CN112980749B

CN112980749B - Rhodococcus erythropolis oxydans ethane and propane and application thereof

Info

Publication number: CN112980749B
Application number: CN202110500828.3A
Authority: CN
Inventors: 全哲学; 邹斌; 张盼盼; 黄莹
Original assignee: Fudan University
Current assignee: Fudan University
Priority date: 2021-05-08
Filing date: 2021-05-08
Publication date: 2022-08-30
Anticipated expiration: 2041-05-08
Also published as: CN112980749A

Abstract

The invention discloses an ethane and propane oxidation strain containing hydrocarbon oxidation related functional genes on plasmids, which belongs to the Rhodococcus, the Latin literature name is Rhodococcus, the strain number is ZPP, and the strain is preserved in Guangdong province microorganism culture Collection (GDMMCC) at 2021, 4 months and 27 days, and the preservation number is as follows: GDMCC NO: 61631. the ethane or propane oxidizing bacteria have a function of decomposing ethane or propane, and can use ethane or propane as a carbon source or energy source required for growth. The ethane and propane oxidizing bacteria contain hydrocarbon monooxygenase gene cluster and similar soluble methane oxygenase gene cluster on the same carrier, and the carrier can be transferred to other strains to ensure that the carrier obtains the ethane or propane oxidizing function.

Description

Rhodococcus erythropolis oxydans ethane and propane and application thereof

Technical Field

The invention relates to Rhodococcus sp.ZPP capable of degrading ethane and propane by using ethane or propane as a carbon source and an energy source and application thereof.

Background

Gaseous short-chain hydrocarbons are naturally sourced from many sources, including the ocean and the soil, and widely distributed oil and gas leaks emit large quantities of oil and gas to the surrounding environment. Ethane and propane are important non-methane hydrocarbon substances in petrochemical production, so that the ethane and propane oxidizing bacteria can be applied to relevant industrial production and air pollution bioremediation.

The copper membrane-bound monooxygenase (CuMMO) superfamily includes ammonia monooxygenase, particulate methane monooxygenase, and Hydrocarbon Monooxygenase (HMO). Some actinomycetes such as Mycobacterium, Nocardioides, etc. have ethane, propane, or butane oxidation function, and hydrocarbon monooxygenase-related genes included in the genomes of these bacteria play an important role in this process. Soluble di-iron monooxygenase (SDIMO), which is similar to soluble methane oxygenase (sMMO), contained in these strains also plays an important role in short-chain alkane oxidation processes. The CuMMO enzyme comprises copper ions, and the SDIMO enzyme comprises iron ions; since Allylthiourea (ATU) can couple with copper ions to inhibit copper-containing functional enzymes, allylthiourea inhibition experiments are commonly used to confirm the action of CuMMO-type enzymes. In previous studies, it was generally considered that the CuMMO functional enzyme gene is on the chromosome, and the phylogenetic tree thereof coincides with the phylogenetic classification based on the 16S rRNA gene, but there have also been studies to suggest the possibility of horizontal gene transfer of part of the CuMMO functional enzyme gene.

Rhodococcus (Rhodococcus) is a rare group of actinomycetes. The rhodococcus has strong environmental adaptability, various organic matters can be used as carbon sources and energy sources, and many rhodococcus have the capability of degrading straight-chain and aromatic hydrocarbon substances. Among the known genomes of many Rhodococcus, CuMMO and SDIMO related genes have been found in only a few strains such as Rhodococcus sp.WAY2, but their activities have not been confirmed.

Disclosure of Invention

The invention aims to provide a Rhodococcus sp.ZPP with ethane and propane oxidation performance, and the strain has key genes related to ethane and propane oxidation on plasmids, and the plasmids can be conjointly transferred to other strains, so that the strains obtain ethane and propane oxidation activity and grow by using the short-chain alkanes as carbon sources and energy sources.

In one aspect, the invention adopts the technical scheme that: the invention provides a Rhodococcus oxydans ethane and propane, which is characterized in that the Rhodococcus oxydans has the Latin literature name of Rhodococcus, and is preserved in Guangdong culture Collection of microorganisms (GDMCC) at 27 days 4 and 4 months 2021, and the preservation number is as follows: GDMCC No.61631, address: the Michelson No. 100 Dazhou building No. 59 in Guangzhou city, Guangdong province microbial analysis and detection center.

