CN107674843B - Rhodobacter xylinum strain RC1 and application thereof - Google Patents

Abstract

The invention relates to a rhodobacter xylinum (Rhodobacter capsulatus strain) RC1, wherein the preservation number of the RC1 in China center for type culture Collection is CCTCC: m2017323, the rhodobacter membranaceus of the invention is a photosynthetic bacterium obtained by taking microorganism in sludge water at the bottom of an artificial wetland anaerobic pool acclimatized by formaldehyde as a material and separating by using an inorganic salt culture medium of the photosynthetic bacterium, and is a gram-negative bacterium, the bacterium liquid is reddish blood, and carotenoid and bacteriochlorophyll a exist in somatic cells. Can grow in a liquid inorganic salt culture medium containing 4mM and 6mM formaldehyde under illumination, the removal rate of the added formaldehyde in the culture medium reaches 100 percent, and the formaldehyde is metabolized and converted into glucose through a photosynthetic carbon assimilation pathway which is a main mechanism for detoxifying the formaldehyde.

Description

Rhodobacter xylinum strain RC1 and application thereof

Technical Field

The invention belongs to the field of environmental microorganisms, and particularly relates to a rhodobacter xylinum with a formaldehyde assimilation way and capable of purifying formaldehyde pollution.

Background

Formaldehyde (HCHO) is widely used in the industrial fields of chemical synthesis, industrial manufacturing, pharmaceutical synthesis and the like, and has a significant role in the field of synthesis of chemical pesticides and intermediates thereof, so that a large amount of production wastewater containing liquid formaldehyde is generated in the production process of modern chemical pesticides. The formaldehyde contains carbonyl groups, so that the formaldehyde has stronger electrophilic property and very active chemical property, and can perform non-specific reaction with various lipids, nucleic acid and proteins to cause macromolecular substances in organisms to lose biological activity. If the production wastewater containing formaldehyde is directly discharged into the environment, the pollution of soil and water bodies can be caused, thereby causing harm to many organisms including animals and human beings. On the other hand, as the quality of life is continuously improved, decoration becomes a part of modern life, formaldehyde is a widely used adhesive in many decoration materials, materials using formaldehyde as an adhesive generally contain some uncrosslinked free formaldehyde, and the free formaldehyde can be continuously released from the materials (for 5-10 years), thereby causing serious indoor air formaldehyde pollution, which has become a serious environmental problem in developing countries. Since the harm of formaldehyde pollution is extensive, many researches have been made to develop technologies and methods for treating formaldehyde pollution, and among the various treatment methods, a biological treatment method developed using the ability of microorganisms to absorb formaldehyde is an effective and economical method, and a bioreactor prepared using microorganisms can be used to purify liquid, solid and gaseous formaldehyde polluted in the environment. Many studies have shown that microorganisms are able to absorb formaldehyde because they all metabolize formaldehyde. The research on the metabolic mechanism of the formaldehyde of the microorganism shows that the formaldehyde can be metabolized through a dissimilatory pathway and an anabolic pathway. In the catabolic pathway, free HCHO within the microorganism first produces an adduct of HCHO by reaction with cofactors such as fungal thiols, reduced Glutathione (GSH), tetrahydromethylpterin, and Tetrahydrofolate (THF), etc., and then is oxidized to CO2 and regenerates the cofactor while providing the energy required for growth of the microorganism. In addition, formaldehyde can also be converted into a component of the cell in the microorganism via an assimilation pathway such as the ribulose monophosphate pathway (RuMP), the serine pathway, the xylulose monophosphate pathway (Xu 5P), or the cyclic oxidation pathway of ribulose monophosphate.

Photosynthetic bacteria, which are the earliest prokaryotes on the earth and have an original light energy synthesis system, can perform photosynthesis in an environment with light and oxygen deficiency, and can assimilate carbon dioxide or other organic matters by using light energy, are different from green plants in that the photosynthesis does not produce oxygen because the photosynthetic bacteria only have one light system PSI in cells, and original hydrogen donors for the photosynthesis are not water but H2S (or some organic matters), so that the photosynthesis performed by the photosynthetic bacteria produces H2, decomposes the organic matters, and simultaneously fixes free nitrogen in the air to produce ammonia. The photosynthetic bacteria complete three extremely important chemical processes of hydrogen production, nitrogen fixation and organic matter decomposition in the assimilation and metabolism process, and the unique physiological characteristics enable the three extremely important chemical processes to have extremely important positions in an ecological system and have wide application prospects in water quality environmental pollution treatment. The photosynthetic bacteria can utilize organic matters as a breathing matrix to carry out aerobic or heterotrophic growth under the dark condition. In aquaculture, photosynthetic bacteria are used to degrade toxic substances such as nitrite and sulfide which are rich in water, so as to achieve the effects of purifying water quality and preventing diseases. In addition, because the photosynthetic bacteria cells are rich in various nutrient substances, the mycoprotein thereof also has the function of a feed additive. The photosynthetic bacteria have strong adaptability, can tolerate high-concentration organic wastewater and have strong metabolic conversion capacity on toxic substances, so that the literature reports on the capability and mechanism of the photosynthetic bacteria for purifying the organic wastewater are very many.

