₃ or ammonia as a nitrogen source; H₂SO₄/KHSO₄ as a sulfate source and H₃PO₄/KH₂PO₄ as a phosphate source.

EFFECT: invention allows to increase the productivity of biomass.

9 cl, 10 ex

Description

TECHNICAL FIELD

The present description relates to a composition, in particular to a nutrient medium composition and to a method for producing a nutrient medium. The description also relates to a method for producing biomass at a higher concentration using a gaseous substrate, such as C1 substrates, in the presence of said nutrient medium composition. The description also relates to a method for producing a value-added product using said culture medium composition, wherein said value-added products are obtained in increased yield.

BACKGROUND OF THE ART

To date, mostly non-homogeneous culture media have been used for the fermentation of C1 substrates. However, inhomogeneous culture media are very difficult to scale up and limit reliable performance. In addition, an inhomogeneous nutrient medium limits the production of biomass at a higher concentration and thereby affects the yield of fermentation products. In addition, the specified nutrient medium is subjected to heat sterilization before use in the bioreactor, which increases additional costs for the fermentation process.

Therefore, there is a need for a growth medium that is homogeneous and does not require heat sterilization, which can increase the concentration of biomass during fermentation and thereby cause an increase in the amount of fermentation products.

The purpose of the present disclosure is to provide a culture medium composition that is homogeneous in nature and self-sterilizing, and that increases the concentration of biomass during fermentation. The present description further develops a method for producing value-added products using a specific nutrient medium composition.

SUMMARY OF THE INVENTION

Accordingly, the present description relates to a nutrient medium composition containing MgSO ₄ .7H ₂ O, CaCl ₂ .2H ₂ O, Fe, Na-EDTA, NaMoO ₄ .2H ₂ O, FeSO ₄ .7H ₂ O, ZnSO ₄ .7H ₂ O, H ₃ BO ₃ , CoCl ₂ .6H ₂ O, Na ₂ -EDTA dihydrate, MnCl2. 4H2O, _NiCl2.6H2O , _CuSO4.5H2O , _{HNO3 , H2SO4 / KHSO4} , _{H3PO4 / KH2PO4} , optionally together with additional nitrogen-containing source, _phosphate -containing source and sulfate _- containing source.

The specified composition of the nutrient medium is homogeneous in nature, self-sterilizing, does not require thermal sterilization and can be used directly in the bioreactor without additional sterilization.

This description additionally relates to a method for preparing the specified nutrient medium composition, including mixing components selected from the group containing MgSO ₄ .7H ₂ O, CaCl ₂ .2H ₂ O, Fe, Na-EDTA, NaMoO ₄ .2H ₂ O, FeSO ₄ .7H ₂ O, ZnSO ₄ .7H ₂ O, H ₃ BO ₃ , CoCl ₂ .6H ₂ O, Na ₂ -EDTA dihydrate, MnCl ₂ .4H ₂ O, NiCl ₂ .6H ₂ O, CuSO ₄ .5H ₂ O , HNO ₃ , H ₂ SO ₄ /KHSO ₄ , H ₃ PO ₄ /KH ₂ PO ₄ , optionally together with an additional nitrogen-containing source, a phosphate-containing source or a sulfate-containing source, or a combination thereof in a predetermined manner to obtain a homogeneous nutrient medium.

The present description further relates to a method for producing biomass, comprising cultivating microorganisms in the specified nutrient medium composition; and biomass collection.

The present description further relates to a method for producing value-added products, comprising cultivating microorganisms in said nutrient medium composition; biomass collection; and separating therefrom value-added products obtained from said microorganism.

DETAILED DESCRIPTION

The present description relates to a composition, in particular to a nutrient medium composition.

In one embodiment of the present disclosure, the nutritional medium composition contains a trace element and a trace element, optionally together with an additional nitrogen-containing source, a phosphate-containing source, and a sulfate-containing source.

In another embodiment of the present disclosure, the culture medium composition contains a trace element and a trace element.

In another embodiment of the present disclosure, the nutritional medium composition includes a trace element, a trace element, a nitrogen-containing source, a phosphate-containing source, and a sulfate-containing source.

In one embodiment of the present disclosure, the trace element is selected from the group consisting of MgSO ₄ .7H ₂ O, CaCl ₂ .2H ₂ O, and combinations thereof.

In one _embodiment of the present _{disclosure , the} trace element is _selected from the _group consisting of FeNa-EDTA, NaMoO4.2H2O, _FeSO4.7H2O , _ZnSO4.7H2O , _H3BO3 , _CoCl2.6H2O , Na ₂ -EDTA dihydrate, MnCl ₂ .4H ₂ O, NiCl ₂ .6H ₂ O, CuSO4⋅5H2O and a combination thereof.

In one embodiment of the present description, the nutrient medium contains MgSO ₄ .7H ₂ O, CaCl ₂ .2H ₂ O, FeNa-EDTA, NaMoO ₄ .2H ₂ O, FeSO ₄ .7H ₂ O, ZnSO ₄ .7H ₂ O, H ₃ BO ₃ , CoCl ₂ .6H ₂ O, Na ₂ -EDTA dihydrate, MnCl ₂ .4H ₂ O, NiCl ₂ .6H ₂ O, CuSO ₄ .5H ₂ O, HNO ₃ , H ₂ SO ₄ /KHSO ₄ , H ₃ PO ₄ /KH ₂ PO ₄ , optionally together with an additional nitrogen-containing source, phosphate-containing source, sulfate-containing source, or a combination thereof.

In one embodiment of the present disclosure, the MgSO ₄ .7H ₂ O in the nutrient medium composition is contained in an amount of from about 0.1% to 1.2%.

In another embodiment of the present disclosure, MgSO ₄ .7H ₂ O in the culture medium composition is contained in an amount of about 0.1%, about 0.2%, about 0.3%, about 0.4%. about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.1%, or about 1.2%.

In one embodiment of the present description, CaCl ₂ .2H ₂ O in the nutrient medium composition is contained in an amount of from about 0.02% to 0.3%.

In another embodiment of the present disclosure, CaCl ₂ .2H ₂ O in the culture medium composition is contained in an amount of about 0.02%, about 0.04%, about 0.06%, about 0.08%, about 0.1%, about 0.12%, approximately 0.14%, approximately 0.16%, approximately 0.18%, approximately 0.20%, approximately 0.22%, approximately 0.24%, approximately 0.26%, approximately 0. 28% or approximately 0.3%.

In one embodiment, the FeNa-EDTA in the culture medium composition is present in an amount ranging from about 0% to 0.013%.

In one embodiment of the present disclosure, the NaMoO ₄ .2H ₂ O in the culture medium composition is contained in an amount ranging from about 0.000026% to 0.0003%.

In one embodiment of the present disclosure, FeSO ₄ .7H ₂ O in the nutrient medium composition is contained in an amount ranging from about 0.00005% to 0.006%.

In one embodiment of the present disclosure, the ZnSO ₄ .7H ₂ O in the culture medium composition is contained in an amount ranging from about 0.00004% to 0.0005%.

In one embodiment of the present disclosure, the H ₃ BO ₃ in the culture medium composition is present in an amount ranging from about 0 to 0.3 ppm.

In one embodiment of the present disclosure, the CoCl ₂ .6H ₂ O in the culture medium composition is contained in an amount ranging from about 0.05 ppm. up to 0.45 ppm

In one embodiment of the present disclosure, the Na ₂ -EDTA dihydrate in the culture medium composition is contained in an amount ranging from about 0 ppm. up to 8 ppm

In one embodiment of the present disclosure, the MnCl ₂ .4H ₂ O in the culture medium composition is contained in an amount ranging from about 0.02 ppm. up to 0.15 ppm

In one embodiment of the present disclosure, NiCl ₂ .6H ₂ O in the culture medium composition is contained in an amount ranging from about 0.01 ppm. up to 0.075 ppm

In one embodiment of the present disclosure, CuSO ₄ .5H ₂ O is present in the culture medium composition in an amount of from about 1 to 50 ppm.

In one embodiment of the present disclosure, the nitrogen-containing source is selected from the group consisting of sodium nitrate, sodium nitrite, potassium nitrate, potassium nitrite, ammonia, ammonium hydroxide, ammonium chloride, ammonium acetate, ammonium sulfate, nitric acid, diammonium phosphate (DAP), and any of them combinations.

In one embodiment of the present disclosure, the phosphate-containing source is selected from the group consisting of potassium dihydrogen phosphate, potassium hydrogen phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, phosphoric acid, diammonium phosphate, and any combinations thereof.

In one embodiment of the present disclosure, the sulfate-containing source is selected from the group consisting of copper sulfate, zinc sulfate, ferrous sulfate, magnesium sulfate, manganese sulfate, sulfuric acid, and any combination thereof.

In one embodiment of the present disclosure, the ratio of elemental nitrogen to phosphate in the culture medium composition ranges from about 1:2 to 10:1.

In another embodiment of the present disclosure, the nutritional medium composition has a nitrogen to phosphate ratio of about 1:2, about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, or about 10:1.

In one embodiment of the present disclosure, the ratio of elemental nitrogen to sulfate in the culture medium composition ranges from about 1:1 to 10:1.

In another embodiment of the present disclosure, the nutritional medium composition has an elemental nitrogen to sulfate ratio of about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1 , about 8:1, about 9:1, or about 10:1.

In one embodiment of the present disclosure, in the culture medium composition, the ratio of elemental nitrogen to phosphate and the ratio of elemental nitrogen to sulfate varies depending on the microorganism cultivated in the specified culture medium composition.

