CN107129949B - Bacillus for promoting enzymolysis of eichhornia crassipes substrate to produce sugar and application thereof - Google Patents

Bacillus for promoting enzymolysis of eichhornia crassipes substrate to produce sugar and application thereof Download PDF

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CN107129949B
CN107129949B CN201710428067.9A CN201710428067A CN107129949B CN 107129949 B CN107129949 B CN 107129949B CN 201710428067 A CN201710428067 A CN 201710428067A CN 107129949 B CN107129949 B CN 107129949B
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eichhornia crassipes
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周文兵
杨庆
肖乃东
朱端卫
肖凯
张超奇
冯伟
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Huazhong Agricultural University
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Abstract

The invention provides a Bacillus (Bacillus axatriensis) CTS-BQ1 for promoting the enzymolysis of eichhornia crassipes to produce sugar, and further provides a method for promoting the enzymolysis of eichhornia crassipes matrix to produce sugar and a microbial agent for promoting the enzymolysis of eichhornia crassipes matrix to produce sugar. The strain provided by the invention enriches the genetic resources of wild degumming bacteria, and expands the backup database of degumming bacteria whole genome breeding. Compared with white rot fungi with better reported effects, the strain can improve the sugar yield of eichhornia crassipes enzymolysis, has high efficiency, is economic and obvious in effect compared with commercial pectinase, and has wide application prospect.

Description

Bacillus for promoting enzymolysis of eichhornia crassipes substrate to produce sugar and application thereof
Technical Field
The invention belongs to the field of solid waste resource utilization, and particularly relates to a bacillus (Bacillus axacquiensis) CTS-BQ1 and a pretreatment effect thereof for promoting enzymolysis of an Eichhornia crassipes matrix to produce sugar, which can be applied to pretreatment of high-value utilization of Eichhornia crassipes.
Background
The water hyacinth is used as an invasive aquatic plant, and on one hand, the water hyacinth grows fast and has strong adaptability, and is easy to pollute water body after putrefaction and deterioration; on the other hand, the biological energy-saving fertilizer grows in water, has large yield, does not occupy land area, has high cellulose and hemicellulose content, and is a production raw material of a potential biological energy substance. However, because of high water content, the lignin wraps the cellulose, so that the cellulose is difficult to release and not easy to utilize, the pretreatment cost is high, and the efficiency of producing sugar and ethanol is low. The biological pretreatment method has the advantages of mild action condition, low energy consumption, little environmental pollution, low treatment cost and the like, and is a pretreatment method with great potential. However, the problems of few lignin-degrading microorganisms, harsh degradation conditions, low enzyme activity of lignin-decomposing enzymes, long action period, bacterial consumption of part of cellulose and hemicellulose in the treatment process and the like still exist in the current biological pretreatment research; the biological enzyme has high economic cost, and the enzyme pretreatment greatly improves the production cost, so that the biological pretreatment is difficult to realize industrial application. How to overcome the above problems and find suitable pretreatment fermentation strains becomes a hotspot of biological pretreatment research.
The degumming bacteria are mainly used for researching ramie, the performance of the ramie fibers is enhanced by removing colloid components in the ramie substances and simultaneously retaining fiber components, and the degumming method is related to other types of plants. By referring to the screening method of ramie degumming bacteria by predecessors, excellent strains for degrading non-cellulose of eichhornia crassipes are screened from different growth environments of eichhornia crassipes, and the enzyme production characteristics of the strains are explored. The degumming strains reported at present include actinomyces, cellulomonas, shigella, Bacillus subtilis and the like, which are degumming strains for hemp, but the reports on Bacillus axarquisis are less.
Disclosure of Invention
In order to improve the defects of the prior art, the invention provides the bacillus and the application thereof, and the bacillus has a remarkable promoting effect on sugar production by enzymolysis of eichhornia crassipes.
The invention provides a Bacillus (Bacillus axarquisis) CTS-BQ1 which is preserved in China Center for Type Culture Collection (CCTCC) in 2017, 5 and 18 months, and the preservation number is CCTCC NO: m2017273, address: china, wuhan university.
The second aspect of the invention provides application of the bacillus CTS-BQ1 in promoting enzymolysis of eichhornia crassipes substrate to produce sugar.
The third aspect of the invention provides a method for promoting the enzymolysis of eichhornia crassipes substrate to produce sugar, which comprises the following steps: the bacillus CTS-BQ1 is adopted, the bacterium mother liquor is obtained after amplification culture, and then the bacterium mother liquor is inoculated into a fermentation bottle containing the fresh eichhornia crassipes sample.
