CN108841765B - Sterobacter and application thereof in production of bioflocculant by transforming sweet potato straws - Google Patents

Abstract

An Empedobacter falsenii BWL1071 with the preservation number: CGMCC No. 13986. The application of the bacillus firmus in the production of the bioflocculant by transforming the hydrolysate of the sweet potato straws comprises the following steps: inoculating the Stephania strain in a seed liquid culture medium to prepare a seed fermentation liquid; transferring the seed fermentation liquid to a fermentation liquid culture medium which takes sweet potato straw hydrolysate as a carbon source for culture to obtain a fermentation culture liquid; heating the fermentation culture solution, centrifuging, and collecting supernatant; and adding absolute ethyl alcohol into the supernatant, centrifuging to collect precipitate, washing with precooled ethyl alcohol, and freeze-drying under vacuum to obtain a solid bioflocculant product. The bacterial strain Empedobacter falsenii BWL1071 can convert sweet potato straw hydrolysate to produce the biological flocculant, is beneficial to reducing the production cost of the biological flocculant, can realize the resource utilization of the sweet potato straw, and the produced biological flocculant can efficiently flocculate and treat mining wastewater.

Description

Sterobacter and application thereof in production of bioflocculant by transforming sweet potato straws

Technical Field

The invention belongs to the technical field of biology, relates to microorganisms and metabolites thereof, and particularly relates to Empedobacter falsenii BWL1071 and application thereof in fermentation production of a bioflocculant by converting sweet potato straw hydrolysate.

Background

With the rapid development of economy in China, environmental problems are increasingly prominent, wherein water pollution is one of the most challenging environmental problems. A large amount of mining wastewater can be generated in the mining and processing processes of mines, and comprises mining process drainage, tailing pond overflow water, mine drainage and the like, the mining wastewater mainly contains solid suspended matters, heavy metal ions, organic pollutants and the like, the concentration of the mining wastewater is far higher than the national emission standard, and particularly the content of the solid suspended matters is seriously exceeded. Flocculants are substances capable of aggregating suspended solid particles in a settled solution, and are widely applied to the fields of sewage treatment and the like. The method is mainly divided into three categories: inorganic flocculant, organic polymer flocculant and biological flocculant. The flocculant can efficiently flocculate and remove solid suspended matters and heavy metal ions in the mining wastewater, and has better application prospect in the fields of recycling of the mining wastewater, ecological environment protection and the like.

The biological flocculant is macromolecular substances such as polysaccharide, protein and the like generated by microbial metabolism, has the advantages of no toxicity, harmlessness, biodegradability, no secondary pollution and the like, can replace the traditional inorganic flocculant and organic polymeric flocculant, and reduces environmental pollution and damage to an ecosystem. However, the production of bioflocculants requires pure sugar as a fermentation medium for microorganisms, and the fermentation production cost is relatively high, which limits the large-scale industrial production and application of bioflocculants. In order to reduce the production cost of the bioflocculant, researchers develop a series of research works, which mainly comprise screening high-yield strains, improving the yield of the existing strains by utilizing a genetic engineering breeding technology, optimizing the fermentation process of the bioflocculant production strains, widening the application field of the bioflocculant and the like. The search for an inexpensive alternative medium is also an effective way to effectively reduce the production cost of bioflocculants, for example, Lievigen et al utilize hot acid hydrolysate of peanut shell as the carbon source of the fermentation medium of the strain Pseudomonas veronii L918 (Bioresource technology,2016,218: 318-.

China is the largest sweet potato producing country in the world, and the planting area is more than 8000 ten thousand mu each year. A large amount of sweet potato straw waste can be generated in the planting process of sweet potatoes, and the sweet potato straw waste is used as livestock and poultry feed or is directly discarded, so that serious biomass resource waste and environmental pollution are caused. Sweet potato straws contain rich protein and lignocellulose substances, and can be decomposed into monosaccharides under the condition of hot acid, so that the monosaccharides can be converted into other high-value-added products.

Disclosure of Invention

The invention provides a strain of Empedobacter falsenii BWL1071, which has the preservation number: CGMCC No.13986, which constitutes the first aspect of the present invention.