In one embodiment of the present disclosure, the culture medium composition is self-sterilizing due to the components used therein. The specified composition of the nutrient medium does not require thermal sterilization or any additional sterilization.

In another embodiment of the present disclosure, the culture medium composition is self-sterilized by components present in the composition that are acidic or basic in nature. A nitrogen-containing source selected from the group consisting of HNO ₃ , NH ₃ and a combination thereof, a phosphate source selected from the group consisting of H ₃ PO ₄ , KH ₂ PO ₄ and a combination thereof), and a sulfate-containing source H ₂ SO ₄ (KHSO ₄ ), and their combination in the nutrient medium composition gives the composition the ability to self-sterilize. In addition, the presence of HNO ₃ , H ₂ SO ₄ /KHSO ₄ and H ₃ PO ₄ /KH ₂ PO ₄ in the culture medium composition makes the medium homogeneous and results in a pH of less than 3, preferably a pH of about 2.5, at which contaminants are not present. can survive. Thus, the requirement for additional sterilization before use of the composition is overcome.

In one embodiment of the present disclosure, the culture medium composition is uniquely designed by synergistically combining HNO ₃ , H ₂ SO ₄ /KHSO ₄ and H ₃ PO ₄ /KH ₂ PO ₄ as a nitrogen source, sulfate source and phosphate source, respectively. These components play a critical role in ensuring a homogeneous mixture and at the same time provide cellular nutrients in optimal ratios to ensure high cell density and biomass growth rate. In addition, the presence of acidic components in the nutrient medium composition eliminates the need to sterilize the nutrient medium composition and neutralizes the growth of contaminants. Thus, the culture medium composition of the present invention overcomes the limitations in reliable process scaling with clear advantages.

In one embodiment of the present description, the composition of the nutrient medium is a homogeneous mixture.

In another embodiment of the present disclosure, the culture medium composition is homogeneous, despite having approximately 10 times the concentration in solution, and does not form any sediment, which plays a role in obtaining high cell densities and biomass growth rates.

In one embodiment of the present disclosure, the nutrient medium composition increases the productivity of the biomass during fermentation.

In another embodiment of the present disclosure, the nutrient medium increases biomass productivity during the fermentation of gaseous substrates.

In one embodiment of the present disclosure, the growth medium composition increases biomass productivity by at least about 2-8 g/L reactor volume per hour during fermentation of gaseous substrates compared to a fermentation process known in the art using a flow reactor with a stirrer (CSTR), whose productivity is approximately 1.8 g/l of reactor working volume per hour.

In one embodiment of the present disclosure, the growth medium composition increases biomass productivity by at least about 2 g/L, about 3 g/L, about 4 g/L, about 5 g/L, about 6 g/L , by about 7 g/L or about 8 g/L reactor volume per hour during fermentation of gaseous substrates compared to a fermentation process known in the art with a continuous stirred tank reactor (CSTR), whose productivity is approximately 1.8 g/l of reactor working volume per hour.

In one embodiment of the present disclosure, the media composition increases biomass productivity by at least about 2-8 g/L of media per hour under batch operation, semi-continuous operation, and continuous operation.

The present description further relates to a method for preparing a nutrient medium composition.

In one embodiment of the present disclosure, a method of preparing a nutritional medium composition includes mixing a trace element and a trace element, optionally together with a nitrogen-containing source, a phosphate-containing source, and a sulfate-containing source, using a predetermined method at a given temperature for a given time to obtain a nutritional media composition.

In another embodiment of the present disclosure, a method of preparing a nutritional medium composition includes mixing a trace element and a trace element by a predetermined method at a given temperature for a given time to obtain a nutritional medium composition.

In another embodiment of the present disclosure, a method of preparing a nutritional medium composition includes mixing a trace element, a trace element, a nitrogen-containing source, a phosphate-containing source, and a sulfate-containing source in a predetermined method at a given temperature for a predetermined time to obtain a culture media composition.

In another embodiment of the present description, a method for preparing a nutrient medium composition includes mixing MgSO ₄ .7H ₂ O, CaCl ₂ .2H ₂ O, Fe, Na-EDTA, NaMoO ₄ .2H ₂ O, FeSO ₄ .7H ₂ O, ZnSO ₄ . 7H ₂ O, H ₃ BO ₃ , CoCl ₂ .6H ₂ O, Na ₂ -EDTA dihydrate, MnCl ₂ .4H ₂ O, NiCl ₂ .6H ₂ O, CuSO ₄ .5H ₂ O, HNO ₃ , H ₂ SO ₄ /KHSO ₄ , H ₃ PO ₄ /KH ₂ PO ₄ and an additional nitrogen-containing source, an additional phosphate-containing source, and an additional sulfate-containing source by a given method at a given temperature for a given time to obtain a nutrient medium composition

In another embodiment of the present description, in the method of preparing a nutrient medium composition, a specified/specified amount of MgSO ₄ .7H ₂ O, CaCl ₂ .2H ₂ O, Fe, Na-EDTA, NaMoO ₄ .2H ₂ O, FeSO ₄ .7H ₂ O, ZnSO ₄ .7H ₂ O, H ₃ BO ₃ , CoCl ₂ .6H ₂ O, Na ₂ -EDTA dihydrate, MnCl ₂ .4H ₂ O, NiCl ₂ .6H ₂ O and CuSO ₄ .5H ₂ O are mixed, then water is added and stir again. Then a given amount of HNO ₃ , H ₂ SO ₄ /KHSO ₄ and/or H ₃ PO ₄ /KH ₂ PO ₄ is added to the mixture and mixed thoroughly to cause the dissolution of all components. Thereafter, additional nitrogen-containing source, additional phosphate-containing source, and/or additional sulfate-containing source are added and mixed for about 10 minutes to obtain a homogeneous solution. The final volume of the carrier is adjusted with water.

The present description also relates to a method for producing biomass using the above-mentioned nutrient medium composition.

In one embodiment of the present disclosure, a method for producing biomass using said nutrient medium composition causes the concentration of biomass to increase during fermentation.

In another embodiment of the present disclosure, a method for producing biomass using said growth medium composition relates to increasing the concentration of biomass during the fermentation of gaseous substrates.

In one embodiment of the present description, a method for producing biomass includes:

cultivating the microorganism in a nutrient medium composition;

adding a nutrient composition having an acidic pH to the culture; And

harvesting biomass to produce biomass.

In one embodiment of the present description, in a method for producing biomass, cultivating a microorganism includes:

inoculating the nutrient medium composition with a microorganism and subsequently providing a gaseous substrate and oxygen; And

adding a nutrient medium composition.

In another embodiment of the present disclosure, in a method for producing biomass, cultivating a microorganism includes:

inoculating the nutrient medium composition with a microorganism and subsequently providing a gaseous substrate and oxygen; And

monitoring microorganism cell density and adding a nutrient medium composition.

In one embodiment of the present disclosure, in a method for producing biomass, the growth media composition has a pH of less than 3, preferably a pH of about 2.5, wherein said pH of the growth media composition allows the media composition to control undesirable microorganisms.

In one embodiment of the present disclosure, the growth medium composition added during the biomass production process contains various components in varying amounts, which are within the scope of the components and their amounts described above for the growth medium composition.

In another embodiment of the present disclosure, in a method for producing biomass, the compositions of the nutrient medium for cultivation and addition are the same or different, and wherein the composition of the nutrient medium for addition to the culture has a pH of less than 3, preferably a pH of about 2.5.

In one embodiment of the present disclosure, in a method for producing biomass, the cultivation of a microorganism is carried out at a temperature in the range of about 5°C to 50°C and under a pressure in the range of about 0 bar to 5 bar.

In one embodiment of the present disclosure, in a method for producing biomass, the microorganism is cultured at a temperature of about 5°C, about 10°C, about 15°C, about 20°C, about 25°C. C, about 30°C, about 35°C, about 40°C, about 45°C or about 50°C, and at a pressure of about 0 bar, about 1 bar, about 2 bar, about 3 bar, about 4 bar or approximately 5 bar.

In one embodiment of the present disclosure, during the biomass production process, the growth medium composition is added once the microorganism cell density reaches a range of about 0.15% to 2%.

In another embodiment of the present disclosure, during the biomass production process, the growth medium composition is added once the microorganism cell density is about 0.15%, about 0.25%, about 0.5%, about 0.75%, about 1%, about 1.25%, about 1.5%, about 1.75%, or about 2%.

In one embodiment of the present disclosure, the biomass production method involves culturing the microorganisms for a period of from about 120 hours to about 900 hours.

In another embodiment of the present disclosure, in a method for producing biomass, the cultivation of microorganisms is carried out for a period of about 120 hours, about 140 hours, about 160 hours, about 180 hours, about 200 hours, about 220 hours, about 240 hours, about 260 hours, about 280 hours, approximately 300 hours, approximately 320 hours, approximately 340 hours, approximately 360 hours, approximately 380 hours, approximately 400 hours, approximately 420 hours, approximately 440 hours, approximately 460 hours, approximately 480 hours, approximately 500 hours, approximately 520 hours, approximately 540 hours, approximately 560 hours, approximately 580 hours, approximately 600 hours, approximately 620 hours, approximately 640 hours, approximately 660 hours, approximately 680 hours, approximately 700 hours, approximately 720 hours, approximately 740 hours, approximately 760 hours, approximately 780 hours, approximately 800 hours, approximately 820 hours, approximately 840 hours, approximately 860 hours, approximately 880 hours or approximately 900 hours.

