An Enterobacter GD-13 with carbon fixation capabilities and its applications

Abstract

The present invention provides an Enterobacter GD‑13 with carbon fixation ability and its application, and relates to the field of microbial technology. The Enterobacter described in the present invention is classified and named Enterobacter huaxiensis, and the preservation number is CGMCC No.31726. The Enterobacter described in the present invention is isolated from the soil between fixed plates in the photovoltaic field area of ​​the rocky desertification region. Experiments have shown that the Enterobacter can maintain good growth activity and carbon fixation ability in an extremely low-nutrient salt culture medium, and its carbon fixation ability can be improved to varying degrees when an inorganic carbon source is added, especially under the condition of adding an inorganic carbon source Na2CO3, its carbon fixation ability is significantly improved, and the addition of an electron donor can further improve its carbon fixation ability. The Enterobacter separated and obtained by the present invention has important scientific significance for improving the soil carbon fixation and sink potential, promoting the high-quality development of the photovoltaic industry in rocky desertification vulnerable areas, and alleviating global climate warming.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and specifically relates to an Enterobacter GD-13 with carbon fixation ability and an application thereof. Background Art

[0002] New energy development has become the core way to deal with climate change, but more than 80% of new energy is distributed in vulnerable areas (such as rocky desertification areas). Rocky desertification vulnerable areas have shallow soil layers, rich in calcium and alkaline, and the ecosystem is very fragile, which seriously restricts the development of regional economy and society. It is urgent to explore a new model of organic integration of energy and ecological restoration and the development of characteristic industries.

[0003] At present, the research on key technologies of ecological photovoltaic in rocky desertification-vulnerable areas mainly focuses on the monitoring and evaluation of meteorological effects in photovoltaic areas and the monitoring and evaluation of soil heterogeneity, but lacks relevant research on the precise identification and screening of functional microorganisms in the soil of photovoltaic areas. It is urgent to analyze the carbon transformation pathways and strategies of key microorganisms and isolate and screen efficient carbon fixation indigenous functional microorganisms in photovoltaic areas, which has important potential application value in stimulating and enhancing the synergistic benefits of indigenous microorganisms in improving soil and fixing carbon. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide an Enterobacter GD-13 with carbon fixation ability and its application. The Enterobacter GD-13 has the ability to fix inorganic carbon sources, especially can efficiently realize carbonate fixation, has strong adaptability under oligotrophic conditions, and has the ability to solubilize phosphate.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The invention provides an Enterobacter GD-13. The classification name of the Enterobacter GD-13 is Enterobacter huaxiensis, and the preservation number is CGMCC No.31726.

[0007] Preferably, the nucleotide sequence of the Enterobacter GD-13 is as shown in SEQ ID NO.1.

[0008] The present invention also provides a soil conditioner, which comprises the Enterobacter GD-13.

[0009] The present invention also provides an application of the Enterobacter GD-13 or the soil conditioner in improving the carbon fixation capacity of soil.

[0010] Preferably, the soil is karst soil.

[0011] The present invention also provides an application of the Enterobacter GD-13 in phosphate solubilization.

[0012] Preferably, the phosphate solubilization is that Enterobacter GD-13 dissolves insoluble inorganic phosphorus into soluble phosphate.

[0013] The present invention also provides an application of the Enterobacter GD-13 in fixing inorganic carbon.

[0014] Preferably, the inorganic carbon comprises one or more of carbon dioxide, sodium carbonate and sodium bicarbonate.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] The present invention provides a strain of Enterobacter huaxiensis GD-13, which can efficiently realize the ability to fix carbonates, has strong adaptability under oligotrophic conditions, and has the ability to solubilize phosphate, with a solubilizing capacity of 407.32 mg / L. Experiments have shown that the present invention inoculates Enterobacter GD-13 in a culture medium containing different inorganic carbon sources, and the bacteria can fix the carbon in the culture medium; by adding an inorganic carbon source, especially sodium carbonate, its carbon fixation ability can be significantly improved. The present invention cultivates Enterobacter GD-13 with a culture medium added with different electron donors, and its carbon fixation process has different degrees of enhancement. The present invention discloses for the first time that Enterobacter has the ability to fix inorganic carbon sources, provides strain resources for improving the carbon fixation and emission reduction capacity of rocky desertification soil, provides a theoretical basis for in-depth understanding of the microbial regulation process of the occurrence form of nutrients in rocky desertification soil, lays the foundation for proposing soil microbial improvement technology in photovoltaic fields, and promotes the high-quality development of the photovoltaic industry in rocky desertification vulnerable areas.

