CN107384839B

CN107384839B - Bacillus siamensis BERC-11 and application thereof

Info

Publication number: CN107384839B
Application number: CN201710789312.9A
Authority: CN
Inventors: 王彦伟; 何明雄; 吴波; 谭芙蓉; 代立春; 祝其丽
Original assignee: Biogas Institute of Ministry of Agriculture
Current assignee: Biogas Institute of Ministry of Agriculture
Priority date: 2017-09-05
Filing date: 2017-09-05
Publication date: 2020-06-30
Anticipated expiration: 2037-09-05
Also published as: CN107384839A

Abstract

The invention discloses a Bacillus siamensis BERC-11 and application thereof, which is preserved in Guangdong province microorganism strain preservation center with the preservation number: GDMCC No. 60223. The screened strain BERC-11 of the invention can tolerate 3.5g L^‑1Furaldehyde at 3.5g L^‑1Under the concentration of the furfural, the enzyme activity of the filter paper reaches 0.1U/ml, the enzyme activity of the CMC reaches 0.21U/ml, the enzyme activity of the cellobiose reaches 0.07U/ml, and the cellose can play a role in the pretreatment process of the lignocellulose.

Description

Bacillus siamensis BERC-11 and application thereof

Technical Field

The invention belongs to the field of microorganisms, and particularly relates to a furfural-resistant cellulose degrading bacterium and application thereof.

Background

The biomass conversion of lignocellulose is one of important means for solving the problems of environmental pollution and energy crisis, and in the last thirty years, researchers focus on the conversion of lignocellulose into fuels, energy sources and chemical products with high added values. However, during the conversion of lignocellulose, a large number of intermediate toxic metabolic inhibitors are produced. Furfural is a metabolite produced during the conversion of lignocellulose that can damage cell membranes and cell walls, degrade DNA, block the synthesis of proteins and RNA, inhibit biological activity, and kill cells. Overcoming these toxic compounds from lignocellulose is a significant challenge in developing low cost, sustainable biomass fuels.

Microbial resources are the basis of industrial fermentation, many researchers have applied furfural-tolerant fermentation strains to industrial fermentation of biomass fuels and have explored some tolerance genes integrated into fermentation strains by screening for tolerant microorganisms, and Sarah j field et al screened Saccharomyces cerevisiae and Saccharomyces paradoxus that can grow at 3g/L of furfural when studying ethanol fermentation systems, but high concentrations of furfural affected ethanol production. Liu et al screened a furan formaldehyde-tolerant lactic acid fermenting strain Bacillus lauguuguigue B38. Genes related to the tolerance of the furan formaldehyde are integrated into Zymomonasmobilis, Escherichia coli, so that the yield of ethanol and the tolerance concentration of the furan formaldehyde are improved. Nevertheless, researchers have focused on fermentation strains, and cellulose-degrading bacteria resistant to furfural have been reported only rarely.

Disclosure of Invention

The technical problem to be solved by the invention is as follows: how to provide a cellulose degrading strain with high furan formaldehyde tolerance.

The technical scheme of the invention is as follows: a Siamese Bacillus (Bacillus siamensis) BERC-11 is preserved in Guangdong province microorganism strain preservation center, the preservation date is 2017, 8 and 17 days, and the preservation address is as follows: the microbial research institute of Guangzhou province, No. 59 building, No. 5 building, Guangdong province, of the Zhonglu-Jieli, Guangzhou city, the preservation number: GDMCC No. 60223. Hereinafter, the strains are simply referred to as BREC-11.

Fermentation broth or culture broth containing the strain BERC-11.

Use of the strain BERC-11 or a fermentation broth or culture broth thereof for the pretreatment of lignocellulosic material.

The application of the strain BERC-11 or fermentation liquor or culture solution thereof in the preparation of microbial fertilizers.

The strain BREC-11 can tolerate high-concentration furfural, maintain high cellulase activity, and degrade lignocellulose, so that the strain BREC-11 can be widely used for pretreatment of lignocellulose-containing substances, and help is provided for industrialization of the lignocellulose-containing substances. Can also be used in the preparation of microbial fertilizers to accelerate the degradation of lignocellulose of organic matters.