In one embodiment of the present disclosure, in a method for producing biomass, cultivating a microorganism involves fermenting a gaseous substrate by microorganisms in the presence of a nutrient medium composition as defined above and air.

In one embodiment of the present disclosure, the gaseous substrate is selected from the group consisting of methane, natural gas, synthesis gas, landfill gas, carbon monoxide, biogas, and any combination thereof.

In another embodiment of the present disclosure, the gaseous substrate is a C1 substrate selected from the group consisting of methane, methanol, carbon dioxide, carbon monoxide, and any combination thereof.

In one embodiment of the present disclosure, the gaseous substrate is contained in a concentration ranging from about 1 mg/L to 8 mg/L.

In another embodiment of the present disclosure, the gaseous substrate is contained at a concentration of about 1 mg/L, about 2 mg/L, about 3 mg/L, about 4 mg/L, about 5 mg/L, about 6 mg/L, about 7 mg /l or approximately 8 mg/l.

In one embodiment of the present disclosure, a gaseous substrate, such as methane, having a purity ranging from about 40% to 100%, is used in the fermentation.

In another embodiment of the present disclosure, the methane has a purity of about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% or about 100%.

In one embodiment, the oxygen used in the biomass production process has a concentration ranging from about 1 mg/L to 10 mg/L and a purity ranging from about 40% to 99.9%.

In another embodiment of the present disclosure, the oxygen used in the biomass production process is present at a concentration of about 1 mg/L, about 2 mg/L, about 3 mg/L, about 4 mg/L, about 5 mg/L, about 6 mg /l, about 7 mg/l, about 8 mg/l, about 9 mg/l or about 10 mg/l, and has a purity of about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99.9%.

In one embodiment of the present description, the microorganism used in the method of producing biomass is selected from the group consisting of Methylococcus capsulatus, Methylobacterium extorquens, Methylomicrobium album, Methylocapsa acidiphila, Methylobacterium organophilum, Methylobacterium mesophilicum, Methylobacterium dichloromethanicum, Methylocella silvestris, Methylosinus trichosporium, Methylobacillus flag ellatus KT , Methylibium petroleiphilum PM1, Methylobacterium nodulans, Methylobacterium populi, Methylobacterium chloromethanicum, Methylacidiphilum infernorum V4, Methylophilus methylotrophus, Methylomonas methanica, Methylobacterium rhodesianum MB 126, Methylobacter tundripaludum, Methylobacterium sp.4-46, Methylovorus glucose trophus SIP3-4, Mycobacterium smegmatis, Methylobacterium rhodesianum , Methylosinus sporium, Methylocella palustris, Methylobacterium fujisawaense, Methylocystis parvus, Methylovulum miyakonense, Methylobacterium rhodinum, Methylocystis echinoides, Methylomonas rubra, Methylococcus thermophilus, Methylobacterium aminovorans, Methylobacterium thiocyanatum, Methylobacterium zatmanii, Acidith iobacillus ferrivorans, Methylobacterium aquaticum, Methylobacterium suomiense, Methylobacterium adhaesivum, Methylobacterium podarium, Methylobacter whittenburyi, Crenothrix polyspora, Clonothrix fusca, Methylobacter bovis, Methylomonas aurantiaca, Methylomonas fodinarum, Methylobacterium variabile, Methylocystis minimus, Methylobacter vinelandii, Methylobacterium hispanicum, Methylomicrobium japanense, Methylococcaceae bacterium, Methylocystis methanolicus and any combination thereof.

In one embodiment of the present disclosure, in a method for producing biomass, the pH is maintained by a basic or acidic compound of a nitrogen-containing source, a phosphate-containing source, a sulfate-containing source, or any combination thereof of a nutrient medium composition.

In another embodiment of the present disclosure, in a biomass production method, the pH is maintained by a basic or acidic compound of a nitrogen-containing source, a phosphate-containing source, a sulfate-containing source, or any combination thereof of a nutrient medium composition and together with compounds selected from the group consisting of sodium hydroxide, hydrochloric acid, and a combination thereof.

In one embodiment of the present disclosure, a method for producing biomass includes a substrate-dependent control strategy that simplifies the process and reduces the costs associated with performing the method, making the method highly efficient and economical in producing biomass at elevated levels.

In one embodiment of the present disclosure, in a biomass production method, the substrate-dependent control strategy includes adjusting or maintaining the pH with a basic or acidic compound of a nitrogen source, a phosphate source, a sulfate source, or any combination thereof in the growth medium composition.

In one embodiment of the present disclosure, said biomass production method causes increased production with a steady state biomass density in the range of about 2% to 5%, wherein the increased productivity is in the range of about 0.5 g*L ^-1 *h ^-1 up to 8 g*l ^-1 *hour ^-1 .

In one embodiment of the present disclosure, in the specified biomass production method, increased levels of biomass are obtained by adjusting the mass flow rate of carbon content to about 30 grams per liter of reactor volume per hour, adjusting the oxygen content to about 130 grams per 1 liter of reactor volume per hour, control nitrogen to approximately 24 grams per liter of reactor volume per day, control phosphate to approximately 14 grams per liter of reactor volume per day, control sulfate to approximately 16 grams per liter of reactor volume per day, control trace elements to approximately 50 grams per liter of reactor volume per day and control trace elements to approximately 1 gram per liter of reactor volume per day.

In one embodiment of the present description, the specified method for producing biomass uses a nutrient medium composition as defined above, which is self-sterilizing and does not require thermal sterilization or any additional means of sterilization. Thus, this method of producing biomass is largely the best economically and energetically.

In one embodiment of the present disclosure, in a method for producing biomass, an important aspect lies in the fact that the cultivation of microorganisms in the presence of a gaseous substrate to produce biomass occurs in the presence of additive/continuous media containing components that allow the media to be stored at a pH of less than 3, preferably at The pH is approximately 2.5, thereby regulating the growth of contaminants. Although the media composition of the present invention is formulated to achieve this result, one skilled in the art will appreciate that any media composition that achieves this requirement of an acidic pH of less than 3, preferably a pH of about 2.5, can alternatively be used to achieve the same.

The present description further relates to a method for producing value-added products using a culture medium composition.

In one embodiment of the present disclosure, a method for producing value-added products includes:

cultivating the microorganism in a nutrient medium composition;

adding a nutrient composition having an acidic pH to the culture;

biomass collection; And

separating there the value-added product derived from biomass.

In another embodiment of the present disclosure, in a method for producing value-added products, cultivating a microorganism includes:

inoculation of the nutrient medium with a microorganism and subsequent provision of a gaseous substrate and oxygen; And

adding nutrient medium.

In another embodiment of the present disclosure, in a method for producing a value-added product, cultivating a microorganism includes:

inoculation of the nutrient medium with a microorganism and subsequent provision of a gaseous substrate and oxygen; And

monitoring microorganism cell density and adding nutrient medium.

In one embodiment of the present disclosure, in the biomass production method, the growth medium composition has a pH of less than 3, preferably a pH of about 2.5. The specified pH of the culture medium composition allows the medium composition to control unwanted microorganisms.

In one embodiment of the present disclosure, the growth medium composition added during the biomass production process contains various components in varying amounts, which are within the scope of the components and their amounts described above for the growth medium composition.

In another embodiment of the present disclosure, in a method for producing a value-added product, the compositions of the culture medium and addition are the same or different; and wherein the nutrient composition to be added to the culture has a pH of less than 3, preferably a pH of about 2.5.

In one embodiment of the present disclosure, in a method for producing value-added products, the cultivation of microorganisms includes fermentation of a gaseous substrate by microorganisms in the presence of a culture medium composition as defined above and air.

In one embodiment of the present disclosure, a method for producing value-added products further includes separating and purifying the value-added products.

In one embodiment of the present disclosure, the method allows for the efficient production of bio-based value-added products from gaseous substrates, where the method can be carried out in a centralized large-scale fermentation facility or in a decentralized smaller-scale fermentation facility.

In one embodiment of the present disclosure, the microorganism used in the method for producing the value-added product may be a wild-type microorganism, a recombinant microorganism, or a combination thereof.

In one embodiment of the present disclosure, in a method for producing a value-added product, the culture medium composition is added once the microorganism cell density reaches a range of about 0.15% to 2%.

In another embodiment of the present disclosure, in a method for producing a value-added product, the culture medium composition is added once the microorganism cell density reaches about 0.15%, about 0.25%, about 0.5%, about 0.75%, about 1 %, about 1.25%, about 1.5%, about 1.75%, or about 2%.

In one embodiment of the present disclosure, in a method for producing a value-added product, the cultivation of the microorganism is carried out at a pressure ranging from about 0 bar to 5 bar.

In one embodiment of the present disclosure, in a method for producing a value-added product, the microorganism is cultured under a pressure of about 0 bar, about 1 bar, about 2 bar, about 3 bar, about 4 bar, or about 5 bar.

In one embodiment of the present disclosure, in a method for producing a value-added product, the microorganism is cultured at a temperature in the range of about 20 to 50°C.

In another embodiment of the present disclosure, in a method for producing a value-added product, the microorganism is cultured at a temperature of about 20°C, about 25°C, about 30°C, about 35°C, about 40°C, about 45°C, or about 50 °C.

In one embodiment of the present disclosure, in a method for producing a value-added product, the microorganism is cultured for a period of from about 120 hours to 900 hours.