[0017] Biological deposit information

[0018] The Enterobacter GD-13 described in the present invention is classified and named Enterobacter huaxiensis, and the depository is the General Microbiology Center of China National Microbiological Culture Collection Administration; the depository address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; the deposit number is CGMCC No. 31726; the deposit date is August 23, 2024. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is a bacterial morphology diagram of Enterobacter GD-13 of the present invention.

[0020] Figure 2 It is a colony characteristic diagram of Enterobacter GD-13 of the present invention.

[0021] Figure 3 It is a phylogenetic tree of the 16S rRNA gene of Enterobacter GD-13 of the present invention.

[0022] Figure 4 It is the RubisCO enzyme activity of the Enterobacter GD-13 of the present invention. In the figure, GD-13 is the Enterobacter of the present invention, and 1 to 4 are other 4 strains of carbon-fixing bacteria.

[0023] Figure 5 2 is the carbon fixation capacity of the Enterobacter GD-13 of the present invention under different conditions. Different letters a, b, and c in the figure indicate significant differences at the p<0.05 level.

[0024] Figure 6 This is the carbon fixation pathway of the Enterobacter GD-13 of the present invention.

[0025] Figure 7 It is the effective phosphorus content of the Enterobacter GD-13 of the present invention after being cultured in a liquid culture medium containing insoluble phosphate salts. In the figure, GD-13 is the Enterobacter of the present invention, 1 to 4 are other 4 strains of carbon-fixing bacteria, and different letters a, b, and c indicate significant differences at the p<0.05 level. DETAILED DESCRIPTION

[0026] The present invention provides an Enterobacter GD-13, wherein the Enterobacter GD-13 is classified and named as Enterobacter huaxiensis, and the deposit number is CGMCC No. 31726. The Enterobacter GD-13 of the present invention is isolated and obtained from soil in a rocky desertification photovoltaic field area in Shilin County, Yunnan Province. The present invention uses a scanning electron microscope to determine that the bacterial morphology of the strain is rod-shaped, and the colonies produced by the strain on a solid culture medium are small, umbilical, white, opaque, smooth and sticky on the surface, and irregular on the edge.

[0027]

[0028] The present invention also provides a soil conditioner, which includes the Enterobacter GD-13. As an operative method, the active ingredient of the soil conditioner of the present invention includes the Enterobacter GD-13; the soil conditioner may also include an acceptable auxiliary material. As another operative method, the sole ingredient of the soil conditioner is the Enterobacter GD-13.

[0029] The present invention also provides an application of the Enterobacter GD-13 or the soil conditioner in improving soil carbon fixation capacity. In the present invention, the soil is preferably karst soil. Experiments have shown that the Enterobacter GD-13 described in the present invention can grow in a very low-nutrient salt culture medium, and its growth activity and carbon fixation capacity can be driven by electron donors. At the same time, it has a higher carbon fixation potential in a soil environment with a high carbonate content, which helps to develop the application of strains in special environments (such as karst soils).

[0030] The present invention also provides an application of the Enterobacter GD-13 in phosphate solubilization. The phosphate solubilization is preferably that the Enterobacter GD-13 dissolves insoluble inorganic phosphorus into soluble phosphate. The present invention determines that the Enterobacter GD-13 of the present invention has the ability to solubilize phosphate by taking the ability to dissolve tricalcium phosphate as an indicator, thereby realizing the utilization of insoluble phosphate. Its phosphate solubilization ability is 407.32 mg / L, which is significantly higher than that of other strains.