The fourth aspect of the invention provides a microbial agent for promoting enzymolysis of eichhornia crassipes substrate to produce sugar, and the active ingredients of the microbial agent comprise the bacillus CTS-BQ 1.
The invention has the beneficial effects that: the strain provided by the invention enriches the genetic resources of wild degumming bacteria, and expands the backup database of degumming bacteria whole genome breeding. Compared with white rot fungi with better reported effects, the strain can improve the sugar yield of eichhornia crassipes enzymolysis, has high efficiency, is economic and obvious in effect compared with commercial pectinase, and has wide application prospect. In the test for detecting the substrate enzymolysis sugar production effect after eichhornia crassipes is pretreated by bacillus CTS-BQ1 by adopting the mode of detecting the substrate enzymolysis sugar production efficiency, the maximum sugar yield in the enzymolysis liquid is 142mg/g raw material dry basis, and the sugar production effect is obviously improved.
Drawings
FIG. 1: and (4) after the eichhornia crassipes is pretreated by the separated strain, sugar yields are compared after 72h of enzymolysis.
FIG. 2: sugar content of pretreatment liquid of isolated strain.
FIG. 3: sugar yield and consumption proportion chart of the isolated strain.
FIG. 4: influence of different pretreatment conditions on the enzymolysis sugar yield of the fresh-like matrix of the eichhornia crassipes pretreated by the strain CTS-BQ 1.
FIG. 5: the sugar yield of enzymolysis of different biological pretreatment eichhornia crassipes substrates is compared.
Detailed Description
The invention provides a Bacillus (Bacillus axarquisis) CTS-BQ1 which is preserved in China Center for Type Culture Collection (CCTCC) in 2017, 5 and 18 months, and the preservation number is CCTCC NO: m2017273, address: china, wuhan university.
The bacillus CTS-BQ1 is a strain obtained by screening water in a pond for growing eichhornia crassipes near a pig farm by using a separation culture medium taking pectin as a unique carbon source. The colony morphology characteristics of the bacillus CTS-BQ1 are as follows: the bacterial colony is dirty white, smooth and moist in surface, irregular in edge and gram-positive bacteria.
The 16S rDNA sequence of the bacillus CTS-BQ1 is shown in a sequence table SEQ ID No.1, and is compared with the sequence in a Genbank database, and finally the strain CTS-BQ1 is determined to be bacillus.
The second aspect of the invention provides application of the bacillus CTS-BQ1 in promoting enzymolysis of eichhornia crassipes substrate to produce sugar.
In one embodiment of the invention, the effect of substrate enzymolysis sugar production after eichhornia crassipes pretreatment by bacillus CTS-BQ1 is detected by detecting the efficiency of substrate enzymolysis sugar production, and the specific method is as follows: the bacillus CTS-BQ1 is adopted, bacterium mother liquor is obtained after amplification culture, the bacterium mother liquor is inoculated into a degumming-containing culture medium according to the inoculum concentration of 2%, shaking culture is carried out for 3 days at 35 ℃ and 145rpm, solid-liquid separation is carried out, the solid is dried at 65 ℃, the solid is dried by enzymolysis with the cellulase load of 30FPU/g substrate for 72 hours, and the yield of reducing sugar in the enzymolysis liquid is detected by a DNS method. The maximum sugar yield in the enzymolysis liquid is 142mg/g raw material dry basis, and the sugar production effect is obviously improved. The bacillus CTS-BQ1 has good application prospect in promoting the enzymolysis of the eichhornia crassipes substrate to produce sugar.
The third aspect of the invention provides a method for promoting the enzymolysis of eichhornia crassipes substrate to produce sugar, which comprises the following steps: the bacillus CTS-BQ1 is adopted, the bacterium mother liquor is obtained after amplification culture, and then the bacterium mother liquor is inoculated into a fermentation bottle containing the fresh eichhornia crassipes sample.
Preferably, the strain mother liquor is inoculated into a fermentation bottle containing the fresh eichhornia crassipes, and the strain mother liquor is cultured for 3 days under the conditions of 35 ℃ and initial pH value of 9.
The fourth aspect of the invention provides a microbial agent for promoting enzymolysis of eichhornia crassipes substrate to produce sugar, and the active ingredients of the microbial agent comprise the bacillus CTS-BQ 1.
The bacillus for promoting the enzymolysis of the eichhornia crassipes substrate to produce sugar and the application thereof are further described in the following by combining specific embodiments. The following examples are illustrative only and are not to be construed as limiting the invention.