The Rhodococcus ZPP strain is characterized in that: the colony is orange yellow, round and opaque, and the 16S rRNA gene sequence is shown in SEQ ID No. 1.

Furthermore, the ethane and propane oxidizing bacteria contain hydrocarbon monooxygenase gene cluster hmoCAB, wherein hmoA is a marker gene, the sequence is shown as SEQ ID NO.2, and the gene is positioned on a plasmid.

Hydrocarbon monooxygenase is the major functional enzyme for the oxidation of ethane or propane, the marker gene for which is hmoA, and this enzyme activity was confirmed by the allylthiourea inhibition assay.

Further, the plasmids containing the hydrocarbon monooxygenase of the ethane and propane oxidizing bacteria are transferred to other strains through conjugation, so that the ethane and propane oxidizing bacteria have ethane and propane oxidizing activity.

Furthermore, the ethane and propane oxidizing bacteria contain a gene cluster smoXYB1C1Z similar to soluble methane oxygenase (sMMO), wherein smoX is a marker gene, the sequence is shown as SEQ ID NO.3, and the gene is located on a plasmid.

Further, the ethane and propane oxidizing bacteria contain hydrocarbon monooxygenase gene cluster and similar soluble methane oxygenase gene cluster on the same plasmid, and the plasmid can be transferred to other strains by conjugation, so that the other strains have ethane and propane oxidizing activity.

In a further aspect, the invention provides the ethane or propane biodegradation effect of the rhodococcus ZPP.

Preferably, the ethane, propane oxidizing bacteria utilize ethane or propane as a carbon and energy source.

Preferably, the ethane, propane oxidizing bacteria degrade ethane or propane in a particular environment.

Preferably, the specific environment includes, but is not limited to, soil, gas, activated sludge, water.

In yet another aspect, the invention provides the use of said ethane, propane oxidizing bacteria in the production of chemicals for use in alkane oxidation.

In another aspect, the present invention provides a method of biodegrading ethane or propane: after a culture medium is washed away by a fermentation liquid, namely a bacterium-containing suspension, obtained by fermenting and culturing rhodococcus ZPP, the fermentation liquid is inoculated into a sealed culture bottle containing ethane or propane and an NMS culture medium, and the fermentation liquid is cultured under the shaking condition of 140-160rpm at the temperature of 30 ℃ by taking the ethane or propane as a carbon source to realize the degradation of the ethane or propane.

Preferably, the doubling time of the strain under ethane oxidation conditions is 16.6 + -2.0 hours, and the doubling time under propane oxidation conditions is 11.5 + -0.3 hours.

Preferably, the final concentration of the NMS culture medium consists of: CaCl ₂ ·2H ₂ O 0.134g，KNO ₃ 0.5g，MgSO ₄ ·7H ₂ O 1g，Na ₂ HPO ₄ ·12H ₂ O 0.717g，KH ₂ PO ₄ 0.272g, 5mg of Ferric ammonium EDTA (ethylenediaminetetraacetic acid Ferric ammonium), 1mL of trace element solution, and deionized water to a constant volume of 1,000mL, adjusting the pH value to 6.8, and sterilizing at 121 ℃ for 15 min. Wherein the microelement solution (Disodium EDTA (Disodium ethylene diamine tetraacetate)) is 0.5g, and FeSO ₄ ·7H ₂ O 0.2g，H ₃ BO ₃ 0.03g，CoCl ₂ ·6H ₂ O 0.02g，CuSO ₄ ·5H ₂ O 0.03g，ZnSO ₄ ·7H ₂ O 0.01g，MnCl ₂ ·4H ₂ O 3mg，Na ₂ MoO ₄ ·2H ₂ O 3mg，NiCl ₂ ·6H ₂ O6 mg, deionized water to a constant volume of 1,000mL, and pH was adjusted to neutral. The preparation method needs to mix well to prevent precipitation. Because of FeSO ₄ ·7H ₂ O is easily oxidized and needs to be prepared separately).

The bacteria-containing suspension is prepared by the following steps:

(1) slant culture: inoculating Rhodococcus ZPP to 50% concentration R2A solid slant culture medium, and culturing at 30 deg.C for 2 days to obtain thallus slant;

(2) seed culture: colonies were picked from the cell slant and inoculated into 50% R2A liquid medium, and cultured at 30 ℃ for 2 days to obtain a seed solution, and a strain harboring an active plasmid was confirmed using a specific hmoA primer.