The invention also provides an application of the bacillus firmus in producing a bioflocculant by transforming sweet potato straw hydrolysate, which forms a second aspect of the invention and specifically comprises the following steps:

step 1, inoculating the Empedobacter in the claim 1 into a seed liquid culture medium, and performing shake culture on a shaker for 10-14h under the conditions of 150-200rpm and 29-31 ℃ to prepare a seed fermentation liquid;

step 2, transferring the seed fermentation liquid obtained in the step 1 to a fermentation liquid culture medium taking sweet potato straw hydrolysate as a carbon source according to the proportion of 1%, and performing shake culture for 23-25h in a shaking table under the conditions of 190rpm at 170 and 33-35 ℃ to obtain a fermentation culture liquid;

step 3, heating the fermentation culture solution obtained in the step 2 in a 80 ℃ constant-temperature water bath kettle for 1h, then centrifuging for 10min at 10000rpm and 4 ℃, and collecting supernatant;

and 4, adding 2 times volume of precooled absolute ethyl alcohol into the supernatant obtained in the step 3, centrifuging to collect precipitates, washing the precipitates for 2 times by using precooled 75% ethyl alcohol, dissolving the precipitates in a small amount of water, freezing and drying in vacuum to obtain a solid bioflocculant product.

Further, the seed liquid culture medium comprises the following components: 5g/L glucose, 20g/L yeast powder, 0.5g/L magnesium sulfate heptahydrate, 2g/L dipotassium hydrogen phosphate, 5g/L potassium dihydrogen phosphate and 2.4g/L sodium chloride, wherein the culture conditions of the seed liquid are 170-190rpm and the overnight culture is carried out at 29-31 ℃.

Further, the culture medium of the fermentation liquid comprises the following components: 300mL/L of sweet potato straw hydrolysate, 20g/L of yeast powder, 0.5g/L of magnesium sulfate heptahydrate, 2g/L of dipotassium hydrogen phosphate, 5g/L of potassium dihydrogen phosphate and 2.4g/L of sodium chloride, wherein the fermentation conditions are 170-190rpm and 33-35 ℃.

Further, the preparation process of the sweet potato straw hydrolysate comprises the following steps: mixing sweet potato straws with 1.7% sulfuric acid solution according to a solid-to-liquid ratio of 1:10, and performing acid hydrolysis in an autoclave at 121 ℃ for 2 h; after cooling, the mixture was centrifuged at 10000rpm for 10min, and the supernatant was collected and Ca (OH) was added₂The pH value is adjusted to 7.0,then centrifuging at 10000rpm for 10min, and collecting supernatant to obtain sweet potato straw hydrolysate.

The invention has the beneficial effects that:

1. the bacterial strain Empedobacter falsenii BWL1071 can convert sweet potato straw hydrolysate to produce the biological flocculant, which is beneficial to reducing the production cost of the biological flocculant and realizing the resource utilization of the sweet potato straw;

2. the biological flocculant produced by the strain Empedobacter falsenii BWL1071 can efficiently flocculate and treat mining wastewater.

The strain preservation date of the invention is 2017, 04 and 5 days, and the preservation number is as follows: CGMCC No. 13986. The classification names are: sterobacter falsenii BWL1071, the name of the preservation unit is China general microbiological culture preservation management center, the address is China academy of sciences microbiology, institute 3, West Lu 1, Beijing, the rising area, North Cheng, China, and the postal code is 100101.

The specific implementation mode is as follows:

example 1: screening process

The process from material drawing to pure strain screening

(1) A rust sample is collected from a certain factory in a copper mountain area of Xuzhou city, Jiangsu province, 1g of the rust sample is suspended in sterile physiological saline, and the sample is diluted by using physiological saline in a gradient manner and is sequentially coated on a screening culture medium (5 g/L of beef extract, 5g/L of sodium chloride, 10g/L of peptone and 20g/L of agar powder).

(2) After the plate was coated, it was cultured in an inverted state in a 30 ℃ incubator. The growth of the strain was checked every 24 h. According to different characteristics of shape, size, color and the like, selecting colonies to an LB liquid culture medium (5 g/L yeast powder, 10g/L NaCl, 10g/L peptone), placing the colonies in a constant temperature shaking table at 30 ℃ for shaking culture, wherein the culture conditions are as follows: culturing at 180rpm for 24 h. After the shaking table is finished, the flocculation activity of the fermentation liquor is measured, and a strain BWL1071 with high flocculation activity is obtained.

Example 2: strain identification

Colony morphology: the colony is milky white, semi-moist and irregular in edge.

The seed tree status of the strain BWL1081 is determined by 16S rRNA sequence, and total DNA is extracted by a bacterial genome DNA extraction kit. The forward primer for the PCR clone was 5'-GAG AGT TTG ATC CTG GCT CAG-3' and the reverse primer was 5'-CTA CGG CTA CCT TGT TAC GA-3'. The PCR reaction was performed in a total volume of 50. mu.L, and added to a 0.2mL Eppendorf tube in the following order: premix 25. mu.L; 2 mu L of forward primer; 2 mu L of reverse primer; 1 μ L of template DNA; 20 μ L of sterile water. The PCR conditions were pre-denaturation at 95 ℃ for 1min, denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 2min for 30 cycles, and extension at 72 ℃ for 10 min. And (3) detecting the 16S rRNA product obtained after amplification by using 1% agarose gel electrophoresis, and sending the product to a sequencing company for sequencing, wherein the sequencing result is shown as SEQ ID No. 1. BLAST comparison is carried out on the measured 16S rRNA sequences in NCBI, a known sequence with the highest similarity is searched, and finally the strain BWL1081 is found to have the highest similarity with Empedobacter falsenii.