The experimental procedures in the following examples are conventional unless otherwise specified. The experimental materials used in the following examples were all commercially available unless otherwise specified.
The media formulations referred to in the examples are as follows:
separating a culture medium: (I) K2SO40.01％0.1g，CaCl2 0.2g，NaCl 0.2g，MgSO4·7H2O 0.3g，KNO30.5g，(NH4)2SO40.5g，K2HPO41g of agar, 20g of agar and 1L of distilled water; (II) 1g of pectin, 5g of agar, pH7.0, and 1L of distilled water. The double-layer plate was poured into a petri dish, the surface layer was 5mL of medium II, and the basal layer was 10mL of medium I.
Hydrolysis loop assay medium: (I) FeSO40.01g，CaCl20.2g，MgSO4·7H2O 0.5g，NaCl 0.5g，KH2PO41g，NH4NO33g of pectin, 5g of agar, 20g of pH7.0, and 1L of distilled water; (II) 20g of agar and 1L of distilled water. The double-layer plate was poured into a petri dish, the surface layer was 10mL of medium I, and the basal layer was 10mL of medium II.
Degumming culture medium: 5g of a fresh eichhornia crassipes sample, 150mL of a salt solution and about 7.0 of pH; salt solution: MgSO (MgSO)4·7H2O0.5g，K2HPO40.5g，(NH4)2SO45g, 1L of sterile water.
Slant culture medium: beef extract peptone solid medium.
White rot fungus pretreatment basic culture solution: MgSO (MgSO)4.7H2O 0.05g/L，KH2PO40.2g/L，CaCl20.01 g/L, vitamin B1100 mg/L, Tween-800.05%, 1mL of inorganic salt solution. Inorganic salt solution: 0.5g/L MnSO4·H2O，0.1g/L FeSO4·7H2O，0.1g/L CoCl2·6H2O，0.1g/LZnSO4·7H2O， 0.01g/L CuSO4·5H2O，0.01g/L AlK(SO4)2·12H2O，0.01g/L H3BO3， 0.01g/LNa2MoO4·2H2O，3.0g/LMgSO4·7H2O。
Example 1
In this example, the primary screening of degumming microorganisms was performed, and the specific screening process was as follows:
collecting pond water, pond bottom sludge, south lake water, south lake bottom sludge, wild angelica lake water, wild angelica lake bottom sludge, fresh pig manure and a vigorous eichhornia crassipes strain in a pond growing eichhornia crassipes near a pig farm of Huazhong agriculture university. Cleaning the eichhornia crassipes strain, cutting the eichhornia crassipes strain into small sections of 1-2 cm, and air-drying the surface moisture. Weighing 10g of bottom mud (containing water) in a sterile triangular flask filled with 90mL of sterile water, oscillating at the normal temperature at the oscillation speed of 125r/min for 1h, and filtering by using two layers of gauze to obtain a bottom mud suspension;
weighing 5g of air-dried eichhornia crassipes fresh sample into an aseptic triangular flask, respectively adding 30mL of water sample and bottom mud suspension, adding 270mL of sterile water, and culturing in a constant-temperature incubator at 35 ℃ until eichhornia crassipes fibers are dispersed for about 5 days to obtain a primary proliferation culture solution. Weighing 5g of eichhornia crassipes fresh sample, putting the fresh eichhornia crassipes fresh sample into a sterile triangular flask, respectively adding 30mL of primary multiplication culture solution of pond water and primary multiplication culture solution of pond bottom mud, adding 270mL of sterile water, and culturing in a constant-temperature incubator at 35 ℃ until eichhornia crassipes fibers are dispersed for about 4 days to obtain secondary multiplication culture solution. Sucking 1mL of secondary proliferation culture solution into a sterilized centrifuge tube containing 9mL of sterile water, repeatedly rinsing the gun head for 3 times, and shaking up to obtain 10-1A dilution of concentration. Sucking 1mL of 10-1 concentration diluent into a sterilized centrifuge tube containing 9mL of sterile water, repeatedly rinsing the gun head for 3 times, and shaking up to obtain 10-2A dilution of concentration. Thus, 10 was repeatedly prepared-1～10-7Dilute bacterial suspension at concentration. 0.1mL 10 of each sample was aspirated-5、10-6、10-7And (3) coating the diluted bacterial liquid with the concentration in a separation culture medium taking pectin as a unique carbon source, and placing the medium in a constant temperature incubator for inverted culture at 35 ℃ for 3d after surface moisture is absorbed. And 3d, taking 4 strains with good growth vigor in the culture medium, and streaking and purifying the 4 strains on a beef extract peptone slant culture medium for 4-10 times. Bacterial colony preliminary screening the separated bacterial strain is inoculated to a hydrolytic ring determination culture medium and cultured for 2d at 35 ℃. Inoculating the strain obtained by enrichment culture and separation to a hydrolysis loop determination culture medium, culturing for 3d at 35 ℃, covering a Congo red solution with the mass concentration of 1mg/mL on the culture medium with a colony, pouring the Congo red solution after 10-15 min, adding a NaCl solution with the mass concentration of lmol/L, and pouring the NaCl solution after 15min, wherein a transparent loop appears around the colony generating pectinase. Measuring the colony diameter and hydrolysis ring diameter of the screened strain,the strains producing the obvious hydrolysis ring are named as XZF-HQ, CTDN-RQB, CTS-BQ1, NHS-S1, CTS-S1, CTDN-HQ, YZHS-BQ1 and YZHS-BQ2 in sequence.
