CN102337236B - Alkaline Bacilluspseudofirmus MC02 and application thereof - Google Patents

Abstract

The invention discloses alkaline Bacilluspseudofirmus MC02 CGMCC (China General Microbiological Culture Collection Center): 4772 and application of the alkaline Bacilluspseudofirmus MC02 in reduction of iron oxides and humus. The Bacilluspseudofirmus MC02 is obtained by separating and purifying an alkaline sludge microbial fuel cell anode biofilm which well operates; and under the alkaline condition, the Bacilluspseudofirmus MC02 has strong Fe(III) and AQDS (anthraquinone-2,6-disulfonic acid) reducing capacity, broad electron utilization spectrum and wide application prospect.

Description

One strain alkaline species bacillus firmus and application thereof

Technical field

The invention belongs to the Environmental Biotechnology field, be specifically related to a strain class bacillus firmus ( Bacillus pseudofirmus), and soil ulmin reduction and the application of iron reductive under alkaline anaerobic condition.

Background technology

Humus respiration is a ubiquitous microbial respiratory metabolisable form in the anaerobic environment.Humus respiration claims that again quinone breathes, refer to mikrobe with soil ulmin as unique terminal electron acceptor, accept the electronics that provides from materials such as organic acids, the process of thalli growth is supported in the generation of coupling energy.Humus respiration and iron reduction are in close relations.Humus respiration acts in the biomass geochemistry working cycle of element significant; Show as: 1) mikrobe is in the process of carrying out humus respiration; Can produce CO directly with organic matter in the environment or environment toxic substance oxidations such as toluene, vinylchlorid ₂2) respiration produces goes back some the oxidation state materials in ortho states humic, the enough reducing environments of mass-energy, like Fe (III), Mn (IV), Cr (VI), U (VI), nitroaromatic and many halos pollutent.The humic acid respiration can influence the biomass geochemistry circulation of C in the environment, N, Fe, Mn and some trace metal elements; And can promote the detoxification of heavy metal and organic pollutant; Have active effect at aspects such as self, the reparation of contaminated soil original position, WWTs, therefore the relevant research of humus respiration in recent years receives much attention.

Since the later stage nineties in last century, multiple with humic acid or humic acid pattern thing anthraquinone-2,6-disulfonic acid (AQDS) also separates in succession for the bacterium of terminal electron acceptor and identifies.Yet alkaline condition is unfavorable for microbial growth relatively, and under this exacting terms, mikrobe possibly tamed out a cover mechanism, with the environment of cope with bad better.At present, do not find to have the class bacillus firmus report of Fe (III) reduction and humic acid reducing activity as yet.

Summary of the invention

The objective of the invention is to find the efficient reduction of the alkalescence bacterium of a strain anaerobic humus and ferriferous oxide.

Another object of the present invention provides the application of above-mentioned type of bacillus firmus in ferriferous oxide and soil ulmin reduction.

The class bacillus firmus of gained of the present invention ( Bacillus pseudofirmus) MC02, being deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center on April 21st, 2011, the preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preserving number is: CGMCC:4772, and in identifying the bacterial classification survival on the same day.

Fe (III) breathes and the bacterial strain of humus respiration capability to the invention provides new the having of a strain; Said bacterial strain is compared with reporting bacterial strain, under alkaline condition, has stronger Fe (III) reduction and humic acid reducing power; Electronics utilization spectrum is wider; Can reduce the higher ferriferous oxide of percent crystallinity, for example lepidocrokile, rhombohedral iron ore are with a wide range of applications.

Description of drawings

Fig. 1 is the design sketch that bacterial strain MC02 utilizes different electron donor reduction AQDS;

Fig. 2 is the design sketch that bacterial strain MC02 utilizes different electron donor reduction Fe (III);

Fig. 3 is that bacterial strain MC02 is a substrate with glucose, reduces the design sketch of different ferriferous oxides;

Fig. 4 is that bacterial strain MC02 is substrate with glucose, under different electron shuttle body mediations, and the design sketch of reduction lepidocrokile.