The final concentration composition of the R2A culture medium is as follows: tryptone 0.125 g.L ^-1 Acid hydrolyzed casein 0.25 g.L ^-1 0.25 g.L yeast extract powder ^-1 0.25 g/L soluble starch ^-1 Dipotassium hydrogen phosphate 0.15 g.L ^-1 Magnesium sulfate 0.05 g. L ^-1 0.15 g.L of sodium pyruvate ^-1 Peptone 0.125 g. L ^-1 0.25 g.L of glucose ^-1 The solvent is water, and the pH value is 7.2 +/-0.2.

The specific hmoA primers e280F (5'-ACCCTATGTGCAGTCGTGTT-3') and e578R (5'-CCGATGTGGAAGGACATTGTG-3') of the invention are used for judging the existence of hmo genes related to the oxidation activity of ethane or propane from the gene level.

The ethane or propane oxidizing bacteria of the present invention have a function of decomposing ethane or propane, and ethane or propane can be used as a carbon source or energy source necessary for growth. The ethane, propane oxidizing bacteria of the present invention contain a hydrocarbon monooxygenase gene cluster and a similar soluble methane oxygenase gene cluster on the same plasmid, which can be transferred to other strains by conjugation, such that they gain ethane or propane oxidizing function. Because the ethane and propane oxidizing bacteria can grow by using organic matters such as ethanol or propanol as a carbon source and an energy source, a large amount of bacteria can be easily obtained in a short time, and the method is applied to industrial or environment-friendly fields. Moreover, the transferability characteristic of the ethane and propane oxidation functional gene cluster of the bacterium can enable indigenous microorganisms in an ethane and propane system to obtain the functional gene clusters and have the ethane and oxidation functions, so that the treatment effect and the stability of the biological treatment system can be obviously improved.

Drawings

FIG. 1 is a colony morphology diagram of ethane and propane oxidizing bacteria ZPP of the invention.

FIG. 2 is the scanning electron microscope image of ethane and propane oxidizing bacteria ZPP of the invention.

FIG. 3 is a line graph of ethane and propane oxidizing bacteria ZPP and zygosity utilizing ethane or propane in accordance with the present invention.

FIG. 4 is a line graph of ethane and propane oxidizing bacteria ZPP of the present invention grown using ethane or propane and inhibited by the inhibitor allylthiourea.

FIG. 5 is a schematic representation of the partial region and transcriptome expression of the plasmids of the ethane, propane oxidizing bacterium ZPP of the present invention containing homoCAB and smoXYB1C 1Z.

Detailed Description

Example 1: strain source and strain separation of ethane and propane oxidizing bacteria ZPP

1. Collecting and enriching samples: collecting a soil sample in organic nutrient soil; weighing 5g of soil sample, placing the soil sample in a sterilized empty culture bottle with the volume of 120mL, covering a rubber plug cover, filling 2mL of ethane or propane, placing the bottle in a 28 ℃ low-temperature constant-temperature tank for water bath culture, detecting the change of gas concentration by using a gas chromatography (GC2020) every 5-8 days, opening the bottle for ventilation after each detection, then filling 2mL of ethane or propane gas for continuous culture, and continuously culturing for four periods.

2. Strain separation and purification: weighing 0.2g of the active enrichment, adding 1mL of sterile water, fully mixing, centrifuging at 5,000rpm for 30s, taking the supernatant, diluting the supernatant with sterile water in a gradient manner, filtering to the front surface (0.2 mu m, Millipore) of a sterile polycarbonate filter membrane with the diameter of 47mm, suspending the front surface of the filter membrane upwards above a small-sized culture dish with the diameter of 5cm and containing 15mL of NMS liquid culture medium, carefully placing the culture dish in a sealed culture tank with the volume of 2.5L, filling 25mL of ethane or propane, and placing the culture dish in a constant-temperature incubator at the temperature of 28 ℃ for culture. After transferring the single colony on the surface of the filter membrane to 50% R2A solid medium, the plate is placed upside down in a constant temperature incubator at 28 ℃ for culture.