Compared with the prior art, the invention has the following beneficial effects:

the invention takes furan formaldehyde as an inhibitor, separates a high-tolerance cellulose degrading bacterium through continuous enrichment culture of a cellulose degrading culture medium, and preliminarily identifies the strain to be a bacillus through heterogeneous taxonomy. Named Siamese Bacillus (Bacillus siamensis) BERC-11. The strain BERC-11 screened by the invention can tolerate 3.5gL^-1Furaldehyde at 3.5g L^-1Under the concentration of the furfural, the enzyme activity of the filter paper reaches 0.1U/ml, the enzyme activity of the CMC reaches 0.21U/ml, the enzyme activity of the cellobiose reaches 0.07U/ml, and the cellose can play a role in the pretreatment process of the lignocellulose.

Drawings

FIG. 1 is a congo red staining chart;

FIG. 2 is an optical microscopic observation of the strain BREC-11;

FIG. 3 is a phylogenetic tree of 16S rDNA-based strains constructed using the NJ method;

FIG. 4 is the total ester content of strain BREC-11;

FIG. 5 shows the growth of BREC-11 strain at different concentrations of furfural;

FIG. 6 shows the cellulase activity of strain BREC-11 under the condition of 3.5g/L furfural.

Detailed Description

Example 1

1. Materials and methods

1.1 sources of insect bodies

Bamboo insect (Omphisafuscidentalis Hampson) larvae were collected from the Dai autonomous State of the Western Banna, Yunnan province.

1.2 reagents and culture media

Carboxymethyl cellulose (CMC) and saligenin (Salicin) were produced by Sigma, microcrystalline cellulose (MCC) by Fluka, furaldehyde by Body chemical Co., Ltd, and all the reagents were domestic analytical pure unless otherwise specified.

Improved Hercinon inorganic salt culture medium (KH)₂PO₄1.0g，NaCl 0.1g，MgSO₄7H₂O 0.3g,NaNO₃2.5gFeCl₃0.01g,CaCl₂0.1g,CMC-Na 5g，H₂O1L), adding furfural with different concentrations, fermenting CMC to produce enzyme culture medium (CMCNa 10g, peptone 5g, yeast powder 1.0g), adding agar 18g to prepare solid culture medium. The fatty acid analysis medium was TSA medium (BD/Difco 236950, USA).

1.3 isolation of Furancarbaldehyde-tolerant cellulose-degrading bacteria

Selecting 10 worms, sterilizing the epidermis, extracting the intestinal tract in a sterile operating platform, placing the worms in sterile water for fully homogenizing, absorbing 5ml of homogenate, placing the homogenate in 100ml of improved Hercinon inorganic salt culture medium added with 0.5g/L of furfural at 30 ℃, culturing for 36h at 150rmp/min, absorbing 5ml of homogenate, transferring the homogenate to a culture medium added with 1g/L of furfural, carrying out shake culture, continuously enriching until the concentration of the furfural in the culture medium reaches 5g/L, separating strains by adopting a plate dilution coating method, uniformly coating 100ul of diluent on a plate of a CMC fermentation culture medium, carrying out 30 ℃ constant temperature inversion culture for 36h, picking out a single colony for streak culture until the single colony is purified to the single strain, inoculating the picked single strain to the CMC fermentation solid plate, and carrying out 30 ℃ culture for 3d, and then carrying out Congo red staining and primary screening on cellulose degradation bacteria.

1.4 identification of the Strain

Morphological observation, physiological and biochemical tests, 16S rRNA gene sequence analysis and cytochemical component analysis are carried out on the separated strains.

The morphological and physiological biochemical characteristics of the strains are described in the literature (Ruan, Z., Wang, Y., Song, J., Jiang, S., Wang, H., Li, Y., & Zhao, B. Kurthia huakukuaki. nov., isolated from biological strain, and expressed description of the gene Kurthia. International journal of systematic and analytical microbiology,2014,64(2), 518-one 521). 16S rRNA gene sequence analysis and reference for cytochemical composition analysis (Xu, X.W., Huo, Y.Y., Wang, C.S., Oren, A., Cui, H.L., Vedler, E., & Wu, M.Pelagi bacillus halologic gen.n., sp.n.and Pelagi bacillus sp.n., and novel members of the family of the microorganisms and the evolution of the microbiology,2011,61(8), 1817-1822).

1.5 Strain Furancarbaldehyde tolerance concentration determination

The screened strains are respectively inoculated into a culture medium containing 0,0.5,1,1.5,2.0,2.5,3.0,3.5,4.0,4.5,5.0,5.5 and 6.0g/L of furfural, each group is repeated three times, one strain without inoculation is used as a blank control, the culture is cultured for 3 days at 30 ℃ under 150rmp/min shaking, and the light absorption value OD is OD₆₀₀The growth of the cells was measured.

