KR102059893B1 - Microbial community for preparation of biosulfur and its use - Google Patents

Abstract

Disclosed in the present invention are a novel microbial community having a conversion capability from high concentration of hydrogen sulfide (H_2S) to biosulfur, and a method for preparing the biosulfur using the same or a method for removing hydrogen sulfide from mixed gas. The microbial community according to the present invention converts 14,000 ppm or more high concentration of hydrogen sulfide to 200 ppm or less of remaining hydrogen sulfide, wherein 200 ppm or less is an environmental safety standard. Also, the microbial community has very excellent sulfur conversion rate (processing rate), 99% or more, and can mass produce biosulfur.

Description

Microbial community for preparation of biosulfur and its use

The present invention relates to a microbial community for producing biosulfur and its use, and more particularly, to a novel microbial community having a high concentration of converting hydrogen sulfide (H ₂ S) into biosulfur, and a method for producing biosulfur using the same. Or to a method for removing hydrogen sulfide from a gas containing hydrogen sulfide.

In the natural environment, microorganisms rarely exist as a single species and form a microbial community that correlates with microorganisms of other species, which characterizes wastewater treatment, water purification systems, survival of pathogenic microorganisms and antibiotics. It is found in almost all microorganisms found in the environment, such as resistance, restoration of contaminated environment and metal corrosion. Since the expression characteristics when the microorganisms are clustered are different from those present in a single species, the microbial community is identified as a functional expression unit, and the microbial phenotype and activity changes according to the dynamic characteristics of the microorganisms constituting it and the environmental changes. Research of industrial applications of microbial communities has been made through the analysis of the interrelationship between, and microbial cells (Patent 10-1109120).

The most common treatment for solid waste is landfilling, and landfill gas (LFG), a mixed gas from these landfills, is harmful to the environment, but methane accounts for 50% to 60% of landfill gas (LFG). (CH ₄ ) can be used as an alternative energy source for power plants, cogeneration plants and district heating and cooling, which is useful not only for environmental issues but also for efficient use of energy. In addition to methane, landfill gas contains harmful components such as hydrogen sulfide and siloxane, along with water, carbon dioxide and nitrogen. Is calculated (registered patent 10-1024969).

The removal of hydrogen sulfide, a hazardous substance contained in landfill gas, has been a biological removal through the process of dissolving hydrogen sulfide gas in water and then oxidizing it into sulfated bacteria to produce sulfur (elemental sulfur, S ⁰ ):

^{_{H 2 S + OH - → HS}} - + H 2 O, HS - + 1/2 O 2 → S 0 + OH -

However, in the landfill site of Korea's metropolitan area, construction waste landfill is made like organic waste landfill, and hydrogen sulfide is gradually increased in the landfill gas to be included in high concentration of 14,000ppm or more. As a result, it was difficult to properly remove high concentrations of hydrogen sulfide in landfill gas by using sulfated bacterial communities introduced from abroad, and the amount of sulfur produced by the biological removal process (also called 'bio sulfur') was not used and was discarded. It was a situation.

Therefore, there is an urgent need for the discovery of microbial communities that can produce biosulfur in large quantities by converting high concentrations of hydrogen sulfide into sulfur.

Accordingly, the present inventors have completed the present invention by separating / identifying a microbial community for converting high concentration of hydrogen sulfide into sulfur in the pretreatment process of landfill gas in a metropolitan landfill while continuing research to meet the demands of the prior art.

It is therefore an object of the present invention to provide a novel microbial community having the ability to convert high concentrations of hydrogen sulfide into biosulfur.

Another object of the present invention is to provide a culture of the microbial community.

Still another object of the present invention is to provide a method for producing biosulfur in large quantities using the microbial community.

Still another object of the present invention is to provide a method for removing high concentration of hydrogen sulfide from a gas containing hydrogen sulfide by using the microbial community.

In order to achieve the above object of the present invention, the present invention is Alkalilimnicola spp., Rhodobaca ( Rhodobaca) spp.), Thioalkalivibrio spp., Aliidiomarina spp.) and Halomonas spp., to provide a microbial community having a high concentration of hydrogen sulfide conversion to biosulfur .