Example 2
In this example, the degumming microorganisms obtained in example 1 were rescreened, and the specific process was as follows:
the strains obtained by primary screening are respectively inoculated into degumming culture medium, cultured for 3 days at 35 ℃ and 145rpm, and meanwhile, liquid phosphate-solubilizing culture medium without any bacteria is used as a control group. And (3) carrying out solid-liquid separation after the culture is finished, measuring the total reducing sugar content of the liquid by using a DNS method, drying the solid at 65 ℃, carrying out enzymolysis for 72 hours by using the cellulase load of 30FPU/g substrate, and detecting the yield of reducing sugar in the enzymolysis liquid by using the DNS method. The results are shown in fig. 1, the 8 strains of bacteria screened by the invention have a promoting effect on the enzymatic hydrolysis sugar production of the substrate after the eichhornia crassipes is pretreated, and the results are shown in fig. 2, wherein the reducing sugar in the pretreatment liquid is consumed to a certain extent; the ratio of the produced sugar to the consumed sugar is shown in figure 3 by taking the consumption of the reducing sugar in the pretreatment as the sugar consumption in the strain treatment and taking the sugar increment in the enzymolysis liquid as the sugar yield after the pretreatment, wherein the ratio of CTS-BQ1 is the largest, and the sugar with the minimum consumption has the largest output.
Example 3
This example identifies the strain CTS-BQ1
(1) Colony morphology and properties of strain CTS-BQ1
The bacterial colony is dirty white, smooth and moist in surface, irregular in edge and gram-positive bacteria.
(2) Molecular biological identification of the Strain CTS-BQ1
The CTS-BQ1 strain obtained by screening is identified by a molecular biology method, the DNA of the strain is amplified by PCR (polymerase chain reaction) by using universal primers 27F and 1492R, the amplified product is subjected to sequence determination by a sequencing company, and the determined sequence is subjected to BLAST (BLAST-specific amplification) comparison with the sequence in a GenBank database, so that the similarity of the strain and bacillus (Bacillus axacquiensis) is more than 99 percent. The strain is determined to be Bacillus (Bacillus axarquisensis) by combining morphological characteristics, culture characteristics and 16S rRNA sequence analysis.
Example 4
The embodiment provides the influence of different culture conditions on the promotion of the bacterial strain CTS-BQ1 on the enzymolysis of the eichhornia crassipes substrate to produce sugar, and the specific process is as follows:
a4-factor 4 horizontal orthogonal test is designed to investigate the influence of temperature (25 ℃, 30 ℃, 35 ℃, 40 ℃), initial pH value (3, 5, 7, 9), inoculation amount (2%, 4%, 6%, 8%) and treatment time (1d, 3d, 5d, 7d) on the substrate enzymolysis sugar production of eichhornia crassipes pretreated by bacillus CTS-BQ 1. The results of the analysis of the difference in the yield of the eichhornia crassipes substrate reducing sugar among different levels of the pretreatment single factor are shown in fig. 4, the yield of the enzymatic hydrolysis reducing sugar tends to increase along with the temperature rise, and no significant difference exists among different temperature levels. The initial pH values of 3, 5 and 7 have no significant influence on the sugar yield of enzymolysis, and when the pH value is 9, the sugar yield of enzymolysis is increased significantly compared with other levels. The optimal conditions of pretreatment of the bacillus CTS-BQ1 obtained after analysis are 35 ℃ of temperature, 9 initial pH, 2% of inoculation amount and 3d of treatment time, and the sugar yield of enzymolysis under the treatment conditions reaches 142 mg/g.