In one embodiment of the present disclosure, in a method for producing a value-added product, the microorganism is cultured for a period of about 120 hours, about 140 hours, about 160 hours, about 180 hours, about 200 hours, about 220 hours, about 240 hours, about 260 hours, approximately 280 hours, approximately 300 hours, approximately 320 hours, approximately 340 hours, approximately 360 hours, approximately 380 hours, approximately 400 hours, approximately 420 hours, approximately 440 hours, approximately 460 hours, approximately 480 hours, approximately 500 hours, approximately 520 hours, approximately 540 hours, approximately 560 hours, approximately 580 hours, approximately 600 hours, approximately 620 hours, approximately 640 hours, approximately 660 hours, approximately 680 hours, approximately 700 hours, approximately 720 hours, approximately 740 hours, approximately 760 hours, approximately 780 hours, approximately 800 hours, approximately 820 hours, approximately 840 hours, approximately 860 hours, approximately 880 hours or approximately 900 hours.

In one embodiment of the present disclosure, in a method for producing a value-added product, culturing microorganisms in a nutrient medium composition causes fermentation of the gaseous substrate.

In one embodiment of the present disclosure, in a method for producing a value-added product, the gaseous substrate is selected from the group consisting of methane, natural gas, synthesis gas, landfill gas, carbon monoxide, biogas, and any combination thereof.

In another embodiment of the present disclosure, the gaseous substrate is a C1 substrate selected from the group consisting of methane, methanol, carbon dioxide, carbon monoxide, and any combination thereof.

In one embodiment of the present disclosure, in a process for producing a value-added product, the gaseous substrate is present at a concentration ranging from about 1 mg/L to 8 mg/L.

In another embodiment of the present disclosure, in a method for producing a value-added product, the gaseous substrate is contained at a concentration of about 1 mg/L, 2 mg/L, 3 mg/L, 4 mg/L, 5 mg/L, 6 mg/L , 7 mg/l or 8 mg/l.

In another embodiment of the present disclosure, in a process for producing a value-added product, the methane has a purity of about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80 %, about 85%, about 90%, about 95%, or about 100%.

In one embodiment of the present disclosure, in a process for producing a value-added product, oxygen is present in a concentration ranging from about 1 mg/L to 10 mg/L and having a purity ranging from about 40% to 99.9%.

In another embodiment of the present disclosure, in a process for producing a value-added product, oxygen is present at a concentration of about 1 mg/L, about 2 mg/L, about 3 mg/L, about 4 mg/L, about 5 mg/L, about 6 mg /L, about 7 mg/L, about 8 mg/L, about 9 mg/L, or about 10 mg/L, and the oxygen has a purity of about 40%, about 45%, about 50%, about 55%, about 60% , about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99.9%.

In one embodiment of the present disclosure, in a process for producing a value-added product, the fermenter and parameters are optimized so that the gas conversion level (C1 carbon) is at least 1 g/l/h. The gas conversion rate refers to the utilization rate of the gaseous substrate that is fed into the reactor.

In another embodiment of the process for producing a value-added product, the gas conversion level (C1 carbon) is in the range of about 0.1 g/L/hr to 20 g/L/hr.

In another embodiment of the present disclosure, in a process for producing a value-added product, the gas conversion level (C1 carbon) is about 0.1 g/L/hr, about 0.5 g/L/hr, about 1 g/L/hr, about 2 g/l/h, approximately 3 g/l/h, approximately 4 g/l/h, approximately 5 g/l/h, approximately 6 g/l/h, approximately 7 g/l/h, approximately 8 g /l/h, approximately 9 g/l/h approximately 10 g/l/h, approximately 11 g/l/h, approximately 12 g/l/h, approximately 13 g/l/h, approximately 14 g/l/ h, about 15 g/l/h, about 16 g/l/h, about 17 g/l/h, about 18 g/l/h, about 19 g/l/h, about 20 g/l/h.

In one embodiment of the present disclosure, in a method for producing a value-added product during the cultivation of microorganisms, process parameters are optimized for ideal fermentation of gaseous substrates. Process parameters to be optimized include, but are not limited to, substrate gas flow rate, air (oxygen) flow rate, substrate gas to air ratio, superficial gas velocity, substrate gas recirculation rate, dissolved carbon dioxide concentration in the medium, gas-liquid separation, specific growth rate, fermentation temperature, agitation, pressure and pH or any combination of parameters.

In another embodiment of the present disclosure, the key parameters that are optimized during fermentation are gas residence time, gas exchange ratio, substrate gas flow rate/methane flow rate, air flow rate, substrate gas:air ratio or methane:air ratio and pressure or any a combination of these parameters.

In one embodiment of the present disclosure, in a method for producing a value-added product, the flow rate of the substrate gas refers to the flow rate of methane or a combination of methane and air (oxygen), which further refers to the volume of gas supplied to the fermentation medium per unit time. The optimal gas flow rate depends on the size of the fermenter. The gas flow rate varies from 0.05 rpm to 3 rpm.

In one embodiment of the present disclosure, in a method for producing a value-added product, a gaseous substrate, such as methane, is fed to a feed composition as defined above, wherein the gaseous substrate may be underutilized and a percentage of the methane may be expelled from the reactor unused. Consequently, the effluent will contain varying amounts of substrate gas, which is released along with carbon dioxide. The ratios of methane and carbon dioxide in the effluent gas stream often vary with higher amounts of carbon dioxide than methane present. This effluent is recirculated to the reactor to further increase the overall substrate gas conversion yield. The rate of substrate gas in the recycle stream is optimized for maximum product conversion. The carbon dioxide in the fermenter effluent stream is purified and the clean gaseous stream is returned to the fermenter. In some cases, the output stream is recirculated as is. As the gaseous substrate is continuously introduced into the medium, methane is also continuously separated from the liquid and forced out of the fermenter.

In one embodiment of the present disclosure, in a method for producing a value-added product, air flow rate refers to the volume of oxygen supplied to the nutrient composition per unit time. The optimal air flow rate depends on the size of the reactor.

In one embodiment of the present disclosure, in a process for producing a value-added product, the oxygen flow rate varies depending on the size of the reactor. The operating range of oxygen flow rate varies from 0.05 rpm to 3 rpm. In some cases it can vary from about 0.1 rpm to 1 rpm.

In one embodiment of the present disclosure, in a process for producing a value-added product, the ratio of substrate gas to oxygen is in the range of about 1:0.1 to 1:5.

In another embodiment of the present disclosure, in a method for producing a value-added product, the ratio of substrate gas to oxygen is about 1:0.1, about 1:0.2, about 1:0.3, about 1:0.4, about 1: 0.5, approximately 1:0.6, approximately 1:0.7, approximately 1:0.8, approximately 1:0.9, approximately 1:1, approximately 1:2, approximately 1:3, approximately 1: 4 or approximately 1:5.

In one embodiment of the present disclosure, in the process for producing a value-added product, the applied superficial gas velocity is from about 0.01 m/s to 0.5 m/s.

In another embodiment of the present disclosure, in a method for producing a value-added product, the superficial gas velocity used is about 0.01 m/s, about 0.02 m/s, about 0.03 m/s, about 0.04 m/s, or approximately 0.05 m/s.

In one of the options for the implementation of this description, in the method of obtaining a product with added, microorganism is selected from a group containing Methylococcus Capsulatus, Methylobacterium Extorquens, Methylomicrobium album, Methylocapsa Acidiphila, Methyloba, Methylolob Acterium Organophilum, Methylobacterium Mesophilicum, Methylobacterium Dichloromethanicum, Methylocellia Silvestris, Methylosinus Trichosporium, Methylobacillus flagellatus KT, Methylibium petroleiphilum PM1, Methylobacterium nodulans, Methylobacterium populi, Methylobacterium chloromethanicum, Methylacidiphilum infernorum V4, Methylophilus methylotrophus, Methylomonas methanica, Methylobacterium rhodesianum MB 126, Methylobacter tundripaludum, Methylobacterium sp.4-46, Methy lovorus glucosetrophus SIP3-4, Mycobacterium smegmatis, Methylobacterium rhodesianum, Methylosinus sporium, Methylocella palustris, Methylobacterium fujisawaense, Methylocystis parvus, Methylovulum miyakonense, Methylobacterium rhodinum, Methylocystis echinoides, Methylomonas rubra, Methylococcus thermophilus, Methylobacterium aminovorans, Methylobacterium thiocyanatum, Methylobacterium zatmanii, Acidithiobacillus ferrivorans, Methylobacterium aquaticum, Methylobacterium suomiense, Methylobacterium adhaesivum , Methylobacterium podarium, Methylobacter whittenburyi, Crenothrix polyspora, Clonothrix fusca, Methylobacter bovis, Methylomonas aurantiaca, Methylomonas fodinarum, Methylobacterium variabile, Methylocystis minimus, Methylobacter vinelandii, Methylobacterium hispanicum, Methylomicrobium japanense, Methylococcaceae bacterium , Methylocystis methanolicus and a combination thereof.

In one embodiment of the present disclosure, in a method for producing a value-added product, the pH is maintained by a basic or acidic compound of a nitrogen-containing source, a phosphate-containing source, a sulfate-containing source, or any combinations thereof of the growth medium composition.

In another embodiment of the present disclosure, in a method for producing a value-added product, the pH is maintained by a basic component or an acid component of a nitrogen-containing source, a phosphate-containing source, a sulfate-containing source, or any combinations thereof of a nutrient medium, along with compounds selected from the group consisting of sodium hydroxide, hydrochloric acid, and their combination.

In another embodiment of the present disclosure, a method for producing a value-added product has a substrate-dependent control strategy that simplifies the nature of the method and reduces the costs associated with performing the method, such that the method is substantially efficient and economical in producing the product with added value.