[0031] The present invention also provides an application of the Enterobacter GD-13 in fixing inorganic carbon. The inorganic carbon preferably includes one or more of carbon dioxide, sodium carbonate and sodium bicarbonate. The present invention performs RubisCO enzyme activity assay on the bacterial solution of Enterobacter GD-13, and its RuBisCO enzyme activity is about 17.02U / mg prot, which has a strong carbon fixation ability. In addition, there are differences in the carbon fixation ability of Enterobacter GD-13 under different inorganic carbon sources and different electron donors. When an inorganic carbon source is added, the strain concentration increases to about 4.5 times and 12.7 times under the addition of NaHCO3 and Na2CO3, respectively. When an electron donor is added, the carbon fixation ability of the strain is further improved. Compared with only adding NaHCO3, the strain concentration increases again by about 2.7 times and 3.0 times when NaNO2 and Na2S2O3 are added, indicating that the electron donor can provide more energy for the strain to fix carbon.

[0032] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0033] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are commercially available products unless otherwise specified.

[0034] Example 1 Isolation and Identification of Carbon-Fixing Bacteria Enterobacterhuaxiensis GD-13

[0035] The target soil is the soil between fixed panels in the rocky desertification photovoltaic field in Shilin County, Yunnan Province. The specific screening plan is as follows:

[0036] 1. Collect soil from a depth of 0 to 20 cm, take 10.0 g of the collected soil, add it to 100 mL of sterilized water, and place it on a shaker for shaking (180 rpm, shaking for 30 min) to make a soil suspension. Then dilute the soil suspension in a 10-fold gradient to 10 -4 times, take 0.2mL of soil dilution, apply it to a solid culture medium without an organic carbon source, and culture it at 28°C for 7 days. The components of the solid culture medium are: Na2HPO4, 0.5g / L; KH2PO4, 0.5g / L; MgSO4, 0.3g / L; CaCl2, 0.2g / L; NaHCO3, 0.5g / L; NH4Cl, 0.5g / L; NaNO3, 0.25g / L; NaCl, 0.4g / L; trace element solution, 10mL / L; agar, 20g / L, pH 7.0. The formula of the trace element solution is: FeCl2, 0.3g / L; FeSO4·7H2O, 0.3g / L, MnSO4·H2O, 0.15g / L; ZnSO4·7H2O, 0.25g / L; CoCl2, 0.2g / L.

[0037] 2. Pick different colonies with better morphology, better growth potential and faster growth rate from the solid culture medium without organic carbon source, and purify them multiple times on a new solid culture medium without organic carbon source by inoculation loop plate streak method until pure culture. In this study, 25 strains of carbon-fixing bacteria were initially isolated and purified by dilution plate method, and the dominant bacteria were selected for carbon fixation function rescreening (refer to Example 2), and the one with the best effect was selected as the research object. Scanning electron microscopy was used to determine that the bacterial morphology of the strain was rod-shaped ( Figure 1 ), the colonies produced on solid culture medium are small, umbilical, white, opaque, smooth and sticky on the surface, and have irregular edges ( Figure 2 ).

[0038] 3. Further 16S rRNA sequencing of the strain: PCR amplification of the genomic DNA of the strain was performed using universal primers 27F: 5'-AGTTTGATCMTGGCTCAG-3' (SEQ ID NO.2) and 1492R primer: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO.3), and then the amplified product was sequenced. The sequencing result is shown in SEQ ID NO.1. Then, BLAST analysis was performed based on the 16S rRNA gene sequence of the strain using the NCBI database, and a phylogenetic tree was constructed. Figure 3 It can be seen that the isolated strain is closest to the genetic evolution of Enterobacter asburiae, and its 16S rRNA gene sequence is homologous to Enterobacter sp., and the homology with Enterobacter asburiae NJBB9 is 99.78%. Combined with its bacterial morphology and colony characteristics, the strain is identified as Enterobacter sp. However, the accurate classification of the strain cannot be determined only by 16S rRNA gene sequencing analysis. Further, whole genome sequencing is performed on it: the extracted whole genome is sent to Shanghai Paisonuo Biotechnology Co., Ltd. for second-generation sequencing, and the obtained sequence result is analyzed by average nucleotide identity (ANI) with the whole genome sequence of the reference strain Enterobacter asburiae 17Nkhm-UP2 in the NCBI database. When the ANI value is greater than 95%, it can be considered that the two are the same species. However, the ANI result showed 88.38%, indicating that the strain was not of this species. The 16SrRNA gene sequence analysis was not accurate enough to classify bacteria because it could not fully reflect the genetic differences between species. Therefore, by comparing the ANI values ​​of the strain with the whole genome sequences of multiple strains, we found that the ANI result of the strain and the whole genome sequence of Enterobacter huaxiensis 090008 was 98.86%, thus confirming that the strain was Enterobacter huaxiensis.