The final concentration composition of the NMS culture medium is as follows: CaCl ₂ ·2H ₂ O 0.134g，KNO ₃ 0.5g，MgSO ₄ ·7H ₂ O 1g，Na ₂ HPO ₄ ·12H ₂ O 0.717g，KH ₂ PO ₄ 0.272g，Ferric ammo5mg of nium EDTA, 1mL of trace element solution, constant volume of deionized water to 1,000mL, adjusting the pH value to 6.8, and sterilizing at 121 ℃ for 15 min. Wherein the microelement solution (Disodium EDTA 0.5g, FeSO) ₄ ·7H ₂ O 0.2g，H ₃ BO ₃ 0.03g，CoCl ₂ ·6H ₂ O 0.02g，CuSO ₄ ·5H ₂ O 0.03g，ZnSO ₄ ·7H ₂ O 0.01g，MnCl ₂ ·4H ₂ O 3mg，Na ₂ MoO ₄ ·2H ₂ O 3mg，NiCl ₂ ·6H ₂ O6 mg, deionized water to a constant volume of 1,000mL, and pH was adjusted to neutral. The preparation method needs to mix well to prevent precipitation. Because of FeSO ₄ ·7H ₂ O is easily oxidized and needs to be prepared separately).

Example 2: identification of strain of ZPP

1. Physiological and biochemical characteristics of strain ZPP

The strain ZPP is gram positive, the colony color is orange yellow, and the round shape is opaque (see figure 1). The shape of the cells was observed as spherical or short rod under a scanning electron microscope (see FIG. 2).

2. 16S rRNA gene identification: total bacterial DNA was extracted using a bacterial genome DNA extraction kit (centrifugal column type) from Tiangen Biochemical technology Ltd, and the extracted and purified DNA was subjected to 16S rRNA gene amplification using bacterial 16S rRNA universal primers 27F (5 '-AGAGTTTGATCMTGGCTCAG-3') and 1492R (5 '-TACGGYTACCTTGTTACGACTT-3') and then sequenced. The sequencing results (gene sequence SEQ ID NO: 1, see sequence Listing) were subjected to nucleotide homology analysis with the 16S rRNA gene sequence disclosed in Genbank using BLAST from NCBI (https:// www.ncbi.nlm.nih.gov).

Combined with physiological and biochemical experiments and 16S rRNA gene identification, this genus is of the genus Rhodococcus, the name Rhodococcus, deposited at 27 months 4 and 2021 at the guangdong collection of microorganisms (GDMCC), address: guangzhou city, Jielizhou 100 large yard, building 59, Guangdong province microbiological analysis and detection center, preservation number: GDMCC NO. 61631.

Example 3: culture of ZPP

1. Slant culture: inoculating ethane and propane oxidizing bacteria ZPP to a 50% concentration R2A slant culture medium, and culturing at 30 deg.C for 2 days to obtain a slant culture medium;

2. seed culture: colonies were picked from the cell slant and inoculated into 50% R2A liquid medium, and cultured at 30 ℃ for 2 days to obtain a seed solution, and the presence of a plasmid containing a gene cluster related to ethane or propane oxidation activity was confirmed using a specific hmoA primer.

The final concentration composition of the R2A culture medium is as follows: tryptone 0.125 g.L ^-1 Acid hydrolyzed casein 0.25 g.L ^-1 0.25 g.L yeast extract powder ^-1 Soluble starch 0.25 g.L ^-1 Dipotassium hydrogen phosphate 0.15 g.L ^-1 Magnesium sulfate 0.05 g. L ^-1 Sodium pyruvate 0.15 g.L ^-1 Peptone 0.125 g.L ^-1 0.25 g.L of glucose ^-1 The solvent is water, and the pH value is 7.2 +/-0.2.

Example 4: growth of ethane and propane oxidizing bacteria ZPP in various alcohols as carbon source and energy source

The strain suspension obtained by liquid culture of Rhodococcus ZPP is washed off with medium and concentrated (OD) ₆₀₀ 1.0), diluted and inoculated in 25mL NMS medium (final OD) ₆₀₀ 0.001) and 0.1% of different alcoholic substrates were added and incubated at 28 ℃. The results show that ethanol or n-propanol is the only carbon source and energy source, and the turbidity (OD) of the culture solution is caused by the growth of the ethane and propane oxidizing bacteria ZPP after 5 days of culture ₆₀₀ ) Increasing to 0.71 + -0.02 or 1.11 + -0.01, growth was slow under isopropanol conditions, but the turbidity of the culture increased to 0.77 + -0.02 after 8 days of culture.