In one embodiment of the present disclosure, the substrate-dependent control strategy includes adjusting or maintaining the pH with a base component or an acid component of a nitrogen source, a phosphate source, a sulfate source, or any combination thereof of the growth media composition.

In one embodiment of the present disclosure, said process for producing a value-added product causes increased production of value-added products such as lactic acid, succinic acid, formic acid, acetic acid, malic acid, beta-carotene, lutein, zeaxanthin, lycopene, astaxanthin , methanobactin annatto, peptides, ectoine, indigo and mandelic acid compared to methods known in the art. The increased level of production of value-added products is particularly attributed to the culture medium composition of the present invention and the tandem operation of the culture medium composition and process parameters used in said process for producing value-added products.

In one embodiment of the present disclosure, lactic acid is produced at a concentration ranging from about 5 g/L to 120 g/L, succinic acid is produced at a concentration ranging from about 5 g/L to 50 g/L, formic acid is produced at a concentration in the range of about 5 g/L to 50 g/L, acetic acid is produced in concentrations in the range of about 5 g/L to 50 g/L, malic acid is produced in concentrations in the range of about 5 g/L to 50 g/L l, beta-carotene is produced in concentrations ranging from about 0.5 g/L to 50 g/L, lutein, zeaxanthin, lycopene, peptides are produced in concentrations ranging from 0.1 to 10 g/L, ectoine is produced in concentrations in the range of 0.1 to 10 g/L, indigo is produced in a concentration ranging from 0.1 to 10 g/L, mandelic acid is produced in a concentration ranging from 0.1 to 20 g/L and annatto is produced in a concentration of range from about 0.5 g/L to 5 g/L, respectively, and methanobactin is produced in a concentration range from about 1 g/L to 5 g/L.

In one embodiment of the present disclosure, increased production of a value-added product in the specified method is achieved by adjusting the mass flow rate of carbon content to about 30 grams per liter of reactor operating volume per hour, adjusting the mass flow rate of oxygen content to approximately 130 grams per liter of operating volume of the reactor per hour, regulating the mass flow rate of nitrogen content to approximately 24 grams per liter of working volume of the reactor per day, regulating the mass flow rate of phosphate content to approximately 14 grams per liter of working volume of the reactor per day, regulating the mass flow rate of sulfate content to approximately 16 grams per liter of working volume reactor per day, regulating the mass flow rate of the content of trace elements to approximately 50 grams per liter of the working volume of the reactor per day and regulating the mass flow rate of the content of trace elements to approximately 1 gram per liter of the working volume of the reactor per day, wherein said regulation of the mass flow rate of the content of various components of the composition of the nutrient medium is achieved by appropriate addition of the nutrient composition to the reactor during the process of producing the value-added product.

In one embodiment of the present disclosure, said method of producing a value-added product uses a culture medium composition as defined above that is self-sterilizing and does not require heat sterilization or any other additional sterilization technique. Generally, the medium will be prepared by adding micronutrients, macronutrients and essential nutrients and hence it acts as a good source for the growth of microorganisms. The environment (water, air, etc.) contains various microorganisms that multiply upon contact with the environment. Because these are unwanted microorganisms, the medium must be sterilized to kill them (Stanbury et al., 2017). Sterilization is carried out primarily by moist heat in the form of saturated steam under pressure in an autoclave. Similarly, NMS medium prepared for methanotroph culture is also steam sterilized before use (Nunes JJ et al., 2016). As the process expands, media sterilization becomes costly because it requires steam and energy. However, the culture medium composition of the present invention does not require steam sterilization because the pH of the culture medium composition is less than 3, preferably the pH is about 2.5, which inhibits unwanted microorganisms. Thus, this method of producing a value-added product is largely economical and energetically better, in addition to producing value-added products to an increased extent.

In one embodiment of the present disclosure, in a method for producing a value-added product, an important aspect is that the cultivation of microorganisms in the presence of a gaseous substrate to obtain a value-added product occurs in the presence of an addition/flow medium containing components with an acidic pH of less than 3, preferably the pH is about 2.5, thereby controlling the growth of contaminants. Although the media composition of the present invention is formulated to achieve this result, one skilled in the art will appreciate that any media composition that achieves this requirement of an acidic pH of less than 3, preferably a pH of about 2.5, can alternatively be used to achieve the same.

The specified biomass production method and/or value-added product production method described herein uses stirred tank reactors for high efficiency of fermentation of the gaseous substrate. Prior art stirred tank reactors are not known to cause highly efficient fermentation of the gaseous substrate to produce increased biomass and/or value-added products. The increased efficiency achieved in stirred tank reactors using the methods of the present disclosure is particularly attributable to the feed composition of the present invention and the tandem operation of the feed composition and process parameters used in said processes for producing biomass and value-added products, respectively.

The media composition described herein is capable of meeting the media requirements for higher biomass productivity and higher productivity of value-added products in stirred tank reactors. The design of the process parameters and the composition of the nutrient medium, excluding thermal sterilization, offer an economically and energetically more advanced process/method for producing increased biomass and value-added products, respectively.

The biomass production process and value-added product process described herein include substrate-dependent control strategies that help facilitate workflow and reduce cost. The use of acidic nutrients or basic nutrients in the culture medium composition to maintain the pH at a predetermined level helps reduce acid/base consumption during the biomass production process and the value-added product production process, respectively. The components of the culture medium composition of the present invention act as sources of nutrients and as pH adjusting agents. The composition of the nutrient medium with the possibility of developing process parameters to effectively carry out the described methods in addition to obtaining biomass and value-added products, respectively, at an increased level, is an advantage of the present description.

Additional embodiments and features of the present disclosure will be apparent to one skilled in the art based on the description provided herein. The embodiments described herein refer in the description to various features and useful details thereof. Descriptions of well known/conventional methods and techniques have been omitted so as not to unduly obscure the understanding of the embodiments presented herein. The examples presented herein are intended merely to facilitate an understanding of the manner in which the embodiments herein may be practiced and to further enable those skilled in the art to practice the embodiments herein. Accordingly, the following examples should not be construed as limiting the scope of the embodiments described herein.

EXAMPLES

EXAMPLE 1: Biomass production by methane fermentation

An approximately 5 liter stirred tank reactor was filled with approximately 4 liters of a growth medium composition containing approximately 0.1% MgSO ₄ .7H ₂ O, approximately 0.02% CaCl ₂ .2H ₂ O, approximately 0.0004% Fe, Na-EDTA , about 0.00003% NaMoO ₄ .2H ₂ O. about 0.00005% FeSO ₄ .7H ₂ O, about 0.00004% ZnSO ₄ .7H ₂ O, about 15 ppb H ₃ BO ₃ , about 50 parts per billion CoCl ₂ .6H ₂ O, approximately 0.25 ppm. Na ₂ -EDTA dihydrate, about 20 ppb MnCl ₂ .4H ₂ O, about 10 ppb NiCl2. 6H2O, approximately 1 ppm CuSO4 ⋅ 5H2O and 0.025% nitrogen in the form of nitrate. A starter culture of M. capsulalus was added to the specified nutrient medium. The reactor was started by bubbling approximately 0.1 L/min of approximately 99.9% pure methane and 0.05 L/min of approximately 99.9% pure oxygen at the bottom of the paddle mixer. The reactor was maintained at a pressure of approximately one atmosphere. Once the cell density in the reactor reached about 0.3% biomass, a culture medium composition comprising about 0.8% MgSO ₄ .7H ₂ O, about 0.2% CaCl ₂ .2H ₂ O, about 0.008% Fe, Na- EDTA, approximately 0.0002% NaMoO4. 2H2O, about 0.0025% FeSO ₄ .7H ₂ O, about 0.0003% ZnSO ₄ .7H ₂ O, about 150 ppb H ₃ BO ₃ , about 300 ppb CoCl ₂ .6H ₂ O, about 4 m.d. Na ₂ -EDTA dihydrate, about 100 ppb MnCl ₂ .4H ₂ O, about 50 ppb NiCl ₂ .6H2O, about 15 ppm. CuSO ₄ .5H ₂ O, about 0.5% HNO ₃ and about 0.5% H ₂ SO ₄ were added to a reactor where the nutrient medium composition is homogeneous and has a pH of less than 3, preferably a pH of about 2.5. A total nitrogen content of 0.5% was obtained by adding NaNO ₃ to the growth medium.

The fermentation broth containing approximately 3.5% solid biomass was continuously withdrawn.

During the reaction, pH 6.8 ± 0.3 was controlled by acidic and basic compounds of nitrogen source, phosphate source and sulfate source together with NaOH/HCl.

As a result, the reactor contained approximately 3.5% biomass.

The composition of the nutrient medium used to obtain the biomass in this Example was as prepared, without additional sterilization. No contamination was observed in the media reservoir during the entire process.