Photosynthetic bacteria are widely distributed in soil, paddy fields, swamps, lakes, rivers, seas and the like in the nature, and are mainly distributed in anoxic zones to which light can be transmitted in aquatic environments. The soil for cultivating plants and the sludge of paddy fields, swamps, lakes and rivers are rich in microorganisms, and some microorganisms have a formaldehyde metabolism mechanism, so that many researches try to assemble bioreactors by using the soil of potted plants or the sludge fished out of water, and the research results prove that the bioreactors have very good effects of removing and degrading various organic pollutants, but the activated sludge contains many types of microorganisms, has undefined compositions and may contain some pathogenic bacteria.

Disclosure of Invention

In view of the above technical problems, the present invention aims to provide a rhodobacter xylinum RC1 (having a formaldehyde assimilation pathway and capable of purifying formaldehyde pollution)Rhodobacter capsulatusstrain RC 1) to provide a new microbial resource for purifying formaldehyde pollution.

In order to achieve the purpose, the invention provides the following technical scheme:

a strain of rhodobacter xylinum RC1 (Rhodobacter capsulatusstrain RC 1), the preservation number of the strain in China center for type culture Collection is CCTCC: m2017323, address: wuhan university in Wuchang district, Wuhan city, Hubei province, preservation date: 6.9.2017, the 16SrRNA gene sequence of the strain is shown as SEQ ID NO. 1.

The invention also discloses rhodobacter xylinum RC1 (Rhodobacter capsulatusstrain RC 1): a photosynthetic carbon assimilation pathway.

The invention also discloses rhodobacter xylinum RC1 (Rhodobacter capsulatusstrain RC 1) as a biological resource in the purification of formaldehyde pollution.

The rhodobacter xylinum RC1 (of the present invention)Rhodobacter capsulatusstrain RC 1) is obtained by the following steps: firstly, collecting bottom layer sludge water from the artificial wetland, adding formaldehyde with different concentrations to domesticate microorganisms in the sludge water, and then separating photosynthetic bacteria which grow fast and have strong resistance to the formaldehyde by using an inorganic salt culture medium of the photosynthetic bacteria.

The invention has the beneficial effects that:

the rhodobacter palmatum RC1 can grow in a liquid inorganic salt culture medium containing 4mM or 6mM of formaldehyde under illumination, has strong resistance to formaldehyde, has a removal rate of 100% when being added into the culture medium, and can metabolize the formaldehyde to be converted into glucose through a photosynthetic carbon assimilation pathway which is a main mechanism for detoxifying the formaldehyde.

Drawings

FIG. 1: strain RC1 andRhodobacter capsulatus16S rRNA sequence homology analysis of strain NBM 67;

FIG. 2: effect of 4mM and 6mM formaldehyde treatment for 1-8 days on growth of rhodobacter capsulatus RC1 and DC-1;

FIG. 3: comparison of the efficiency of uptake of 4 and 6mM formaldehyde by rhodobacter capsulatus RC1 and DC-1;

FIG. 4: comparison of the Formaldehyde metabolism profiles of rhodobacter capsulatus RC1 and DC-1.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention and the accompanying drawings, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1 screening, identification and detection of strains

The invention firstly collects bottom layer sludge water from the artificial wetland, adds formaldehyde with different concentrations to domesticate microorganisms in the sludge water, and then utilizes an inorganic salt culture medium of photosynthetic bacteria to separate the photosynthetic bacteria which grow fast and have strong resistance to the formaldehyde, and the specific screening steps are as follows:

1. acclimatization of activated sludge

Collecting water containing sludge at the bottom layer of the artificial wetland anaerobic tank, filtering by using gauze to remove large-particle impurities, adding formaldehyde into filtrate to acclimate microorganisms in the sludge water, gradually increasing the formaldehyde concentration from initial 0.5mM to final 10mM day by day, and continuously treating for 20 days.