Example 5: ethane, propane oxidizing bacteria ZPP ethane or propane oxidizing property detection

The strain suspension obtained by liquid culture of Rhodococcus ZPP is washed off with culture medium and concentrated, and then inoculated into 25mL NMS culture medium in 125mL sealed culture flask until the absorbance OD corresponding to the thallus concentration ₆₀₀ 0.1, 1% by volume of ethane or propane was added to the head space, and the mixture was incubated at 28 ℃ with standing. The change in ethane or propane concentration is detected by gas chromatography. The Rhodococcus ZPP degrades ethane or propane completely in 6 days or 16 days, i.e. the Rhodococcus ZPP degrades 0.23 + -0.03 vol% of ethane or 0.09 + -0.02 vol% of ethane effectively every dayVolumetric ratio of propane (see fig. 3).

Example 6: detection of ZPP inhibitor and functional gene of ethane and propane oxidizing bacteria

The strain suspension obtained by liquid culture of Rhodococcus ZPP is washed off with medium and concentrated (OD) ₆₀₀ 1.0), 25mL NMS medium (final OD) inoculated in 125mL sealed culture flasks was diluted ₆₀₀ 0.001) at 28 ℃ and 160rpm at 140-. The doubling time was calculated to be 16.6 + -2.0 hours under ethane and 11.5 + -0.3 hours under propane conditions based on the change in absorbance of the amount of Rhodococcus ZPP cells (see FIG. 4). The inhibitor allylthiourea (0.1mM) almost completely inhibited propane oxidation, but not ethane oxidation. Indicating that the role of HMO enzyme is more important in the oxidation of propane.

Because the oxidation function of ethane and propane is inhibited to different degrees after using the inhibitor allyl thiourea, the functional gene hmoCAB can be considered as the basis of the oxidation function of the strain ZPP for producing ethane and propane, the whole genome sequencing shows that the gene is positioned on a plasmid, and the existence of the functional gene hmoCAB is determined by using a specific hmoA primer e280F (5'-ACCCTATGTGCAGTCGTGTT-3') and a primer e578R (5'-CCGATGTGGAAGGACATTGTG-3'); primers tcpC256R (5'-GACAACGCCACCGAAGACA-3') and tcpC481R (5'-CGTATGTCCGTGACTTCTCCT-3') were used to determine the presence of the plasmid. The PCR conditions were all pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 30s for 35 cycles; finally, extension is carried out for 10min at 72 ℃.

Since the ethane oxidation function was not completely inhibited by the inhibitor allylthiourea, it was considered that the functional gene smoXYB1C1Z is also an important basis for the oxidation of ethane by rhodococcus ZPP, and whole genome sequencing revealed that this gene and hmoCAB are located on the same plasmid (see fig. 5).

Example 7: conjugal transfer and expression of hydrocarbon monooxygenase-containing plasmids from ethane, propane oxidizing bacteria

The bacterial conjugation experiment was performed by liquid conjugation using Rhodococcus erythropolis ATCC 25544 (CGMCC 1.2362 ═ NBRC 15567) as donor bacteria and Rhodococcus erythropolis ATCC 25544 as acceptor bacteria. The method specifically comprises the following steps: selecting single colonies of donor and acceptor bacteria, and performing shaking culture in 40mL of R2A liquid culture medium at 28 deg.C and 120rpm for 48 h; respectively adding 1mL of donor bacterium and recipient bacterium solution into 40mL of R2A liquid culture medium, mixing, shaking uniformly, standing for culture, and culturing in a constant-temperature incubator at 28 ℃ for 24 h; taking out 0.01mL of mixed bacterial liquid, coating the mixed bacterial liquid on NMS solid culture medium containing ampicillin with final concentration of 0.010mg/mL to ensure that Rhodococcus ZPP is inhibited by antibiotics, putting the mixed bacterial liquid into a 2.5L sealed tank, filling 125mL of propane, and performing inverted culture at 28 ℃ for 7 days; after single colonies grow out, picking out single colonies, inoculating the single colonies into 25mL of NMS liquid culture medium containing ampicillin with the final concentration of 0.010mg/mL, filling 5% of propane, and placing the single colonies in a low-temperature constant-temperature tank at 28 ℃ for water bath culture for 7 days; taking out 0.01mL of bacterial liquid, streaking and inoculating the bacterial liquid on an NMS solid culture medium, putting the NMS solid culture medium into a 2.5L sealed tank, filling 125mL of propane, carrying out inverted culture at 28 ℃ for 7 days, and determining that the receptor strain is successfully jointed through morphological identification and 16S and functional gene PCR experiments; a single colony was picked up and subjected to scale-up culture to obtain the zygote Rhodococcus erythropolis ATCC 25544(pRZP 1). The conjugant Rhodococcus erythropolis ATCC 25544(pRZP1) was cultured according to the method described in example 1. The results showed that the zygote Rhodococcus erythropolis ATCC 25544(pRZP1) had substantially the same ethane and propane oxidation activity as Rhodococcus ZPP, while Rhodococcus erythropolis ATCC 25544(pRZP1) did not consume ethane or propane (see FIG. 3).