EXAMPLE 2: Production of biomass by fermentation of methane using ammonia as a source of nitrogen in the composition of the nutrient medium

An approximately 5 liter stirred tank reactor was filled with approximately 4 liters of a growth medium composition containing approximately 0.1% MgSO ₄ .7H ₂ O, approximately 0.02% CaCl ₂ .2H ₂ O, approximately 0.0004% Fe, Na-EDTA , about 0.00003% NaMoO ₄ .2H ₂ O. about 0.00005% FeSO ₄ .7H ₂ O, about 0.00004% ZnSO ₄ .7H ₂ O, about 15 ppb H ₃ BO ₃ , about 50 parts per billion CoCl ₂ .6H ₂ O, approximately 0.25 ppm. Na ₂ -EDTA dihydrate, about 20 ppb MnCl ₂ .4H ₂ O, about 10 ppb NiCl ₂ .6H ₂ O, about 1 ppm. CuSO ₄ .5H ₂ O and 0.025% nitrogen in the form of liquid ammonia. A starter culture of M. capsulalus was inoculated into the specified nutrient medium composition. The reactor was started by bubbling approximately 0.1 L/min of approximately 99.9% pure methane and approximately 0.06 L/min of approximately 90% pure oxygen at the bottom of the paddle mixer. The reactor was maintained at a pressure of approximately one atmosphere. When the cell density in the reactor reached approximately 0.5% biomass, a nutrient medium containing approximately 0.4% MgSO ₄ .7H ₂ O, approximately 0.1% CaCl ₂ .2H ₂ O, approximately 0.0045% Fe, Na- EDTA, about 0.0001% _NaMoO4.2H2O , about 0.0012% FeSO4.7H2O _, about 0.00015 _{% ZnSO4.7H2O ,} about ₈₅ ppb _H3BO3 , about ₁₅₀ parts per billion CoCl ₂ .6H ₂ O, approximately 2.5 ppm. Na ₂ -EDTA dihydrate, about 50 ppb MnCl ₂ .4H ₂ O, about 25 ppb NiCl ₂ .6H ₂ O, about 7 ppm. CuSO ₄ .5H ₂ O, about 0.3% H ₂ SO ₄ and about 0.1% H ₃ PO ₄ were added to a reactor where the feed composition is homogeneous and has a pH of less than 3, preferably a pH of about 2.5 . Liquid ammonia was added to the reactor separately to bring the nitrogen content of the medium to approximately 0.25%.

Fermentation broth containing approximately 2 to 2.5% solid biomass was continuously withdrawn.

During the reaction, the pH was adjusted using acidic and basic compounds of nitrogen source, phosphate source and sulfate source together with NaOH/HCl.

As a result, approximately 2.2±0.3% of biomass was obtained in the reactor.

The composition of the nutrient medium used to obtain the biomass in this Example was as prepared, without additional sterilization.

EXAMPLE 3: Biomass production by methane fermentation

Approximately 10 liters of a stirred tank reactor was filled with approximately 8 liters of a growth medium composition containing approximately 0.1% MgSO ₄ .7H ₂ O, approximately 0.02% CaCl ₂ .2H ₂ O, approximately 0.0004% Fe, Na-EDTA , about 0.00003% NaMoO ₄ .2H ₂ O, about 0.00005% FeSO ₄ .7H ₂ O, about 0.00004% ZnSO ₄ .7H ₂ O, about 15 ppb H ₃ BO ₃ , about 50 parts per billion CoCl ₂ .6H ₂ O, approximately 0.25 ppm. Na ₂ -EDTA dihydrate, about 20 ppb MnCl ₂ .4H ₂ O, about 10 ppb NiCl ₂ .6H ₂ O, about 1 ppm. CuSO ₄ .5H ₂ O and 0.025% nitrogen in the form of potassium nitrate and sodium nitrate. A starter culture of M. trichosposrium was inoculated into the specified composition of the nutrient medium. The reactor was started by bubbling approximately 0.1 L/min of approximately 99.9% pure methane and approximately 0.1 L/min of approximately 70% pure oxygen. Once the total solid biomass in the fermentation broth reached approximately 0.25%, continuous feeding of the nutrient medium was started. The nutrient medium contained approximately 0.4% MgSO ₄ .7H ₂ O, approximately 0.1% CaCl ₂ .2H ₂ O, approximately 0.0045% Fe, Na-EDTA, approximately 0.0001% NaMoO ₄ .2H ₂ O, about 0.0012% FeSO ₄ .7H ₂ O, about 0.00015% ZnSO ₄ .7H ₂ O, about 85 ppb H ₃ BO ₃ , about 150 ppb CoCl ₂ .6H ₂ O, about 2.5 ppb Na ₂ -EDTA dihydrate, approximately 50 ppb MnCl ₂ .4H ₂ O, approximately 25 ppb NiCl ₂ .6H ₂ O, approximately 7 ppb CuSO ₄ .5H ₂ O, approximately 0.2% H ₂ SO ₄ and about 0.08% H ₃ PO ₄ and a total nitrogen content of 0.25% were prepared by adding NaNO ₃ to a nutrient medium where the nutrient medium composition is homogeneous and has a pH of less than 3, preferably about 2.5. The copper content in the nutrient medium varied from 3 to 10 ppm, where it was found that 7 ppm. best to achieve good growth of M. trichosposrium and good mycobactin production. During the reaction, the pH of the medium was maintained at approximately 6.5 ± 0.3 using dilute NaOH and H ₂ SO ₄ .

Growth of M. trichosposrium was monitored by assessing reactor samples for 6 to 8 hours. Methane and carbon dioxide in the exhaust gas were monitored using a biogas analyzer. The composition of the nutrient medium used to obtain the biomass in this Example was as prepared, without additional sterilization. There was no contamination observed in the media reservoir throughout the entire process.

EXAMPLE 4: Preparation of a value-added product (lactic acid)

An approximately 5 liter stirred tank reactor was filled with approximately 4 liters of a growth medium composition containing approximately 0.1% MgSO ₄ .7H ₂ O, approximately 0.02% CaCl ₂ .2H ₂ O, approximately 0.0004% Fe, Na-EDTA , about 0.00003% NaMoO ₄ .2H ₂ O. about 0.00005% FeSO ₄ .7H ₂ O, about 0.00004% ZnSO ₄ .7H ₂ O, about 15 ppb H ₃ BO ₃ , about 50 parts per billion CoCl ₂ .6H ₂ O, approximately 0.25 ppm. Na ₂ -EDTA dihydrate, about 20 ppb MnCl ₂ .4H ₂ O, about 10 ppb NiCl ₂ .6H ₂ O, about 1 ppm. CuSO ₄ .5H ₂ O and 0.025% nitrogen in the form of nitrate. A starter culture of M. capsulalus, which was developed for the production of lactic acid, was inoculated into the specified nutrient medium composition. The reactor was started by bubbling approximately 0.1 L/min of approximately 99.9% pure methane and approximately 0.05 L/min of approximately 99.9% pure oxygen at the bottom of the paddle mixer. The reactor was maintained at a pressure of approximately one atmosphere. Once the cell density in the reactor reached approximately 0.15% - 1% biomass, a growth medium containing approximately 0.4% MgSO ₄ .7H ₂ O, approximately 0.1% CaCl ₂ .2H ₂ O, approximately 0.0045% Fe, Na _{- EDTA} , about 0.0001% _NaMoO4.2H2O , about 0.0012% _FeSO4.7H2O , about 0.00015% _{ZnSO4.7H2O ,} about 85 ppb _{H3BO 3} , about 150 ppb CoCl ₂ .6H ₂ O, about 2.5 ppm. Na ₂ -EDTA dihydrate, about 50 ppb MnCl ₂ .4H ₂ O, about 25 ppb NiCl ₂ .6H ₂ O, about 7 ppm. CuSO ₄ .5H ₂ O and about 0.25% nitrogen in the form of nitrate were added to a reactor where the nutrient medium composition is homogeneous and has a pH of less than 3, preferably about 2.5.

During the reaction, the pH of 6.3 was controlled using acidic and basic nitrogen sources, phosphate sources, and sulfate sources along with NaOH/HCl.

The reactor temperature was maintained at approximately 45°C.

As a result, approximately 1±0.2% of biomass was obtained in the reactor.

The content of lactic acid in the medium was measured using HPLC. The amount of lactic acid formed in the reaction was approximately 3 g/l.

EXAMPLE 5: Biomass production by biogas fermentation

An approximately 5 liter stirred tank reactor was filled with approximately 4 liters of a growth medium composition containing approximately 0.1% MgSO ₄ .7H ₂ O, approximately 0.02% CaCl ₂ .2H ₂ O, approximately 0.0004% Fe, Na-EDTA , about 0.00003% NaMoO ₄ .2H ₂ O, about 0.00005% FeSO ₄ .7H ₂ O, about 0.00004% ZnSO ₄ .7H ₂ O, about 15 ppb H ₃ BO ₃ , about 50 parts per billion CoCl ₂ .6H ₂ O, approximately 0.25 ppm. Na ₂ -EDTA dihydrate, about 20 ppb MnCl ₂ .4H ₂ O, about 10 ppb NiCl ₂ .6H ₂ O, about 1 ppm. CuSO ₄ .5H ₂ O and 0.025% nitrogen. A starter culture of M. capsulalus was inoculated into the specified nutrient medium composition. The reactor was started by bubbling approximately 0.18 L/min of approximately 60% purity biogas and approximately 0.09 L/min of approximately 90% purity oxygen into the bottom of a paddle mixer. The reactor was maintained at a pressure of approximately one atmosphere. When the cell density in the reactor reached approximately 0.2% biomass, a nutrient medium containing approximately 0.4% MgSO ₄ .7H ₂ O, approximately 0.1% CaCl ₂ .2H ₂ O, approximately 0.0045% Fe, Na- EDTA _, about 0.0001% _{NaMoO4.2H2O .} , about 0.0012% _FeSO4.7H2O , about 0.00015% _ZnSO4.7H2O , about ₈₅ ppb _{H3BO3 ,} about 150 ppb CoCl ₂ .6H ₂ O, approximately 2.5 ppm. Na ₂ -EDTA dihydrate, about 50 ppb MnCl ₂ .4H ₂ O, about 25 ppb NiCl ₂ .6H ₂ O, about 7 ppm. CuSO ₄ .5H ₂ O and about 0.25% nitrogen were added to a reactor where the nutrient medium composition is homogeneous and has a pH of less than 3, preferably a pH of about 2.5.