2. Isolation of photosynthetic bacteria

After the acclimatization treatment is finished, a proper amount of sludge aqueous solution is taken to be inoculated into 500mL of photosynthetic bacteria culture medium (1L of culture medium component is CH)₃CH₂COONa 5g、NaHCO₃3.0g、NH₄Cl 1.5g，MgSO₄•7H₂O 0.5g、K₂HPO₄2.8 g, NaCl 2.5g and yeast powder 1.5g) in a mineral water bottle, mixing uniformly and covering the bottle mouth; culturing at 28 deg.C under 60W incandescent lamp for 7-8 days; sucking 50mL of red liquid when the culture solution turns red or blood red, pouring into mineral water bottle containing fresh culture solution, and continuously performing enrichment culture for 2-3 times. And (3) separating and purifying by adopting a double-layer plate method to obtain a single colony, and carrying out morphological observation and subsequent identification on the purified single strain on the plate.

3. Identification of isolated strains

Picking up pure culture on the plate with sterilized toothpick, transferring into 25mL anaerobic tube filled with culture solution, mixing well, sealing after filling with culture solution, culturing at 25 deg.C under 60W incandescent lamp for 7-8 days, and making the culture solution become blood red. And (3) taking a thallus smear, carrying out gram staining, and observing the thallus morphology under a microscope, wherein the result shows that the separated strain is a gram-negative bacterium, and the size of thallus cells is about 0.5-0.7 mu m. 2mL of the culture medium was put into a centrifuge tube, centrifuged at 8000 rpm for 15 minutes, and the viable cells were washed 2 to 3 times by centrifugation with physiological saline and resuspended in 60% (600 g/L) sucrose solution. The absorbance of the isolate was measured in the wavelength range of 200-800nm using a 60% sucrose solution as a blank, and the results indicated that the isolate had 4 characteristic absorption peaks in the wavelength range of 200-800nm, which were located at 375, 475, 509, 590nm, respectively. There was a special absorption doublet at 475nm and 509nm, indicating the presence of carotenoids in the isolated bacterial cells. There are also absorption peaks at 375nm and 590nm, indicating the presence of bacteriochlorophyll a in living cells. The 16S rDNA is amplified by PCR, the PCR product is detected by 1.2 percent agarose gel electrophoresis, and then the 16S rDNA sequencing data obtained by sequencing is shown as SEQ ID NO. 1.

Carrying out homologous alignment on the measured 16S rDNA sequence in a GenBank database, and determining the separated strain and the strainRhodobacter capsulatusStrain NBM67 strain 16S rRNA sequence homology 97% (figure 1), identified isolate as rhodobacter capsulatus of Rhodospirillaceae, RC1 for short.

Example 2

Resistance of RC1 to formaldehyde, effect of removing formaldehyde and metabolic pathway analysis

1. Resistance analysis of RC1 to Formaldehyde

The applied invention patent is a strain of rhodobacter capsulatus (Rhodobacter capsulatusstrain) DC-1 (201710038574.1), which has a preservation number of CCTCC NO: m2016676 that the strain has formaldehyde removing ability and can be used for treating sewage polluted by formaldehyde. For comparison with physiological and biochemical characteristics of the strain, DC-1 and the isolated photosynthetic strain RC1 were inoculated into an anaerobic tube containing 25ml of a photosynthetic bacteria liquid medium, 4mM and 6mM of formaldehyde were added to each bacterium when OD375 of each bacterium was 0.5 to 0.6, the mixture was cultured at 25 ℃ under light, OD375 of each bacterium was measured on 1, 2, 3, 4, 5, 6, 7, and 8 days after inoculation, and the growth of each bacterium was observed. The results show that under 4mM formaldehyde stress, there was no significant difference in OD375 between the two strains on days 1-2, both rising with time (FIG. 2, left); OD375 of DC-1 was greater than RC1 at 3-5 days (FIG. 2, left), indicating that the formaldehyde resistance of DC-1 was greater than RC1 at this time; however, the OD375 of DC-1 was less than RC1 (FIG. 2, left) at 5-8 days, indicating that the formaldehyde resistance of DC-1 was less than RC1 at this time period. The OD375 of DC-1 remained essentially unchanged at days 1-4 under 6mM formaldehyde stress, and the OD375 of DC-1 appeared gradually decreasing at days 5-8 thereafter (FIG. 2 right), indicating that DC-1 was less resistant to 6mM formaldehyde stress, 6mM formaldehydeThe flank forces part of the bacterial cells to die, so that the OD375 gradually decreases. In contrast, the OD375 of RC1 appeared in a pattern opposite to that of DC-1 (right in FIG. 2), gradually increasing at 1-5 days of 6mM formaldehyde stress (right in FIG. 2), and still slowly increasing at 5-8 days (right in FIG. 2), so that the OD375 of RC1 was significantly greater than that of DC-1 (right in FIG. 2) throughout the treatment period, which indicates that the resistance of the RC1 photosynthetic bacteria to 6mM formaldehyde was significantly greater than that of DC-1.