In the present invention, 'hydrogen sulfide' uses hydrogen sulfide recovered from a gas in which hydrogen sulfide is mixed. Gases in which hydrogen sulfide is mixed include landfill gas (LFG), digestive gas (ADG) generated from an anaerobic digester, by-product gas during crude oil refining, natural gas, and by-product gas generated in industrial facilities. (LFG).

In the present invention, 'high concentration hydrogen sulfide' means 14,000 ppm or more, preferably 18,000 ppm or more, and most preferably 20,000 ppm or more.

In the present invention, bio-sulfur (biosulfur) "refers to Water suspensions containing elemental sulfur (S ^o), or it is produced through a biological sulfur conversion process. Biosulfur is hydrophilic in comparison with chemically produced sulfur and is in a stable aqueous suspension in which elemental sulfur is suspended with a particle size of 10 µm or less. Biosulfur is also very toxic and can be used without the legalization required for chemically produced sulfur.

In the present invention, 'conversion ability' means a characteristic capable of finally converting hydrogen sulfide and sulfur compounds derived therefrom into elemental sulfur.

The present inventors have identified a microbial community showing excellent conversion of high concentrations of hydrogen sulfide to sulfur (biosulfur) among samples obtained in a landfill gas pretreatment process in the Seoul metropolitan landfill, and using next generation sequencing (NGS) analysis The microbial community was isolated / identified (confirmed diversity), and was deposited with the Korea Institute of Biotechnology and Biotechnology Center (KCTC) on December 11, 2018, and was given accession number KCTC13771BP (Example 1).

Alkalilimnicola ( Alkalilimnicola) in a microbial community according to the present invention spp.), Rhodobaca spp.), Thioalkalivibrio spp.), Aliidiomarina spp. and Halomonas spp.) was dominant (FIGS. 1-4). More specifically Alkalilimnicola spp.) from 35.0 to 63.0%, Rhodobaca spp.) 7.0 to 20.0%, Thioalkalivibrio spp. 9.0 to 18.0%, Aliidiomarina spp.) is 8.0-15.0 %, and Halomonas spp.) from 5.5 to 6.5%.

Alkalilimnicola spp.) is mostly Alkalilimnicola ehrlichii , Rhodobaca spp.) is mostly Rhodobaca bogoriensis ), Thioalkalivibrio spp.) are mainly thioalkalivibrio ( Thioalkalivibrio) halophilus ) and Halomonas spp.) is mainly Halomonas campaniensis (FIGS. 1 and 3).

Alkalilimnicola spp., The most dominant species in the microbial community of the present invention, is a heterotrophic bacterium that utilizes nitrate, thiosulfur, etc. as heterotrophic bacteria, and converts sulfur compounds into elemental sulfur and discharges them extracellularly. This is completely different from Thiobacillus spp., A sulfur conversion strain used to remove hydrogen sulfide from landfill gas.

The microbial community of the present invention comprising the above-mentioned predominant species was inoculated in a bioreactor for removing hydrogen sulfide during a landfill gas purification process at a landfill site in Seoul, and produced biosulfur for 2 months (April and October 2017). By removing the high concentration of hydrogen sulfide of 14,000ppm or more in the gas to convert the residual hydrogen sulfide to less than 200ppm, the sulfur conversion rate (treatment rate) was about 99% or more and confirmed that biosulfur can be produced in large quantities (Example 2).

According to another object of the present invention, the present invention provides a culture of the microbial community.

The culture of the microbial community according to the present invention can be used as a spawn for the production of bio-sulfur or hydrogen sulfide removal of landfill gas.

According to another object of the present invention, the present invention provides a method for producing biosulfur using the microbial community. The method is

i) dissolving hydrogen sulfide in an aqueous sodium hydroxide (NaOH) solution to prepare an aqueous hydrogen sulfide solution;

Ii) converting the sulfur compound into elemental sulfur by introducing an aqueous hydrogen sulfide solution into the viro reactor inoculated with the microbial community; And

Iii) precipitation of the elemental sulfur containing solution from step ii) in a precipitator to obtain biosulfur.

The method may further comprise dehydrating the biosulfur from step iii) for the commercialization of biosulfur, if necessary.

Biosulfur prepared according to the production method of the present invention is a stable aqueous suspension containing at least about 38% of elemental sulfur, and at least 90% of which is suspended in a particle size of less than 10 μm, and is hydrophilic and nontoxic.