Embodiment

The enrichment of bacterial strain MC02 with separate

1) from pH is 9.0 sludge microbe fuel cell, tears and get the anode carbon felt and be soaked in the 50mL liquid enrichment medium; (configuration such as the CN200910041235.4 of sludge microbe fuel cell are said), the composition of liquid concentration and separation substratum is: 16g AQDS (electron acceptor(EA)), 0.25g NH ₄Cl, 0.678g NaH ₂PO ₄2H ₂O, 0.1g KCl, 2.94g NaHCO ₃, 1.59g Na ₂CO ₃, 10.0mL vitamin solution, 10.0mL trace element solution (pH 9.0).Wherein, vitamin solution is to contain 2.0mg vitamin H, 2.0mg folic acid, VB6 10.0mg VITAMINs, 5.0mg VitB1,5.0mg vitamin G, 5.0mg nicotinic acid, 5.0mg VA, 0.1mg cobalamin, 5.0mg para-amino benzoic acid, 5.0mg Thioctic Acid in every liter of deionized water; Trace element solution is to contain 1.5g nitrilotriacetic acid(NTA), 3.0g MgSO in every liter of deionized water ₄7H ₂O, 0.5g MnSO ₄H ₂O, 1.0g NaCl, 0.1g FeSO ₄7H ₂O, 0.1g CoCl ₂6H ₂O, 0.1g CaCl ₂, 0.1g ZnSO ₄7H ₂O, 0.01g CuSO ₄5H ₂O, 0.01g AlK (SO ₄) ₂12H ₂O, 0.01g H ₃BO ₃, 0.01g Na ₂MoO ₄2H ₂O;

2) inflated with nitrogen is no less than 30 minutes in the vaccinated enrichment medium, and air-blowing finishes and covers serum cap and add the aluminium lid sealing, places anaerobism workstation (N ₂/ CO ₂=80/20), 30 ℃ leave standstill cultivation, observe the colour-change situation of nutrient solution;

3) become when orange-yellow from colourless when the nutrient solution color, the inoculum size with 10% is forwarded to another fresh liquid separation culture medium with nutrient solution, and anaerobic operation and anaerobism culture condition are with 2), so enrichment culture is three times;

4) reacted nutrient solution employing of the 4th generation dilution-plate method is suitably diluted, nutrient solution 0.1 mL after the dilution is evenly coated on the LB solid medium, place anaerobism workstation (N ₂/ CO ₂=80/20) cultivate 48h for 30 ℃, picking list bacterium colony carries out single bacterium colony isolation and purification, and wherein, the composition of said LB solid medium is: peptone 10g/L, and yeast extract 5g/L, NaCl 10 g/L, agar powder 20 g/L, pH are 9.0.

Obtained strains type of being bacillus firmus ( Bacillus pseudofirmus) MC02, on April 21st, 2011 in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, preserving number is: CGMCC 4772, and in identifying bacterial classification survival on the same day.

Bacterial strain( Bacillus pseudofirmus) MC02 Morphological specificity

1) thalli morphology characteristic

Bacterial strain MC02 Gram-positive, shaft-like, gemma and middle life are arranged, the paired or catenation of thalline.

) the colonial morphology characteristic

After aerobic LB agar plate was cultivated 48h, the bacterium colony smooth surface was opaque, and the edge is irregular, the about 2-3mm of colony diameter.

Bacterial strain( Bacillus pseudofirmus) MC02 Physiological and biochemical property

According to the physiological and biochemical property of " the outstanding Bacteria Identification identification handbook of uncle " the 9th edition described standard program test strain, its biochemical character is seen table 1.

Bacterial strain( Bacillus pseudofirmus) MC02 The molecular classification status

The 16SrRNA of the bacterial strain that separation is obtained is through pcr amplification, order-checking.The Genebank number of landing of the 16S rRNA gene order of bacterial strain PAH-1 is JN566125, and concrete sequence is seen SEQ ID NO:1.