2. Analysis of effect of RC1 on removing formaldehyde

Introducing DC-1 and RC1 into an anaerobic tube containing 25ml of photosynthetic bacteria liquid culture medium, respectively adding 4mM and 6mM formaldehyde when the OD375 of each bacteria is 0.5-0.6, culturing at 25 ℃ under illumination, measuring the concentration of the residual formaldehyde in the treatment solution when the bacteria are inoculated for 1, 2, 3, 4, 5, 6, 7 and 8 days, detecting the volatilization amount of the formaldehyde in the treatment system by using a formaldehyde solution which is not added with bacteria liquid but contains the same concentration, and determining the formaldehyde removal rate according to the formula: 100% (initial formaldehyde of treatment liquid) -residual formaldehyde% of treatment liquid-volatile formaldehyde% calculation. The results show that the absorption efficiency of RC1 on 4mM (FIG. 3 left) and 6mM (FIG. 3 right) formaldehyde is greater than DC-1 at all time points, and that the absorption efficiency of RC1 on 4mM reaches 100% at day 7 (FIG. 3 left), the formaldehyde absorption efficiency of DC-1 is only 60% (FIG. 3 left); at day 8, when the absorption efficiency of RC1 on 6mM formaldehyde reached 100% (right in FIG. 3), the formaldehyde absorption efficiency of DC-1 was only-35% (right in FIG. 3). These data indicate that the absorption efficiency of RC1 for 4mM, 6mM formaldehyde is significantly greater than DC-1.

3. Pathway analysis for metabolic conversion of RC1 into formaldehyde

2g of somatic cells of rhodobacter capsulatus DC-1 and RC1 were collected by centrifugation and washed with 4mM H¹³CHO solution (5 ml, containing 5mM NaHCO)₃0.1% MES (2-N-Morpholino ethanesulfonic acid, W/V) was treated for 24 h under incandescent light, with no treatment of the sample as a background control. After the treatment, the thalli are collected centrifugally, 3ml of sterile water is added for suspension, the soluble metabolite is extracted by ultrasonic crushing,¹³C-NMR Nuclear magnetic resonance analysis H¹³CHO metabolic profile (fig. 4). By reacting with a known compound¹³The C-NMR spectra were compared to estimate the assignment of the resonance peak. Integrating the target formants with formamide as internal reference, and calculating each sampleRelative content of metabolites. The results show that the formaldehyde metabolism mechanism of DC-1 and RC1 is the same, and H of DC-1 and RC1 is treated for 24H¹³Multiple [ U-¹³C]Gluc (glucose) formant shows that the two photosynthetic bacteria can process H¹³CHO assimilation to glucose, but several [ U-¹³C]The intensity of the Gluc formant is obviously greater than that of DC-1, and the integral calculation result of the formant shows that the H of the DC-1¹³CHO treatment of [ U-¹³C]The relative content of Gluc is about 1.5 times that of CK control, while H of RC1¹³CHO treatment of samples [ U-¹³C]The relative content of Gluc reaches 12.5 times of that of the CK of the control, and is higher than that of H of DC-1¹³CHO treated samples were 3.3 times higher, indicating that RC1 assimilated H¹³The CHO glucose capacity is significantly greater than DC-1, thus making RC1 substantially more resistant to and efficient at absorbing formaldehyde than DC-1.

The rhodobacter palmatum RC1 can grow in a liquid inorganic salt culture medium containing 4mM or 6mM of formaldehyde under illumination, has strong resistance to formaldehyde, has a removal rate of 100% when being added into the culture medium, and can metabolize the formaldehyde to be converted into glucose through a photosynthetic carbon assimilation pathway which is a main mechanism for detoxifying the formaldehyde.