Fermentation broth containing from about 2% to 2.5% solid biomass was continuously withdrawn.

During the reaction, the pH of 6.8 was controlled using acidic and basic nitrogen sources, phosphate sources, and sulfate sources along with NaOH/HCl.

As a result, approximately 2.2±0.3% of biomass was obtained in the reactor.

The composition of the nutrient medium used to obtain the biomass in this Example was as prepared, without additional sterilization. No contamination was observed in the media reservoir during the entire process.

EXAMPLE 6: Biomass production by fermentation of natural gas

An approximately 5 liter stirred tank reactor was filled with approximately 4 liters of a growth medium composition containing approximately 0.1% MgSO ₄ .7H ₂ O, approximately 0.02% CaCl ₂ .2H ₂ O, approximately 0.0004% Fe, Na-EDTA , about 0.00003% NaMoO ₄ .2H ₂ O, about 0.00005% FeSO ₄ .7H ₂ O, about 0.00004% ZnSO ₄ .7H ₂ O, about 15 ppb H ₃ BO ₃ , about 50 parts per billion CoCl ₂ .6H ₂ O, approximately 0.25 ppm. Na ₂ -EDTA dihydrate, about 20 ppb MnCl ₂ .4H ₂ O, about 10 ppb NiCl ₂ .6H ₂ O, about 1 ppm. CuSO ₄ .5H ₂ O and 0.025% nitrogen. A starter culture of M. capsulalus was inoculated into the specified nutrient medium composition. The reactor was started by bubbling about 0.13 L/min of natural gas containing about 90.5% methane, about 5.5% ethane, about 1.75% propane, about 0.5% butane, about 0.25% pentane, about 0.25% carbon dioxide and approximately 1.25% nitrogen and 0.07 l/min oxygen approximately 90% pure at the bottom of the paddle mixer. The reactor was maintained at a pressure of approximately one atmosphere. When the cell density in the reactor reached approximately 0.15-2% biomass, a growth medium containing approximately 0.4% MgSO ₄ .7H ₂ O, approximately 0.1% CaCl ₂ .2H ₂ O, approximately 0.0045% Fe, Na-EDTA, about 0.0001% _NaMoO4.2H2O , about 0.0012% _FeSO4.7H2O , about 0.00015 _{% ZnSO4.7H2O ,} about ₈₅ ppb _{H3BO3 ,} about 150 ppb CoCl ₂ .6H ₂ O, about 2.5 ppb Na ₂ -EDTA dihydrate, about 50 ppb MnCl ₂ .4H ₂ O, about 25 ppb NiCl ₂ .6H ₂ O, about 7 ppm CuSO ₄ .5H ₂ O, about 0.3% HNO ₃ , about 0.3% H ₂ SO ₄ and about 0.09% H ₃ PO ₄ were added to a reactor in which the nutrient composition is homogeneous and has a pH less than than 3, preferably a pH of about 2.5. A total nitrogen content of 0.5% was obtained by adding NaNO ₃ to the growth medium.

Fermentation broth containing from about 2% to 2.5% solid biomass was continuously withdrawn.

During the reaction, the pH was adjusted using acidic and basic compounds of nitrogen source, phosphate source and sulfate source together with NaOH/HCl.

As a result, approximately 2.2±0.3% of biomass was obtained in the reactor.

The composition of the nutrient medium used to obtain the biomass in this Example was as prepared, without additional sterilization. No contamination was observed in the media reservoir during the entire process.

EXAMPLE 7: Biomass production by methane fermentation using a pH stat

Approximately 5 liters of Sartorius B plus automatic reactor filled with approximately 4 liters of a growth medium composition containing approximately 0.1% MgSO ₄ .7H ₂ O, approximately 0.02% CaCl ₂ .2H ₂ O, approximately 0.0004% Fe, Na -EDTA, about 0.00003% NaMoO ₄ .2H ₂ O, about 0.00005% FeSO ₄ .7H ₂ O, about 0.00004% ZnSO ₄ .7H ₂ O, about 15 ppb H ₃ BO ₃ , about 50 parts per billion CoCl ₂ .6H ₂ O, approximately 0.25 ppm. Na ₂ -EDTA dihydrate, about 20 ppb MnCl ₂ .4H ₂ O, about 10 ppb NiCl ₂ .6H ₂ O, about 1 ppm. CuSO ₄ .5H ₂ O and 0.025% nitrogen, inoculated with M. capsulalus. The reactor was started by continuously bubbling approximately 0.1 L/min of approximately 99.9% pure methane and approximately 0.06 L/min of approximately 99.9% pure oxygen at the bottom of the paddle mixer. The input of a nutrient medium containing approximately 0.4% MgSO ₄ .7H ₂ O, approximately 0.1% CaCl ₂ .2H ₂ O, approximately 0.0045% Fe, Na-EDTA, and approximately 0.0001% NaMoO ₄ was gradually increased. 2H ₂ O, about 0.0012% FeSO ₄ .7H ₂ O., about 0.00015% ZnSO ₄ .7H ₂ O, about 85 ppb H ₃ BO ₃ , about 150 ppb CoCl ₂ .6H ₂ O , about 2.5 ppb Na ₂ -EDTA dihydrate, about 50 ppb MnCl ₂ .4H ₂ O, about 25 ppb NiCl ₂ .6H ₂ O, about 7 ppm. CuSO ₄ .5H ₂ O about 0.5% HNO ₃ , about 0.5% H ₂ SO ₄ , about 0.15% H ₃ PO ₄ , and a total nitrogen content of 0.25% was obtained by adding NaNO ₃ to the nutrient environment where the composition of the nutrient medium is homogeneous and has a pH of less than 3, preferably a pH of about 2.5. HNO ₃ , H ₂ SO ₄ and H ₃ PO ₄ act as acids to maintain pH, acids to dissolve all nutrients and nutrients for the growth of the body. This strategy of maintaining a constant pH of 6.8 during a reaction with a change of 0.01 is called pH stat. Possible mass flow rates of carbon, oxygen, nitrogen, phosphates, sulfates, trace elements and trace elements gradually increased within the range of 0.2-9 g/l/h, 0.2-32 g/l/h, 0.2-9 g/h l/day, 0.1-4 g/l/day, 0.1-5 g/l/day, 0.3-17 g/l/day and 0.002-0.39 g/l/day, respectively, to maintain a high specific growth rate, as well as their optimal concentrations in the reactor. The culture medium was continuously fed into the reactor through a Watson and Marlow peristaltic pump, and the fermentation broth was removed using a Masterflex peristaltic pump.

Approximately 2% of the total solid biomass in the broth was reached, which was removed at a rate of approximately 0.5 l/h.

In this method, the pH was kept constant at approximately 6.8 ± 0.4 by using the acidic and basic compounds of a nitrogen source, a phosphate source, and a sulfate source along with dilute solutions of NaOH and HCl.

The reactor temperature was maintained at approximately 45±0.5°C using a reactor thermostat.

The reactor operated successfully for approximately 600 hours with a maintained productivity of 2.5 g/l/hour.

The composition of the nutrient medium used to obtain the biomass in this Example was as prepared, without additional sterilization. There was no contamination observed in the media reservoir throughout the entire process.

EXAMPLE 8: Increased biomass productivity through fermentation of methane and nitrate as a nitrogen source

An approximately 5 liter stirred tank reactor was filled with approximately 4 liters of a growth medium composition containing approximately 0.1% MgSO ₄ .7H ₂ O, approximately 0.02% CaCl ₂ .2H ₂ O, approximately 0.0004% Fe, Na-EDTA , about 0.00003% NaMoO ₄ .2H ₂ O. about 0.00005% FeSO ₄ .7H ₂ O, about 0.00004% ZnSO ₄ .7H ₂ O, about 15 ppb H ₃ BO ₃ , about 50 parts per billion CoCl ₂ .6H ₂ O, approximately 0.25 ppm. Na ₂ -EDTA dihydrate, about 20 ppb MnCl ₂ .4H ₂ O, about 10 ppb NiCl ₂ .6H ₂ O, about 1 ppm. CuSO ₄ .5H ₂ O and approximately 0.025% nitrogen in the form of nitrate. A starter culture of M. capsulalus was inoculated into the specified nutrient medium composition. The reactor was started by bubbling approximately 0.1 L/min of approximately 99.9% pure methane and 0.05 L/min of approximately 99.9% pure oxygen at the bottom of the paddle mixer. The reactor was maintained at a pressure of approximately one atmosphere. Once the cell density in the reactor reached about 0.15-2% biomass, a culture medium composition containing about 0.4% MgSO ₄ .7H ₂ O, about 0.1% CaCl ₂ .2H ₂ O, about 0.0045% Fe, Na _{- EDTA} , about 0.0001% _NaMoO4.2H2O , about 0.0012% _FeSO4.7H2O , about 0.00015% _{ZnSO4.7H2O ,} about 85 ppb _{H3BO 3} , about 150 ppb CoCl ₂ .6H ₂ O, about 2.5 ppb Na ₂ -EDTA dihydrate, about 50 ppb MnCl ₂ .4H ₂ O, about 25 ppb NiCl ₂ .6H ₂ O , approximately 7 ppm CuSO ₄ .5H ₂ O, about 0.3% HNO ₃ , about 0.3% H ₂ SO ₄ and about 0.09% H ₃ PO ₄ were added to a reactor where the culture medium composition is homogeneous and has a pH less than 3 preferably a pH of about 2.5. A total nitrogen content of 0.25% was obtained by adding NaNO ₃ to the culture medium composition. The dilution was gradually increased until a productivity of approximately 4.5 ± 0.1 g/l/h was achieved during continuous operation at steady state.