[0039] Example 2 Carbon-fixing enzyme activity of Enterobacter huaxiensis GD-13

[0040] 1. Materials and methods

[0041] 1.1 Test strain: Enterobacter huaxiensis GD-13, isolated and identified in Example 1.

[0042] 1.2 Detection of RubisCO carbon fixation enzyme activity in Enterobacter huaxiensis GD-13

[0043] In order to study the carbon fixation ability of Enterobacter huaxiensis GD-13, the colonies purified from the solid culture medium without organic carbon source in Example 1 were picked and inoculated into 10 mL of liquid culture medium without organic carbon source, and cultured for 5 days at 28°C and 160r / min. The bacterial liquid at the end of the culture was taken for RubisCO enzyme activity determination, and the ribulose diphosphate carboxylase / oxygenase (RuBisCO) kit provided by Beijing Bo Ao Sen Biotechnology Co., Ltd. was used for determination. At the same time, the total protein content of the bacterial liquid was measured using the BCA protein concentration determination kit, and the RuBisCO enzyme activity determination results were standardized. The specific measurement method refers to the kit instructions, and the determination results are shown in Figure 4 In order to highlight the strong carbon fixation ability of Enterobacter huaxiensis GD-13, this example uses the other 4 carbon fixation bacteria separated and purified in Example 1 for comparative study.

[0044] 1.3 Data processing

[0045] The raw data of this experiment were preliminarily sorted using Microsoft Excel Office 2016. The experimental results were plotted using Graphpad Pism 9.5.

[0046] 2. Results and Analysis

[0047] The carbon fixation capacity of Enterobacter huaxiensis GD-13 is as follows Figure 4 As shown, its RuBisCO enzyme activity was about 17.02U / mg prot, which was much greater than that of the other four carbon-fixing bacteria, indicating that it had good carbon fixation ability.

[0048] Example 3 Study on the carbon fixation ability of Enterobacter huaxiensis under different conditions

[0049] 1. Materials and methods

[0050] 1.1 Test strain: Enterobacter huaxiensis, isolated and identified in Example 1.

[0051] Carbon-free liquid culture medium: Na2HPO4, 0.5g / L; KH2PO4, 0.5g / L; MgSO4, 0.3g / L; CaCl2, 0.2g / L; NH4Cl, 0.5g / L; NaNO3, 0.25g / L; NaCl, 0.4g / L; trace element solution, 10mL / L; pH 7.0. The formula of the trace element solution is: FeCl2, 0.3g / L; FeSO4·7H2O, 0.3g / L, MnSO4·H2O, 0.15g / L; ZnSO4·7H2O, 0.25g / L; CoCl2, 0.2g / L.

[0052] Na2CO3 liquid culture medium: Add Na2CO3, 0.63g / L, to the carbon-free liquid culture medium.

[0053] NaHCO3 liquid culture medium: Add NaHCO3, 0.5g / L, to the carbon-free liquid culture medium.

[0054] NaNO2 liquid culture medium: NaNO2, 4.6g / L; Na2HPO4, 0.5g / L; KH2PO4, 0.5g / L; MgSO4, 0.3g / L; CaCl2, 0.2g / L; NaHCO3, 0.5g / L; NH4Cl, 0.5g / L; NaNO3, 0.25g / L; NaCl, 0.4g / L; pH 7.0.

[0055] Na2S2O3 liquid culture medium: Replace NaNO2, 4.6 g / L in NaNO2 liquid culture medium with Na2S2O3, 5 g / L.