According to still another object of the present invention, there is provided a method for removing hydrogen sulfide from a gas containing hydrogen sulfide by using the microbial community. The method is

i) washing the landfill gas with an aqueous sodium hydroxide solution to prepare an aqueous hydrogen sulfide solution; And

Ii) converting the elemental sulfur into a hydrogen sulfide solution in a bioreactor inoculated with a microbial community.

Optimal conditions for the production of bio-sulfur (hydrogen sulfide removal; elemental sulfur conversion from hydrogen sulfide) of the microbial community of the present invention is pH of 9.0 or less, preferably 7.5 to 8.5, temperature is less than 37 ℃, preferably 32.0 to 36.5 ℃, The redox potential (ORP) is -340 to -410 mV, the alkalinity is 0.3 to 1.1 mol / L, the electrical conductivity is 40 to 90 mS / cm, preferably 65 mS / cm, and the inoculation concentration is 5.0 × 10 ⁶ to 8.0 × 10 ⁷ cfu / ml.

The microbial community according to the present invention is a new microbial community composed of completely different dominant species from the microbial community based on thiobacilli species, which has been used for conventional hydrogen sulfide removal. In addition, the microbial community of the present invention converts high-density hydrogen sulfide of 14,000 ppm or more in the gas containing hydrogen sulfide into elemental sulfur so that residual hydrogen sulfide reaches 200 ppm or less, which is an environmental safety standard, and the sulfur conversion rate (processing rate) is about 99% or more. great. In addition, the microbial community of the present invention can produce a large amount of bio sulfur.

In the method for removing hydrogen sulfide using the microbial community of the present invention, it is possible to supply a safe gas by removing high concentration of hydrogen sulfide having high corrosiveness to a facility using a gas containing hydrogen sulfide in a high yield.

The method for producing bio-sulfur by using the microbial community of the present invention can convert high concentrations of hydrogen sulfide into elemental sulfur, thereby producing bio-sulfur in large quantities, and excellent economical efficiency.

Biosulfur prepared according to the present invention, unlike sulfur conventionally produced chemically, has high solubility in water, harmless to humans and harmless to animals and plants, has high utility as a material and a product in various industries.

Figure 1 shows the bacterial taxa that make up the microbial community (Amplicon Library A) of the present invention as confirmed by Illumina Next Generation Sequencing (NGS).
2 is a graph showing the distribution of bacterial taxa constituting the microbial community (Amplicon Library A) of the present invention.
Figure 3 shows the bacterial taxon making up the microbial community (Amplicon Library B) of the present invention as confirmed by NGS.
4 is a graph showing the distribution of bacterial taxa constituting the microbial community (Amplicon Library B) of the present invention.
5 is a graph showing the distribution of the dominant species classification group constituting the microbial community of the present invention.
6 is a schematic view showing an example of a biosulfur production process using the microbial community of the present invention.

Hereinafter, the present invention will be described in more detail with reference to specific examples in order to help the understanding of the present invention. However, the following examples are only illustrated to more clearly understand the present invention, and the scope of the present invention is not limited by the following examples.

Example  One. Illumina NGS  analysis

<Sample collection>

April 10 (LFG within the average H ₂ S concentration of about 17,000ppm) and 2017 2017 pre-treatment step (purification) of the gas filled in each Incheon Western Landfill to (embedded within the gas H ₂ S the average concentration of about 20,000ppm) Samples A and B were collected from the bioreactor-derived process water used for storage and stored at 4 ° C., and then microorganisms (bacteria) constituting the microbial community by Illumina next generation sequencing (NGS) based on 16S rRNA sequences. The type and distribution of was analyzed by analyzing.

<DNA Extraction>

DNA was extracted from each sample using the FastDNA Spin Kit (MP Biomedicals) for soil. The amount of extracted DNA was measured by an Epoch spectrometer (BioTek), and the DNA concentration used for analysis was at least 200 ng.