Example 3:

the method for producing the bioflocculant by transforming sweet potato straws with the bacterial strain Empedobacter falsenii BWL1071 has the following specific implementation mode:

step 1, inoculating an Empedobacter falsenii BWL1071 strain into a seed liquid culture medium, and performing shake cultivation at 150-200rpm and 29-31 ℃ for 10-14h to prepare seed fermentation liquid;

step 2, transferring the seed liquid in the step 1 to a fermentation liquid culture medium taking sweet potato straw powder as a unique carbon source according to the proportion of 1%, and carrying out shaking culture at the constant temperature of between 33 and 35 ℃ at 190rpm for 23 to 25 hours;

step 3, heating the fermentation liquor in a water bath kettle at 80 ℃ for 1h, then centrifuging for 10min at 10000rpm and 4 ℃, and collecting supernatant;

and 4, adding 2 times volume of precooled absolute ethyl alcohol into the supernatant obtained in the step 3, centrifuging to collect precipitates, washing the precipitates for 2 times by using 75% ethyl alcohol, dissolving the precipitates in a small amount of water, and performing vacuum freeze drying to obtain a solid bioflocculant product.

The seed liquid culture medium in the step 1 is 5g/L of glucose, 20g/L of yeast powder, 0.5g/L of magnesium sulfate heptahydrate, 2g/L of dipotassium phosphate, 5g/L of potassium dihydrogen phosphate and 2.4g/L of sodium chloride, and the culture conditions of the seed liquid are 170-190rpm and overnight culture at 29-31 ℃;

in the step 2, the fermentation medium is 300mL/L of sweet potato straw hydrolysate, 20g/L of yeast powder, 0.5g/L of magnesium sulfate heptahydrate, 2g/L of dipotassium phosphate, 5g/L of potassium dihydrogen phosphate and 2.4g/L of sodium chloride, and the fermentation condition is 190rpm at 170 ℃ and is 33-35 ℃.

The preparation method of the sweet potato straw hydrolysate in the step 2 comprises the following steps: mixing sweet potato straws with 1.7% sulfuric acid solution according to a solid-to-liquid ratio of 1:10, and performing acid hydrolysis in an autoclave at 121 ℃ for 2 h; after cooling, the mixture was centrifuged at 10000rpm for 10min, and the supernatant was collected and Ca (OH) was added₂Adjusting the pH value to 7.0, then centrifuging at 10000rpm for 10min, and collecting the supernatant, thereby obtaining the sweet potato straw hydrolysate.

Example 4

The method for treating mining wastewater by flocculating a biological flocculant generated by the strain Empedobacter falsenii BWL1071 comprises the following specific implementation steps:

taking 60mL of mining wastewater, and adding 100uL of fermentation liquor of Empedobacter falsenii BWL1071 into the mining wastewater; rapidly stirring for 2min, slowly stirring for 1min, standing for 1min, collecting supernatant, and measuring OD with spectrophotometer₅₅₀The flocculation activity of the bioflocculant on mining wastewater was expressed by calculating the flocculation rate with the addition of 100. mu.L of distilled water as a control. Flocculation rate (A-B)/A × 100%, A represents OD of supernatant when distilled water is added₅₅₀The value B represents the OD of the supernatant when the fermentation broth is added₅₅₀The value is obtained. The application effect of the flocculant produced by the strain Empedobacter falsenii BWL1071 on mining wastewater is measured, and the result shows that the obtained biological flocculant has an obvious flocculation effect on the mining wastewater, and the flocculation rate reaches more than 90%.

Sequence listing

<110> university of Jiangsu profession

<120> Sterculia and application thereof in production of bioflocculant by transforming sweet potato straws

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1343

<212> DNA

<213> Steganbacillus (Empedobacter falsenii)