[0056] 1.2 Study on the carbon fixation ability of Enterobacter huaxiensis under different inorganic carbon sources and different electron donors

[0057] The colonies purified from the solid culture medium without organic carbon source in Example 1 were picked and inoculated into 5 mL carbon-free liquid culture medium. After culturing for 5 days at 28°C and 160 r / min, 400 μL was inoculated into 5 mL carbon-free liquid culture medium, Na2CO3 liquid culture medium, NaHCO3 liquid culture medium, NaNO2 liquid culture medium and Na2S2O3 liquid culture medium respectively, and three parallels were made. After culturing for 5 days at 28°C, the abundance of strains in the bacterial solution was detected. The specific method for measuring microbial abundance is as follows: SYBR-Green (10000×, Solebol) was diluted 100 times, and then mixed with propidium iodide (PI, Solebol) at 50:1 (SYBR-Green:PI, V:V) to obtain a fluorescent dye. After 1 mL of bacterial solution was passed through a 300-mesh nylon sieve to remove large particles of impurities, 10 μL of fluorescent dye was added to avoid light and dye for 15 minutes, and the number of viable bacteria was determined after vortexing.

[0058] 1.3 Data processing

[0059] The experimental data were analyzed in CytExpert SRT software and then preliminarily sorted using Microsoft ExcelOffice 2016. The experimental results were plotted using Graphpad Pism 9.5.

[0060] 2. Results and Analysis

[0061] Since the only carbon sources available to the strain during cultivation are atmospheric carbon dioxide and inorganic carbon sources in the culture medium, the increase in strain biomass is all from carbon fixation. Figure 5. The results showed that after 5 days of cultivation under the condition of atmospheric carbon dioxide alone, the strain concentration of Enterobacter huaxiensis was only 2267.01Events / μL, indicating that it has a weak fixation ability for atmospheric carbon dioxide. When an inorganic carbon source was added, the strain concentration increased, indicating that its carbon fixation ability was improved, which may be the result of the strain fixing carbon through other pathways. However, its utilization of two different forms of inorganic carbon sources was quite different, and its strain concentration increased to about 4.5 times and 12.7 times with the addition of NaHCO3 and Na2CO3, respectively. When an electron donor was added, the carbon fixation ability of the strain was further improved. Compared with the addition of only NaHCO3, the strain concentration increased again by about 2.7 times and 3.0 times when NaNO2 and Na2S2O3 were added, indicating that the electron donor can provide more energy for the strain to fix carbon, and Na2S2O3 has a better effect on improving the carbon fixation ability of the strain. The results showed that the strain was able to grow in extremely low-nutrient salt culture media, and its growth activity and carbon fixation ability could be driven by electron donors. At the same time, it had a higher carbon fixation potential in soil environments with higher carbonate content, which would help develop the application of the strain in special environments (such as karst soils).

[0062] Example 4 Analysis of Carbon Fixation Pathway of Enterobacter huaxiensis GD-13

[0063] 1. Materials and methods

[0064] The Enterobacter huaxiensis GD-13 isolated and identified in Example 1 was sent to Shanghai Paisonno Biotechnology Co., Ltd. for second-generation sequencing using the Illumina Novasek platform, and the whole genome framework map was assembled and spliced. The bacteria were annotated and functionally identified using bioinformatics software and databases, and the carbon fixation pathway of strain GD-13 was inferred through the KEGG database to provide a basis for studying the carbon fixation ability of the strain.

[0065] 2. Results and Analysis

[0066] Based on the KEGG database, the CO2 fixation pathway of strain GD-13 was analyzed. First, acetyl-CoA and CO2 generate pyruvate under the action of pyruvate ferrodoxin oxidoreductase, and phosphoenolpyruvate is formed by phosphoenolpyruvate synthetase to fix CO2 to generate oxaloacetate, which is then converted into L-aspartate by aspartate aminotransferase and then converted into threonine. Threonine is cleaved into glycine by threonine aldolase, and glycine is then converted into serine by glycine hydroxymethyltransferase, and finally converted into pyruvate by serine dehydrogenase to form a carbon fixation cycle ( Figure 6). The first stable product produced when this pathway fixes CO2 is the four-carbon compound oxaloacetate, which is converted into phosphoenolpyruvate through the conversion of multiple amino acids. The synthesis and degradation of threonine is the core process in this cycle. In addition, when oxaloacetate is produced when CO2 is fixed, oxaloacetate is converted into malic acid under the catalysis of malate dehydrogenase, and malic acid is directly converted into pyruvate under the catalysis of malic enzyme to complete the carbon fixation cycle. At the same time, strain GD-13 also has an enzyme with a high carbon fixation efficiency, acetyl-CoA carboxylase (ACC), which catalyzes the reaction of acetyl-CoA with HCO3 - The reaction of producing malonyl-CoA can to some extent explain the phenomenon that the addition of inorganic carbon source NaHCO3 increases the carbon fixation capacity of the strain.