< Amplicon  Library Production>

Using the extracted DNA as a template, forward and reverse amplicon primers were prepared to target the V3 to V4 regions of the 16S rRNA gene as shown in Table 1 below. First PCR amplification was performed:

Forward
1st_341F TCGTCGGCAGCGTC-AGATGTGTATAAGAGACAG-CCTACGGGNGGCWGCAG
(50mer) Reverse
1st_805R GTCTCGTGGGCTCGG-AGATGTGTATAAGAGACAG-GACTACHVGGGTATCTAATCC
(55mer) Configuration: NexTera concensus-Sequencing adapter-Target sequence
N: G or A or T or C, W: A or T,
H: A or T or C, V: G or A or C

10 μl PCR buffer 2.5 μl, 10 mM dNTP Mix 2.5 μl, Taq polymerase (Dr. Max DNA polymerase, 500U) 0.25 μl, each forward primer (10 pmol; SEQ ID NO: 1) 1 μl, reverse primer (10 pmol; SEQ ID NO: 2) 1 16.75 μl of distilled water (DW) was mixed in 2 μl and 2 μl of template DNA, followed by a 3-minute initial denaturation at 95 ° C., 30 second denaturation at 95 ° C., 30 second primer annealing at 55 ° C., and extension at 72 ° C. using a Thermocycler 30 Second, the last extension was carried out 25 cycles of 5 minutes at 72 ° C. After the first PCR was completed, the PCR product was confirmed by agarose gel electrophoresis (about 500bp). Then, a secondary PCR primer set, a forward primer (Index i5; SEQ ID NO: 3), and a reverse primer (Index i7;) as shown in Table 2 were used as a template to attach an Illumina Index (NexTera barcode) as a template. Secondary PCR amplification was performed using SEQ ID NO: 4):

Index i5 (S522)
51mer AATGATACGGCGACCACCGAGATCTACAC- TTATGCGA -TCGTCGGCAGCGTC
(Configuration: Left-i5 ( XXXXXXXX )-Right) Index i7 (N724)
47mer CAAGCAGAAGACGGCATACGAGAT- CGCTCAGT -GTCTCGTGGGCTCGG
(Configuration: Left-i7 ( XXXXXXXX )-Right)  X represents barcode area

The conditions of the second PCR amplification were performed in the same manner as the conditions of the first PCR amplification except that eight amplification cycles were set.

After completion of PCR amplification, PCR products were identified using 1% agarose gel electrophoresis (approximately 600 bp) and visualized under Gel Doc system (BioRad, Hercules, CA, USA). Then, each PCR product was purified with Agencourt AMPure XP (A63880, Beckman Coulter, Inc.), quantified by Picogreen method using Quanti-iT PicoGreen dsDNA Assay Kit (P11496, Invitrogen), and all samples were collected in equal amounts ( pooling) Amplicon libraries were prepared, and the libraries were quantified by Picogreen method to confirm library size using Agilent 2100 Bioanalyzer System (Agilent technologies, US). Short fragments up to 200 bp were removed using Agencourt AMPure XP (A63880, Beckman Coulter, Inc.). The quality and product size of PCR products were determined on the Bioanalyzer 2100 (Agilent, Palo Alto, CA, USA) using a DNA 7500 chip, and the amplicon libraries A and B constructed for each of Samples A and B were used in the following experiments. It was.

<Illumina MiSeq Sequencing and Analysis>

The constructed amplicon libraries A and B were subjected to next generation sequencing (NGS) using the Illumina MiSeq Sequencing system (Illumina, USA) according to the manufacturer's instructions to obtain 16S metanome sequencing data (raw read data). Sequencing data were analyzed by the MiSeq pipeline method as follows.

The raw read data processing obtained in the sequencing process is a quality check using Trimmomatic 0.32 to filter out low quality (<Q25) with a quality score of less than 25, and after the QC pass, the sequence data of the paired ends are paired with PANDAseq. Merged. Then, after trimming the primers at the similarity cutoff 0.8, nonspecific amplicons not encoding 16S rRNA were detected by HMMER's hmmsearch program with 16S rRNA profile. DUDE-Seq was used to remove noise and non-redundant reads were extracted with UCLUST-clustering.