Example 5
The embodiment provides the influence of different biological pretreatment methods on the sugar production by enzymolysis of eichhornia crassipes, and the specific process is as follows:
weighing 15g (water content is 92.6%) of the ground fresh eichhornia crassipes sample in a 250mL conical flask, adding the basic culture solution according to the solid-to-liquid ratio of 1:2.5, naturally adjusting pH, sealing, and sterilizing at 121 ℃. Cooling to room temperature, inoculating 5 pieces of Phanerochaete chrysosporium strain block with diameter of 1cm, and culturing at 28 deg.C for 10 days. And (4) taking out the inoculation block after the culture is finished, cleaning the sample, drying and weighing at 65 ℃, and measuring the yield of the enzymatic reducing sugar for 72 h. The control was performed without inoculating Phanerochaete chrysosporium samples, and 3 replicates were set for each treatment.
Weighing 15g (water content is 92.6%) of the ground fresh eichhornia crassipes sample into a 250mL triangular flask, adding 30mL of citric acid-sodium citrate buffer solution with pH of 3.5 according to the solid-to-liquid ratio of 1:30, sealing, and sterilizing at 121 ℃. Cooling to room temperature, preheating in 50 deg.C water bath, adding pectinase enzyme solution according to enzyme load of 20U/g substrate, reacting at 125r/min oscillation speed, and pretreating for 72 h. After the treatment, solid-liquid separation is carried out, the solid matters are washed to be neutral by ultrapure water, and dried and weighed at 65 ℃. No enzyme solution was added as a control treatment, and 3 replicates were set for each treatment.
Weighing 15g (water content is 92.6%) of the ground fresh eichhornia crassipes sample into a 250mL conical flask, adding 150mL of salt solution, sterilizing at 121 ℃ for 30min, and cooling to room temperature. The pH was adjusted to 9, and the CTS-BQ1 strain stock solution (2% LB medium added as a control) was inoculated at 2% and treated for 3d on a water-bath shaker at 35 ℃ under a shaking speed of 145 r/min. After the treatment was completed, solid-liquid separation was performed with a 0.45 μm filter, the solid was washed to neutrality, dried at 65 ℃ and weighed, and each treatment was repeated 3 times.
The effect of sugar production by enzymolysis of eichhornia crassipes substrate after three biological pretreatments is shown in figure 5. Among the 3 kinds of biological pretreatment, the pectinase pretreatment has the highest sugar yield, the CTS-BQ1 times and the Phanerochaete chrysosporium has the lowest yield. Compared with different treatments and the comparison, the sugar yield of the raw materials is obviously improved, the Phanerochaete chrysosporium and the comparison thereof have no obvious difference, the CTS-BQ1 and the comparison thereof have obvious sugar yield difference, and the pectinase and the comparison thereof have no obvious sugar yield difference. The improvement effect is most remarkable after the CTS-BQ1 pretreatment.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
<110> university of agriculture in Huazhong
<120> bacillus for promoting enzymolysis of eichhornia crassipes to produce sugar and application thereof
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Claims (5)

1. Bacillus aryabhattai (A)Bacillus axarquiensis) CTS-BQ1, which is preserved in China center for type culture Collection with the preservation number of CCTCC NO: m2017273.
2. The Bacillus aryabhattai of claim 1, (b), (c), (dBacillus axarquiensis) Application of CTS-BQ1 in promoting enzymolysis of Eichhornia crassipes matrix to produce sugar.
3. A method for promoting the enzymolysis of eichhornia crassipes substrate to produce sugar is characterized by comprising the following steps: the method comprises the following steps: the use of the Bacillus salbihtea strain of claim 1 (b), (c), (d), (Bacillus axarquiensis) CTS-BQ1, obtaining bacterium mother liquor after amplification culture, and then inoculating the bacterium mother liquor into a fermentation bottle containing the eichhornia crassipes.
4. The method for promoting the enzymolysis of eichhornia crassipes substrate to produce sugar as claimed in claim 3, wherein: inoculating the strain mother liquor into a fermentation bottle containing the eichhornia crassipes according to the inoculation amount of 2%, and culturing for 3 days at the temperature of 35 ℃ and under the condition that the initial pH value is 9.
5. A microbial agent for promoting the enzymolysis of eichhornia crassipes substrate to produce sugar is characterized in that: the active ingredient of the microbial agent comprises the Bacillus assayi (Bacillus) of claim 1Bacillus axarquiensis)CTS-BQ1。
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