1.6 determination of cellulase of strain.

The strains were cultured in a medium supplemented with 3.5G/L of modified Hodgson medium (KH2PO41.0G, NaCl 0.1G, MgSO47H2O 0.3G, NaNO32.5g FeCl30.01g, CaCl20.1g, CMC-Na 5G, H2O L) and a medium supplemented with no furfural at 30 ℃ for 3d, centrifuged at 4 ℃ and 8000rpm for 10min, the supernatant was used as a crude enzyme solution, the cellulase activity was measured by the DNS method (Oppert et al, 2010; Shi et al, 2011), the activity of the cellulase (FPA), endo- β -1, 4-glucanase (CMC) and β -glucosidase (β -G) was measured by comparison, the enzyme activity of the filter paper was measured by using Whatman1 (1cm x 6cm) strip as a substrate, the enzyme was measured by 1% sodium carboxymethylcellulose (CMC), the substrate was measured by 25-2% CMC, the substrate was measured by sodium dihydrogenphosphate (3.5L), the substrate was used by sodium citrate was measured by sodium phosphate (3.8 mol), the pH of the filter paper was measured by sodium phosphate buffer, and the substrate was measured by the same method^-1) And a glycine-sodium hydroxide buffer solution (0.2 mol. L) having a pH of 9^-1) And (4) preparing. Mixing 0.1mL of diluted enzyme solution with 0.5mL of the above substrate, warm-bathing in 50 deg.C water bath for 30min, 40min, 60min, adding 2mL of DNS developer, developing in boiling water bath for 10min, cooling with running water, measuring OD at 540nm, repeating the measurement for 3 times, averaging, and setting blank control. The enzyme activity was calculated as the amount (. mu. mol.) of reducing sugar (glucose) produced by the enzymatic reaction per unit time (min) in the enzyme solution protein content (mg) under the test conditions, i.e., μmol. min^-1mg^-1(Shi et al.，2011)。

2. Results and analysis

2.1 isolation of Furfural Formaldehyde-tolerant cellulose-degrading bacteria

Three degrading strains are screened after Congo red dyeing primary screening, and are shown in figure 1. Among them, the strain with the number O-2 had the largest clearing circle/colony diameter and the colony diameter was 2.2, which was designated as the strain BREC-11.

2.2 identification of Strain BREC-11

The strain BREC-11 is gram-positive bacteria, facultative anaerobic and mobile. Colonies on TSA plates were off-white, opaque, smooth-edged, and slightly rough-surfaced. The shape of the cells is shown in FIG. 2, and the cells are rod-shaped and have a size of 0.3-0.6 x 1.5-3.5. mu.m. The growth temperature is 10-50 ℃, the pH range is 5-9, and the NaCl concentration is 8% (W/V) tolerance. Glucose, sucrose, xylose, fructose, lactose, glycogen, glycerol, ribose, inositol, mannitol, raffinose, maltose can be used as the carbon source. The results of part of the physiological and biochemical experiments of the strain BREC-11 are shown in Table 1.

Table 1: physiological and biochemical results of part of the strain BERC-11

Obtaining a 1430bp genome sequence, comparing and analyzing the 16S rDNA of the strain BERC-11 on an eztamte website, and displaying the results of the strain BREC-11 and Bacillus siemensisssis KCTC13613^TThe similarity reaches 99.93 percent, a phylogenetic tree is constructed, and as shown in figure 3, the strain BERC-11 and the Bacillus siamensis KCTC13613^TIn the same subcircuit, the stability is higher and the evolutionary relationship is similar. Phylogenetic tree and sequence analysis results show that the strain BERC-11 is classified into Bacillus (Bacillus).

The fatty acid type of the strain BREC-11 mainly comprises anteiso-C_15:0(41.36%) and anteiso-C_17:0(23.31%) in iso-C_15:0(10.3%) content of Bacillus siemensis KCTC13613 as determined by PunnaeeSumpadapol^THas a difference in the main fatty acid types, Bacillus siamensis KCTC13613^Tiso-C in fatty acids of_15:0The content of (a) is 9.8% less iso-C in the fatty acids of the strain BERC-11_15:0. The polar lipid type of strain BERC-11 includes diphosphodydiglycol, diphosphodylethanolamine, lysylphosphatidydiglycol, glycolipd, 1 unknown aminophosphatid, 4 unknown aminophylipids and 2 unknown lipds as shown in FIG. 4.