Taxonomically assigned using EzBioCloud data base and USEARCH (8.1.1861_i86linux32), non-chimeric 16S rRNA databases from UCHIME and EzBioCloudare detected chimeras in reads with less than 97% similarity, and finally CD-HIT and UCLUST Sequence data was clustered using to classify into taxons, and more than 30 bacterial taxa were identified, and bacterial taxonomic distribution results compared to NCBI BLAST and sequences are shown in FIGS. 1 and 2 (Amplicon Library A) and FIG. 3. And FIG. 4 (Amplicon Library B). The microbial community isolated / identified as described above (Amplicon Library B) was named 'Bacteria Mixture ICN' and was deposited on December 11, 2018 to the Korea Institute of Bioscience and Biotechnology (KCTC), and assigned accession number KCTC13771P. received.

Looking at the results shown in Figures 1 to 4 in detail, Alkalilimnicola spp., Rhodobaca in the microbial community of the present invention spp.), Thioalkalivibrio spp.), Aliidiomarina spp. and Halomonas Five dominant taxons of spp.) were identified in common and their distribution is shown in Table 3 and FIG.

Dominant Species Amplicon Library A Amplicon Library B Alkalilimnicola ssp. 43.1 57.1 Thioalkalivibrio ssp. 17.4 11.0 Halomonas ssp. 5.9 5.8 Rhodobaca ssp. 19.0 8.4 Aliidiomarina ssp. 9.2 11.7 etc 5.4 6.0 sum 100.0 (%) 100.0 (%)

1 and 3, Alkalilimnicola spp. Was identified as Alkalilimnicola ehrlichii , and Rhodobaca spp. Was mainly Rhodobaca bogoriensis . Thioalkalivibrio spp. Is mainly Thioalkalivibrio halophilus , and Halomonas spp. Is mainly Halomonas campaniensis .

Alkalilimnicola spp., The most dominant species of the microbial community of the present invention, had a share of more than 43%, but not at all with Thiobacillus spp., A sulfur conversion strain used to remove hydrogen sulfide from landfill gas. It is a different species.

In addition, the share of the dominant species in the microbial community of the present invention showed some changes in the two amplicon libraries, which appeared to be due to the concentration of H ₂ S to be treated, compared to Sample A (H ₂ S about 17,000 ppm). H ₂ S about 20,000ppm) no bar was under the environment of high concentration of H ₂ S, alkali rim Nicolas (Alkalilimnicola spp.) and alriyi video Marina (Aliidiomarina spp.) are at higher the H ₂ S concentration increases the share, and also Rhodobaca spp . And Thioalkalivibrio spp.) showed a decrease in occupancy as the concentration of H ₂ S increased.

Example 2 Biosulfuric Production (Hydrogen Sulfide Removal from Landfill Gas)

Using the microbial community of the present invention, biosulfuric acid in the biosulfuric production (removing hydrogen sulfide from landfill gas) plant of Ecobio Holdings Co., Ltd. located in Seo-gu, Incheon on April 20, 2017 and October 20, 2017, respectively. Prepared (FIG. 6).

<Use of landfill gas with H ₂ S concentration of 16,036ppm>

Specifically, in April 20, 2017, a 25% caustic soda solution was introduced while introducing landfill gas (H ₂ S concentration: 16,036 ppm) derived from the second landfill site in the Seo-gu metropolitan area of Incheon at a flow rate of about 24,180 m ³ / hr. After washing at about 1.25 ton / hr to prepare an aqueous hydrogen sulfide solution (S-load, which is converted to the amount of sulfur atoms in the aqueous hydrogen sulfide solution, is about 594.1 Kg-S / hr), the microbial community and neutrimix of the present invention are shown in the table. The aqueous hydrosulfide solution was introduced into a bioreactor (bioreactor) having a working volume of 636 m ³ in which the aqueous solution inoculated as shown in FIG. 4 and the reactor conditions were set was converted into elemental sulfur by introducing a circulation flow of 1,150 m ³ / hr. Then, it was introduced into a precipitator and settled for 2 hours to obtain biosulfur (see FIG. 6). The residual amount of hydrogen sulfide in the methane gas discharged during the manufacturing process was measured to be about 90 ppm (about 99.4% hydrogen sulfide removal efficiency).