The sequence that obtains is compared with the relevant 16S rRNA sequence on the Genebank.Compare with Cluster X method with the disclosed corresponding bacterial strain sequence of Genebank.Based on 1000 multiple sampling analyses, use MEGA 4.0 to press adjacent method constructing system tree.The 16S rRNA phylogenetic tree that structure obtains is as shown in Figure 1.

Theoretical according to phylogeny, similarity just can be thought unified bacterial strain only greater than 97% between bacterial strain.Aimed strain reaches 99.8% with a type bacillus firmus similarity, and therefore, bacterial strain MC02 is defined as Bacillus pseudofirmus

Bacterial strainMC02 Utilize the test of different electron donor reduction AQDS

Liquid culture based formulas: 0.25g NH in every liter of deionized water ₄Cl, 0.678g NaH ₂PO ₄2H ₂O, 0.1g KCl, 2.94g NaHCO ₃, 1.59g Na ₂CO ₃Each 10.0 mL (vitamin solution and the same isolation medium of trace element solution composition) of vitamin solution and trace element solution; (carbonic acid buffering, pH 9.0) was in 121 ℃ of sterilizations 20 minutes; Different electron donors such as the sodium acetate of sterilization back interpolation sterilization, glucose, sucrose, USP Kosher, lactic acid, final concentration is 10 mmol/L.Be seeded to the beef extract-peptone liquid nutrient medium from the inclined-plane picking lawn of preserving bacterial strain MC02, in 30 ℃, 180 rev/mins of shaking table activation thalline 18 hours make bacterial number reach exponential phase of growth.4 ℃, the centrifugal 10min of 8000r/min removes supernatant, and carbonic acid buffer suspends again.Repeat aforesaid operations 2 times.About 1.2 (λ=600 nm) of concentration of final suspension-s.Add 1ml (CFU=1.0 * 10 ⁷) as handling, the contrast that does not add bacterium is set simultaneously.Every separated about 12h gets the 4mL nutrient solution, adopts ultraviolet-visible spectrophotometer to measure AH at the 510nm place ₂DS content is with AH ₂DS concentration is index, and the checking bacterial strain utilizes the ability of different electron donor reduction AQDS, and test-results is as shown in Figure 1.Can know that from Fig. 1 when being electron donor with sucrose, glucose, USP Kosher and lactic acid in the system, the reduction efficiency of AQDS is higher, wherein, when being substrate with glucose, reduction efficiency is the highest, has 85% AQDS to be reduced approximately.The electronics utilization spectrum of MC02 is wider, and reduction efficiency is higher.

Bacterial strainMC02 Utilize the test of different electron donor reduction Fe (III)

The prescription of liquid nutrient medium is the same, contains the Fe (III) (soluble state iron) of 25mM in the system.Other operation is the same.Every got at a distance from 5 days the 4ml nutrient solution that mixes in 16ml 0.5mol/l HCl solution 180 rev/mins vibrated 1.5 hours; Refilter; Adopt adjacent phenanthroline method to give the Fe (II) in the filtrating colour developing; Adopt ultraviolet spectrophotometer to measure the content of Fe (II) at 510nm place, and be contrast, verify the iron reducing power of bacterial strain with the nutrient solution that does not add bacterium.As shown in table 2, MC02 can utilize sucrose, USP Kosher, Hydrocerol A and glucose reduction Fe (III).Wherein, when being substrate with sucrose, reduction efficiency is the highest, has the iron of 0.5mM to be reduced approximately.

Bacterial strainMC02 With glucose is donor, reduces the test of different ferriferous oxides

The prescription of liquid nutrient medium is the same, contains the different ferriferous oxide (pyrrhosiderite, lepidocrokile and rhombohedral iron ore) of 25mM in the system.Other operation and testing method are the same.The result is as shown in Figure 3, is electron acceptor(EA) with glucose, and bacterial strain is the highest to the reduction efficiency of lepidocrokile, secondly is pyrrhosiderite.