During the reaction, the pH of approximately 6.8 ± 0.3 was controlled with acidic and basic compounds of the nitrogen source, phosphate sources and sulfate sources of the growth medium composition, optionally together with NaOH/HCl. The reactor operated for approximately 250 hours.

The composition of the nutrient medium used to obtain the biomass in this Example was as prepared, without additional sterilization. There was no contamination observed in the media reservoir throughout the entire process.

EXAMPLE 9: Increased biomass productivity through fermentation of methane and ammonia as a nitrogen source

An approximately 5 liter stirred tank reactor was filled with approximately 4 liters of a growth medium composition containing approximately 0.1% MgSO ₄ .7H ₂ O, approximately 0.02% CaCl ₂ .2H ₂ O, approximately 0.0004% Fe, Na-EDTA , about 0.00003% NaMoO ₄ .2H ₂ O, about 0.00005% FeSO ₄ .7H ₂ O, about 0.00004% ZnSO ₄ .7H ₂ O, about 15 ppb H ₃ BO ₃ , about 50 parts per billion CoCl ₂ .6H ₂ O, approximately 0.25 ppm. Na ₂ -EDTA dihydrate, about 20 ppb MnCl ₂ .4H ₂ O, about 10 ppb NiCl ₂ .6H ₂ O, about 1 ppm. CuSO ₄ .5H ₂ O and approximately 0.025% nitrogen in the form of liquid ammonia. The specified composition of the nutrient medium was inoculated with a starter culture of M. capsulalus. The reactor was started by bubbling approximately 0.1 L/min of approximately 99.9% pure methane and approximately 0.06 L/min of approximately 90% pure oxygen at the bottom of the paddle mixer. The reactor was maintained at a pressure of approximately one atmosphere. Once the cell density in the reactor reached approximately 0.1% biomass, a culture medium composition containing approximately 0.5% MgSO ₄ .7H ₂ O, approximately 0.14% CaCl ₂ .2H ₂ O, approximately 0.006% Fe, Na- EDTA, about 0.00012% _NaMoO4.2H2O , _about 0.0015% FeSO4.7H2O _, about 0.00019 _{% ZnSO4.7H2O} , about _{106 ppb H3BO3} , about 188 parts per billion CoCl ₂ .6H ₂ O, approximately 3.1 ppm. Na ₂ -EDTA dihydrate, about 63 ppb MnCl ₂ .4H ₂ O, about 31 ppb NiCl ₂ .6H ₂ O, about 8.6 ppm. CuSO ₄ .5H ₂ O, about 0.37% H ₂ SO ₄ and about 0.12% H ₃ PO ₄ were added to a reactor where the feed composition is homogeneous and has a pH of less than 3, preferably a pH of about 2.5 . Liquid ammonia was added to the reactor separately to bring the nitrogen content of the medium to approximately 0.3%.

The dilution was gradually increased until a productivity of 6±0.1 g/l/hour was achieved in a continuous stationary mode. During the reaction, the pH was controlled using acidic and basic compounds of the nitrogen source, phosphate sources and sulfate sources of the nutrient composition, optionally together with NaOH/HCl. The reactor operated for approximately 120 hours.

EXAMPLE 10: Increase (10x) biomass production by methane fermentation

Approximately 50 liters of a stirred tank reactor (Biogenic Engineering Chennai) was filled with approximately 45 liters of a growth medium composition containing approximately 0.1% MgSO ₄ .7H ₂ O, approximately 0.02% CaCl ₂ .2H ₂ O, approximately 0.0004% Fe, Na _{- EDTA} , about 0.00003% _NaMoO4.2H2O , about 0.00005% _FeSO4.7H2O , about 0.00004% _{ZnSO4.7H2O ,} about 15 ppb _{H3BO 3} , about 50 ppb CoCl ₂ .6H ₂ O, about 0.25 ppm. Na ₂ -EDTA dihydrate, about 20 ppb MnCl ₂ .4H ₂ O, about 10 ppb NiCl ₂ .6H ₂ O, about 1 ppm. CuSO ₄ .5H ₂ O and 0.025% nitrogen in the form of liquid ammonia. A starter culture of M. capsulalus was inoculated into the specified nutrient medium composition. The reactor was started by bubbling biogas containing approximately 76% methane and 99% pure oxygen into the bottom of a paddle mixer. The reactor was maintained at a pressure of approximately one atmosphere. When the cell density in the reactor reached approximately 0.25% biomass, a culture medium composition containing approximately 0.4% MgSO ₄ .7H ₂ O, approximately 0.1% CaCl ₂ .2H ₂ O, approximately 0.0045% Fe, Na -EDTA _, about 0.0001% _NaMoO4.2H2O , about 0.0012% _FeSO4.7H2O , about 0.00015 _{% ZnSO4.7H2O ,} about 85 ppb _{H3BO3 ,} about 150 ppb CoCl ₂ .6H ₂ O, approximately 2.5 ppm. Na ₂ -EDTA dihydrate, about 50 ppb MnCl ₂ .4H ₂ O, about 25 ppb NiCl ₂ .6H ₂ O, about 7 ppm CuSO ₄ .5H ₂ O, about 0.3% H ₂ SO ₄ and about 0.1% H ₃ PO ₄ were added to a reactor where the feed composition is homogeneous and has a pH of less than 3, preferably a pH of about 2.5 . Liquid ammonia was added to the reactor separately to bring the nitrogen content of the medium to approximately 0.25%. Fermentation broth containing approximately 2 to 2.5% solid biomass was continuously withdrawn.

During the reaction, the pH was adjusted with acidic and basic compounds of the nitrogen source, phosphate sources and sulfate sources of the nutrient composition, optionally together with NaOH/HCl. The reactor operated for approximately 120 hours.

The composition of the nutrient medium used to obtain the biomass in this Example was as prepared, without additional sterilization. There was no contamination observed in the media reservoir throughout the entire process.

Claims (16)

1. A method for producing biomass, including: cultivating a microorganism selected from methanotrophs in a medium composition, adding to the culture a nutrient medium composition containing a microelement selected from the group consisting of MgSO4.7H2O, CaCl2.2H2O and their combinations; and a trace element selected from the group consisting of FeNa-EDTA, NaMoO4.2H2O, FeSO4.7H2O, ZnSO4.7H2O, H3BO3, CoCl2.6H2O, Na2-EDTA dihydrate, MnCl2.4H2O, NiCl2.6H2O, CuSO4.5H2O and their combinations, and HNO ₃ or ammonia as a nitrogen source, H ₂ SO ₄ /KHSO ₄ as a sulfate source and H ₃ PO ₄ /KH ₂ PO ₄ as a phosphate source, wherein the nutrient medium composition has a pH of less than 3; And biomass collection. 2. The method according to claim 1, where the cultivation of the microorganism includes: inoculating the medium composition with a microorganism and subsequently providing a gaseous substrate and oxygen; And monitoring microorganism cell density and adding a nutrient medium composition. 3. Method according to any one of paragraphs. 1-2, wherein the cultivation of the microorganism is carried out at a temperature ranging from about 5°C to 50°C, at a pressure ranging from about 0 bar to 5 bar for a period of time from about 120 hours to 900 hours. 4. The method of claim 2, wherein the culture medium composition is added when the microorganism cell density is in the range of about 0.15% to 2%. 5. The method according to claim 2, where the gaseous substrate is methane and the specified substrate further contains natural gas, synthesis gas, landfill gas, carbon monoxide, biogas and any combinations thereof; and the gaseous substrate is present in a concentration ranging from about 1 mg/L to 8 mg/L; and oxygen is present in a concentration ranging from about 1 mg/L to 10 mg/L. 6. Method according to any one of paragraphs. 1-4, where the microorganism is selected from the group consisting of Methylococcus capsulatus, Methylomicrobium album, Methylocapsa acidiphila, Methylocella silvestris, Methylosinus trichosporium, Methylacidiphilum infernorum V4, Methylomonas methanica, Methylobacter tundripaludum, Methylosinus sporium, Methylocella palustris, Methylocystis parvus , Methylovulum miyakonense, Methylocystis echinoides , Methylomonas rubra, Methylobacter whittenburyi, Crenothrix polyspora, Clonothrix fusca, Methylobacter bovis, Methylomonas aurantiaca, Methylomonas fodinarum, Methylocystis minimus, Methylobacter vinelandii, Methylomicrobium japanense, Methylocystis methanolicus and a combination thereof. 7. Method according to any one of paragraphs. 1-6, where the biomass is obtained with an increased level of productivity in the range from about 0.5 g*l ^-1 *hour ^-1 to 8 g*l ^-1 *hour ^-1 . 8. The method of claim 1, wherein the nutrient medium composition contains an additional nitrogen-containing source, a phosphate-containing source, a sulfate-containing source, or a combination thereof. 9. The method of claim 8, wherein the nitrogen-containing source is selected from the group consisting of sodium nitrate, sodium nitrite, potassium nitrate, potassium nitrite, ammonium hydroxide, ammonium chloride, ammonium acetate, ammonium sulfate, diammonium phosphate (DAP) and any combinations thereof; the phosphate-containing source is selected from the group consisting of potassium hydrogen phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, diammonium phosphate, and any combinations thereof; and the sulfate-containing source is selected from the group consisting of copper sulfate, zinc sulfate, ferrous sulfate, magnesium sulfate, manganese sulfate, and any combinations thereof.