Item Condition Inoculation concentration 1.08 × 10 ⁷ cfu / ml Nutrimix Nutrimix (Thiopaq) 3 ml / L, Na ₂ CO ₃ 30 g / L Redox potential (ORP)   -340 to -410mV pH   8.5 Electrical conductivity   65 mS / cm Temperature   35 ℃ Alkaline   0.7 mol / L Total suspended solids   10 g / L

The biosulfur produced according to the invention exiting the settler was biosulfur in suspension containing about 38% elemental sulfur (S-load is about 594 Kg-S / hr).

In order to commercialize bio-sulfur, the product was further processed into a suspension containing about 50% of elemental sulfur through a further dehydration process, and finally, about 516 kg-S / hr except for 13% of the filtrate which was removed during the dehydration process. Biosulfur products were produced.

<Use of landfill gas with H ₂ S concentration 20,365ppm>

On October 20, 2017, landfill gas (H ₂ S concentration: 20,365ppm) derived from the 2nd landfill in Seo-gu, Incheon Metropolitan City was introduced with an inflow of 23,460 m ³ / hr (S-load is about 683.41 Kg-S / hr) Except that it was carried out in the same manner as the manufacturing process to produce a bio-sulfur.

The residual amount of hydrogen sulfide in the methane gas discharged during the manufacturing process was measured to be about 140 ppm (about 99.3% hydrogen sulfide removal efficiency).

 The produced biosulfur released from the precipitator was biosulfur in suspension containing about 38% elemental sulfur (S-load about 680 Kg-S / hr). For further commercialization, the biosulfur product was produced at a final yield of about 590 kg-S / hr when further processed into biosulfur (about 13% desorption) in suspension containing about 50% elemental sulfur.

From the above results, it can be seen that the microbial community of the present invention can produce a large amount of bio-sulfur, and is highly functional capable of removing high-concentration hydrogen sulfide from landfill gas by converting it to 99% or more elemental sulfur (biosulfur). .

Korea Research Institute of Bioscience and Biotechnology KACC13771BP 20181211

<110> ECO BIO HOLDINGS CO., LTD <120> Microbial community for preparation of biosulfur and its use <130> P-10273 <160> 4 <170> KoPatentIn 3.0 <210> 1 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> Forward primer <400> 1 tcgtcggcag cgtcagatgt gtataagaga cagcctacgg gnggcwgcag 50 <210> 2 <211> 55 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 2 gtctcgtggg ctcggagatg tgtataagag acaggactac hvgggtatct aatcc 55 <210> 3 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> Forward primer <400> 3 aatgatacgg cgaccaccga gatctacacc tctctattcg tcggcagcgt c 51 <210> 4 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 4 caagcagaag acggcatacg agattcgcct tagtctcgtg ggctcgg 47

Claims (10)

Microbial community deposited with accession number KCTC13771BP having the ability to convert hydrogen sulfide to biosulfur, the microbial community is
The bacterial community comprises alkali rim Nicolas (Alkalilimnicola spp.), Also bakka (Rhodobaca spp.), Thio alkali Vibrio (Thioalkalivibrio spp.), Alriyi video Marina (Aliidiomarina spp.) And halo Pseudomonas (Halomonas spp.).
The microbial community according to claim 1, wherein the hydrogen sulfide has a high concentration of 14,000 ppm or more.
The microbial community according to claim 1, wherein the hydrogen sulfide has a high concentration of 18,000 ppm or more.
The microbial community according to claim 1, wherein the hydrogen sulfide has a high concentration of 20,000 ppm or more.
The method of claim 1, wherein the Alkalilimnicola spp. Has a share of 35.0 to 63.0%, the Rhodobaca spp. Has a share of 7.0 to 20%, and the Thioalkalivibrio spp. Has a share of 9.0 to 18.0. % of share, alriyi video Marina (Aliidiomarina spp.) of 8.0 to 15.0 percent market share, and halo Monastir (Halomonas spp.) is a microbial community, which will occupy a market share of 5.5 to 6.5 percent.
Culture of the microbial community according to claim 1.
The culture of the microbial community according to claim 6, which is for producing biosulfur or for removing hydrogen sulfide from a gas containing hydrogen sulfide.
A method for producing biosulfur by using the microbial community according to claim 1.
A method for removing hydrogen sulfide from a gas containing hydrogen sulfide by using the microbial community according to claim 1.
10. The method of claim 9, wherein the gas containing hydrogen sulfide is landfill gas (LFG).

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