Bacterial strainMC02 With glucose is donor, under different electron shuttle body mediations, and the test of reduction lepidocrokile

The prescription of liquid nutrient medium is the same, contains the lepidocrokile of 25 mM in the system, and the electron shuttle body adds the AQDS of 0.5 mM and the humic acid (HA) of 0.2 g/l respectively.Result such as Fig. 4 add AQDS and HA and can obviously improve the reduction efficiency of bacterial strain to ferriferous oxide, and reduction of a fraction you can well imagine high 16% and 38%.

< 110>Guangdong Prov. Inst. of Ecological Environment & Soil Science

 

< 120>one strain alkaline species bacillus firmus and application thereof

 

<130> MC02

 

<160> 1

 

<170> PatentIn?version?3.5

 

<210> 1

<211> 1408

<212> DNA

< 213>type bacillus firmus (Bacillus pseudofirmus)

 

<400> 1

acataaaggt?tacctcaccg?acttcgggtg?ttacaaactc?tcgtggtgtg?acgggcggtg 60

 

tgtacaaggc?ccgggaacgt?attcaccgcg?gcatgctgat?ccgcgattac?tagcaattcc 120

 

ggcttcatgt?aggcgagttg?cagcctacaa?tccgaactga?gaatggcttt?atgggattcg 180

 

ctcaacctcg?cggttttgca?gccctttgta?ccatccattg?tagcacgtgt?gtagcccagg 240

 

tcataagggg?catgatgatt?tgacgtcatc?cccaccttcc?tccggtttgt?caccggcagt 300

 

caccttagag?tgcccaactg?aatgctggca?actaagatca?agggttgcgc?tcgttgcggg 360

 

acttaaccca?acatctcacg?acacgagctg?acgacaacca?tgcaccacct?gtcactttgt 420

 

cccccgaagg?ggaaagccct?atctctaggg?tggtcaaagg?atgtcaagac?ctggtaaagg 480

 

ttcttcgcgt?tgcttcgaat?taaaccacat?gctccactgc?ttgtgcgggc?ccccgtcaat 540

 

tcctttgagt?ttcaaccttg?cggtcgtact?ccccaggcgg?agtgcttaat?gtgttaactt 600

 

cggcactaag?ggcatcgaaa?cccctaacac?ctagcactca?tcgtttacgg?cgtggactac 660

 

cagggtatct?aatcctgttt?gctccccacg?ctttcgcgcc?tcagcgtcag?ttacagacca 720

 

gagagtcgcc?ttcgccactg?gtgttcctcc?acatatctac?gcatttcacc?gctacacgtg 780

 

gaattccact?ctcctcttct?gtactcaagt?ctcccagttt?ccaatgaccc?tccacggttg 840

 

agccgtgggc?tttcacatca?gacttaagag?accgcctgcg?cgcgctttac?gcccaataat 900

 

tccggacaac?gcttgccacc?tacgtattac?cgcggctgct?ggcacgtagt?tagccgtggc 960

 

tttctggtta?ggtaccgtca?aggtgccgcc?ttattcaaac?ggcacttgtt?cttccctaac 1020

 

aacagagctt?tacgatccga?aaaccttcat?cactcacgcg?gcgttgctcc?gtcagacttt 1080

 

cgtccattgc?ggaagattcc?ctactgctgc?ctcccgtagg?agtctgggcc?gtgtctcagt 1140

 

cccagtgtgg?ccgatcaccc?tctcaggtcg?gctacgcatc?gtcgccttgg?taagccgtta 1200

 

ccttaccaac?tagctaatgc?gccgcgggcc?catctgtaag?tgatagccag?aggccatctt 1260

 

ttaccgctcc?accatgaggt?ggaacgggtt?atccggtatt?agccccggtt?tcccggagtt 1320

 

atcccagtct?tacaggcagg?ttgcccacgt?gttactcacc?cgtccgccgc?taacatcagg 1380

 

gagcaagctc?ccatcagtcc?gctcgact 1408

Claims (2)

1. Type bacillus firmus ( Bacillus pseudofirmus) bacterial strain MC02, its preserving number is CGMCC:4772.
2. 2. the application of said type of bacillus firmus MC02 of claim 1 in ferriferous oxide and soil ulmin reduction.

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