[0067] Example 5 Study on the Phosphate Solubilization Capacity of Carbon-Fixing Bacteria Enterobacterhuaxiensis GD-13

[0068] 1. Materials and methods

[0069] 1.1 Test strain: Enterobacter GD-13, isolated and identified in Example 1.

[0070] Pikovskaya liquid medium: yeast extract, 0.5 g / L; glucose, 10 g / L; Ca3(PO4)2, 5 g / L; (NH4)2SO4, 0.5 g / L; KCl, 0.2 g / L; Mg3(PO4)2, 0.1 g / L; MgSO4, 0.0001 g / L; FeSO4, 0.0001 g / L; pH 7.0.

[0071] 1.2 Detection of phosphate solubilization ability of carbon-fixing bacteria Enterobacter GD-13

[0072] The 5 g / L KH2PO4 standard solution was diluted to concentrations of 0, 2, 2.5, 250, 500, and 1000 mg / L, and the absorbance was determined using the molybdenum antimony antichromogenic method. The standard curve equation was y=0.0028x+0.048, and R2=0.9996.

[0073] The colony purified from the solid culture medium without an organic carbon source in Example 1 was inoculated into 10 mL of Pikovskaya liquid culture medium and cultured at 28°C, 160 r / min, for 5 days. A control group without inoculation of the strain was set up. At the end of the culture, the bacterial solution was centrifuged, 1 mL of sterile water was added to the bacterial precipitate, ground with a tissue crusher, and mixed with the supernatant, and NaHCO3 was added and filtered by vibration. The organic phosphorus content was determined by the molybdenum antimony colorimetric method. The results are shown in Figure 7 In order to highlight the strong phosphorus-solubilizing ability of Enterobacter GD-13, this example used the other 4 carbon-fixing bacteria separated and purified in Example 1 for comparative study.

[0074] 1.3 Data processing

[0075] The experimental data were preliminarily sorted using Microsoft Excel Office 2016. The experimental results were plotted using Graphpad Pism 9.5.

[0076] 2. Results and Analysis

[0077] By using the ability to dissolve tricalcium phosphate as an indicator, it was determined that the Enterobacter GD-13 had the ability to dissolve phosphate and achieved the utilization of insoluble phosphates. Its phosphate dissolving capacity was 407.32 mg / L, while the phosphate dissolving capacity of the other four carbon-fixing bacteria was as low as 115.18 mg / L and as high as 301.25 mg / L, all lower than that of the carbon-fixing bacteria.

[0078] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An Enterobacter huaxiensis GD-13, characterized in that: The deposit number is CGMCC No.31726.

2. A soil conditioner, characterized in that: The soil conditioner contains the Enterobacter sinoecidis GD-13 described in claim 1.

3. Use of the Enterobacter sphenanthera GD-13 described in claim 1 or the soil conditioner described in claim 2 in soil carbon fixation.

4. The use according to claim 3, characterized in that: The soil is karst soil.

5. Use of the Enterobacter sphenanthedis GD-13 described in claim 1 in phosphate solubilization.

6. The use according to claim 5, characterized in that: The phosphate solubilization is that Enterobacter sphenanthenicum GD-13 dissolves insoluble inorganic phosphorus into soluble phosphate.

7. Use of the Enterobacter sphenanthera GD-13 described in claim 1 in fixing inorganic carbon.

8. The use according to claim 7, characterized in that: The inorganic carbon is one or more of carbon dioxide, sodium carbonate and sodium bicarbonate.

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