Fermentation broth obtained by fermentation culture of Rhodococcus ZPP and the conjugant Rhodococcus erythropolis ATCC 25544(pRZP1), namely bacterial suspension, is washed out of the culture medium, concentrated and then inoculated into a sealed culture flask containing propane and NMS culture medium, propane is used as a carbon source, the culture is carried out at 30 ℃ and 150rpm, samples in logarithmic phase are taken for carrying out transcriptome experiment, and sequencing of the transcriptome shows that both hmoCAB and smoXYB1C1Z have expression far higher than that of conserved genes (see figure 5).

Sequence listing

<110> university of Compound Dan

<120> Rhodococcus Oxirae and propane and application thereof

<141> 2021-05-08

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aagtctcccc ggaatctgtc tgacgcaaag aaggaagcct actggagcca ccacttcacg 1020

tttgcggctg cgtacgcgct gtggcccctg ctcggcattc gtatggaaat gccaaacagc 1080

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actctgatgc agatgggcgc aaacgtctac ttctgccgtg tatgccagtt cccgatcatc 1260

acccccatgc cttctgccgg cgagaacctg cgaatcatcg aacatggagg acgccgacac 1320

gcgctgtgct cgcaatggtg tgaacgaatg ttcctcaccg agccggagcg cttcgacggt 1380

cagaatctct tcgaagtatt cgacggctgg gaactttctg aggtcgtcaa ggcaggccgg 1440

gcggtgcgca gtgacgaaag cactctcctt gcccaaccac atctcagcga cgagcggatg 1500

tggaccctag aggacctcaa ggcatgcaac ctcgtaatcc gagatcccct gactgcggga 1560

gtgttcctgg agaagcgctg a 1581

Claims

1. An ethane and propane oxidizing bacterium, which is characterized in that: the strain ZPP, whose scientific name is Rhodococcus and Latin literature is Rhodococcus sp, was deposited in Guangdong province culture Collection (GDMCC) at 27 days 4 months 2021, with the following deposit numbers: GDMCC NO. 61631.

2. Use of an ethane, propane oxidizing bacterium according to claim 1 for the biodegradation of ethane or propane.

3. Use according to claim 2, characterized in that: the ethane, propane oxidizing bacteria utilize ethane or propane as a carbon source.

4. Use according to claim 2, characterized in that: the ethane, propane oxidizing bacteria degrade ethane or propane in a particular environment.

5. The use of claim 4, wherein: the specific environment is soil, gas, activated sludge and water.

6. Use of ethane, propane oxidizing bacteria according to claim 1 in the production of chemicals based on alkane oxidation.

7. A method for biodegrading ethane or propane, characterized in that, after washing off the culture medium from the fermentation broth obtained by the fermentation culture of the ethane or propane oxidizing bacteria of claim 1, the fermentation broth is inoculated into a sealed culture flask containing ethane or propane and NMS culture medium, and the ethane or propane is used as the carbon source, and the fermentation broth is cultured under the shaking conditions of 30 ℃, 140 ℃ and 160rpm, so as to realize the degradation of ethane or propane, wherein the doubling time of the ethane or propane oxidizing bacteria under the ethane oxidizing condition is 16.6 +/-2.0 hours, and the doubling time of the propane oxidizing bacteria under the propane oxidizing condition is 11.5 +/-0.3 hours.