Finally, the above embodiments and the accompanying drawings are only intended to illustrate the technical solution of the present invention and not to limit, and although the present invention has been described in detail by the above embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the present invention as defined by the claims.

Sequence listing

<110> Wankui Yun-Nan Biotech Co., Ltd

<120> rhodobacter xylinum strain RC1 and application thereof

<141>2017-07-26

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<170>SIPOSequenceListing 1.0

<210>1

<211>1319

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<213> Rhodobacter xylinum (Rhodobacter capsulatus strain NBM 67)

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GTCGACGAAC TCTTCGGAGT TAGTGGCGGA CGGGTGAGTA ACACGTGGGA ACGTGCCTTT 60

AGGTTCGGAA TAACTCAGGG AAACTTGTGC TAATACCGAA TGTGCCCTTC GGGGGAAAGA 120

TTTATCGCCT TTAGAGCGGC CCGCGTCTGA TTAGCTAGTT GGTGAGGTAA AGGCTCACCA 180

AGGCGACGAT CAGTAGCTGG TCTGAGAGGA TGATCAGCCA CATTGGGACT GAGACACGGC 240

CCAAACTCCT ACGGGAGGCA GCAGTGGGGA ATCTTGCGCA ATGGGCGAAA GCCTGACGCA 300

GCCATGCCGC GTGAATGATG AAGGTCTTAG GATTGTAAAA TTCTTTCACC GGGGACGATA 360

ATGACGGTAC CCGGAGAAGA AGCCCCGGCT AACTTCGTGC CAGCAGCCGC GGTAATACGA 420

AGGGGGCTAG CGTTGCTCGG AATTACTGGG CGTAAAGGGA GCGTAGGCGG ACATTTAAGT 480

CAGGGGTGAA ATCCCGGGGC TCAACCTCGG AATTGCCTTT GATACTGGGT GTCTTGAGTA 540

TGAGAGAGGT GTGTGGAACT CCGAGTGTAG AGGTGAAATT CGTAGATATT CGGAAGAACA 600

CCAGTGGCGA AGGCGACACA CTGGCTCATT ACTGACGCTG AGGCTCGAAA GCGTGGGGAG 660

CAAACAGGAT TAGATACCCT GGTAGTCCAC GCCGTAAACG ATGATTGCTA GTTGTCGGGA 720

TGCATGCATT TCGGTGACGC AGCTAACGCA TTAAGCAATC CGCCTGGGGA GTACGGTCGC 780

AAGATTAAAA CTCAAAGGAA TTGACGGGGG CCCGCACAAG CGGTGGAGCA TGTGGTTTAA 840

TTCGAAGCAA CGCGCAGAAC CTTACCACCT TTTGACATGC CTGGACCGCC AGAGAGATCT 900

GGCTTTCCCT TCGGGGACTA GGACACAGGT GCTGCATGGC TGTCGTCAGC TCGTGTCGTG 960

AGATGTTGGG TTAAGTCCCG CAACGAGCGC AACCCTCGCC ATTAGTTGCC ATCATTTAGT 1020

TGGGAACTCT AATGGGACTG CCGGTGCTAA GCCGGAGGAA GGTGGGGATG ACGTCAAGTC 1080

CTCATGGCCC TTACAGGGTG GGCTACACAC GTGCTACAAT GGCGACTACA GAGGGTTAAT 1140

CCTTAAAAGT CGTCTCAGTT CGGATTGTCC TCTGCAACTC GAGGGCATGA AGTTGGAATC 1200

GCTAGTAATC GCGGATCAGC ATGCCGCGGT GAATACGTTC CCGGGCCTTG TACACACCGC 1260

CCGTCACACC ATGGGAGTTG GTTCTACCCG AAGGCGCTGC GCTGACCGCA AGGAGGCAG 1319

Claims (4)

1. A Rhodobacter sphaeroides RC1 (Rhodobacter capsulatus strain RC 1) is characterized in that: the preservation number of the strain in China center for type culture Collection is CCTCC: m2017323.

2. The enveloped rhodobacter RC1 of claim 1, wherein: the 16SRNA gene sequence of the strain is shown as SEQID NO. 1.

3. The enveloped rhodobacter RC1 of claim 1, wherein: the strain has resistance to formaldehyde and has a formaldehyde photosynthetic carbon assimilation way.

4. The enveloped rhodobacter RC1 of claim 1, wherein: the strain is applied as a biological resource in the purification of formaldehyde pollution.

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