Through morphology, physiological and biochemical tests, cytochemical analysis and 16r RNA sequence analysis, the strain BERC-11 is a kind of Bacillus (Bacillus) and is named as Siamese Bacillus (Bacillus simensis) BERC-11.

2.3 Furancarbaldehyde-tolerant concentration determination of Strain BERC-11

Analysis of Furanormaldehyde tolerance of Strain BERC-11 As shown in FIG. 5, when the concentration of Furanormaldehyde in the medium reached 1.5g/L, the growth was affected, but normal growth was also possible, and until the concentration of Furanormaldehyde reached 4.0g/L, the absorbance decreased and Furanormaldehyde inhibited the growth of Strain BREC-11. Furfural formaldehyde can destroy cell wall and cell membrane, cause damage to nucleic acid structure, and inhibit synthesis of protein and RNA. IrnayuSitepu researches the furan formaldehyde tolerance of 45 strains of yeast, and the result shows that most strains can only grow in a culture medium of 0-0.5g/L of furan formaldehyde, and only a small part of strains can grow in 1g/L of furan formaldehyde. The growth of microorganisms is inhibited by the high-concentration furfural, and the strain BREC-11 can tolerate furfural at high concentration, so that microbial resources and tolerant gene resources can be provided for biorefinery.

2.4 Strain BREC-11 cellulase

The strain BREC-11 is cultured for 2 days in an improved Hocqueson culture medium added with 3.5g/L of furfural at 30 ℃ under 150rmp, the cellulase activity is determined, the enzyme activity of filter paper is determined to reach 0.1U/ml, the enzyme activity of CMC is determined to reach 0.21U/ml, and the enzyme activity of cellobiose is determined to reach 0.07U/ml. The results are shown in FIG. 6.

3. Conclusion

In the pretreatment process of cellulose biorefinery, some inhibiting substances (furan formaldehyde, phenol inhibitors and the like) with toxic effects exist, and can cause toxic effects on microorganisms, so that the fermentation efficiency is reduced. To overcome this dilemma, researchers have screened a large number of tolerant strains and related tolerant genes, but these studies are based on fermentation strains such as E.coli, Saccharomyces cerevisiae, Bacillus coagulans, Z.mobilis, etc. The invention takes furan formaldehyde as an inhibitor, separates a cellulose degradation bacterium BREC-11 with high tolerance by continuous enrichment culture of a cellulose degradation culture medium, and preliminarily identifies the strain BREC-11 as a bacillus species through heterogeneous taxonomy.

The biological refining of cellulose is inhibited by high-concentration furaldehyde, and Peng finds that Bacillus coagulans P38 tolerates furaldehyde with concentration of 10g L in the process of researching lactic acid producing bacteria Bacillus coagulans P38^-1But at a furan formaldehyde concentration of 6g L^-1The lactic acid production is severely inhibited. Sarah J Field studied the tolerated concentrations of furfural between 71 strains of Saccharomyces cerevisiae and Saccharomyces paradoxus, only five strains at 3g L^-1The yield of ethanol is reduced, and the screened Bacillus BREC-11 can tolerate 3.5g L^-1Furaldehyde at 3.5g L^-1Under the concentration of the furfural, the enzyme activity of the filter paper reaches 0.1U/ml, the enzyme activity of the CMC reaches 0.21U/ml, and the enzyme activity of the cellobiose reaches 0.07U/ml. Can play a role in the pretreatment process of straws and provide strain resources for the pretreatment process of biorefinery.

The above-mentioned embodiments only express the specific embodiments of the present application, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present application. It should be noted that, for those skilled in the art, without departing from the technical idea of the present application, several changes and modifications can be made, which are all within the protection scope of the present application.

Claims

1. A Siamese Bacillus (Bacillus siamensis) BERC-11 is preserved in Guangdong province microorganism strain preservation center, and the preservation number is as follows: GDMCC No. 60223.

2. A fermentation broth or culture comprising the siamese bacillus BERC-11 of claim 1.

3. Use of the Siamese Bacillus BERC-11 of claim 1 or the fermentation broth or culture solution of claim 2 for the pretreatment of lignocellulosic material.

4. The use of bacillus siamensis BERC-11 according to claim 1 or the fermentation broth or culture solution according to claim 2 in the preparation of a microbial fertilizer.