<400> 1

agttctttcg ggaactagag accggcgcac gggtgcgtaa cgcgtatgca acttgcccta 60

ctgaaaagga tagcccttcg aaaggaggat taatacttta taacagattt aatggcatca 120

ttagattttg aaagatttat cgcagtagga taggcatgcg taagattagt tagttggtga 180

ggtaacggct caccaagacg atgatcttta gggggcctga gagggtgaac ccccacactg 240

gtactgagac acggaccaga ctcctacggg aggcagcagt gaggaatatt ggacaatggg 300

tggaagcctg atccagccat cccgcgtgta ggatgacggc cttatgggtt gtaaactact 360

tttatctggg gataaaccta cttacgtgta agtagctgaa ggtaccagaa gaataagcac 420

cggctaactc cgtgccagca gccgcggtaa tacggagggt gcaagcgtta tccggattta 480

ttgggtttaa agggtccgta ggcggattaa tcagtcagtg gtgaaatctc atagcttaac 540

tatgaaactg ccattgatac tgttagtctt gagtgatgtt gaagttgctg gaatgtgtag 600

tgtagcggtg aaatgcttag atattacgca gaacaccaat tgcgaaggca ggtgactaaa 660

cattaactga cgctgatgga cgaaagcgtg gggagcgaac aggattagat accctggtag 720

tccacgccgt aaacgatgga tacttgctgt tggattttcg gattcagtgg ctaagcgaaa 780

gttataagta tcccacctgg ggagtacgtt cgcaagaatg aaactcaaag gaattgacgg 840

gggcccgcac aagcggtgga gcatgtggtt taattcgatg atacgcgagg aaccttacca 900

aggcttaaat gcaatttgac agaactagaa atagtttttt cttcggacag aatgcaaggt 960

gctgcatggc tgtcgtcagc tcgtgccgtg aggtgttagg ttaagtcctg caacgagcgc 1020

aacccctatc attagttgcc agcgtttaaa gacggggact ctaatgagac tgccggtgca 1080

aaccgcgagg aaggtgggga cgacgtcaag tcatcacggc ccttacgtct tgggctacac 1140

acgtgctaca atggtaagta cagagggcag ctacttggca acaagatgcg aatctcaaaa 1200

acttatctca gttcggattg gagtctgcaa ctcgactcta tgaagctgga atcgctagta 1260

atcgcatatc agccatgatg cggtgaatac gttcccgggc cttgtacaca ccgcccgtca 1320

agccatggaa gctgggggta cct 1343

Claims (3)

1. Sterobacter (II)Empedobacter falsenii) BWL1071 strain, its deposit number is: CGMCC No. 13986.

2. The use of the Empedobacter of claim 1 in the production of bioflocculants by transforming sweet potato straws.

3. The application of the Empedobacter in producing the bioflocculant by transforming sweet potato straws as claimed in claim 2, wherein the application specifically comprises:

step 1, inoculating the Empedobacter in the claim 1 into a seed liquid culture medium, and performing shake culture on a shaker for 10-14h under the conditions of 150-200rpm and 29-31 ℃ to prepare a seed fermentation liquid; the seed liquid culture medium comprises the following components: 5g/L glucose, 20g/L yeast powder, 0.5g/L magnesium sulfate heptahydrate, 2g/L dipotassium hydrogen phosphate, 5g/L potassium dihydrogen phosphate and 2.4g/L sodium chloride, wherein the culture conditions of the seed liquid are 170-190rpm and overnight culture at 29-31 ℃;

step 2, transferring the seed fermentation liquid obtained in the step 1 to a fermentation liquid culture medium taking sweet potato straw hydrolysate as a carbon source according to the proportion of 1%, and performing shake culture for 23-25h in a shaking table under the conditions of 190rpm at 170 and 33-35 ℃ to obtain a fermentation culture liquid; the preparation process of the sweet potato straw hydrolysate comprises the following steps: mixing sweet potato straws with 1.7% sulfuric acid solution according to a solid-to-liquid ratio of 1:10, and performing acid hydrolysis in an autoclave at 121 ℃ for 2 h; after cooling, the mixture was centrifuged at 10000rpm for 10min, and the supernatant was collected and Ca (OH) was added₂Adjusting the pH value to 7.0, then centrifuging at 10000rpm for 10min, and collecting supernatant to obtain sweet potato straw hydrolysate; the fermentation liquid culture medium comprises the following components: hydrolysis of sweet potato straw300mL/L of the compound, 20g/L of yeast powder, 0.5g/L of magnesium sulfate heptahydrate, 2g/L of dipotassium hydrogen phosphate, 5g/L of potassium dihydrogen phosphate and 2.4g/L of sodium chloride, wherein the fermentation condition is 190rpm at 170 ℃ and 33-35 ℃;

step 3, heating the fermentation culture solution obtained in the step 2 in a 80 ℃ constant-temperature water bath kettle for 1h, then centrifuging for 10min at 10000rpm and 4 ℃, and collecting supernatant;

and 4, adding 2 times volume of precooled absolute ethyl alcohol into the supernatant obtained in the step 3, centrifuging to collect precipitates, washing the precipitates for 2 times by using precooled 75% ethyl alcohol, dissolving the precipitates in a small amount of water, freezing and drying in vacuum to obtain a solid bioflocculant product.