Novel artificial algae crust construction method based on community characteristics and application

Abstract

The invention discloses a novel artificial algae crust construction method and application based on community characteristics, through OTU co-occurrence network topology analysis (Zi / Pi value screening module hubs, connection hubs and network hubs), hub algae species and peripheral nodes (such as Nodolinean OTU160 and Phormides OTU53) which have no competitive relationship with basic algae species (Nostoc, Microcoleus and Scytonema) or have a positive effect are accurately screened, inter-species resource competition is avoided, and the construction method of the novel artificial algae crust based on the community characteristics has the advantages that the construction efficiency is improved, and the construction cost is reduced. The crust forming speed is obviously improved. The obtained artificial algae crust has a crust coverage degree of 75% within 30 days, which is increased by 40% compared with that of a single algae species, and the problem of neck clamping caused by slow field growth is solved. The depth of the algae crust water-holding soil layer obtained through the method reaches 7.2 cm, and water evaporation in the high-cold region can be effectively relieved; the content of organic carbon is increased by 2.1 times, the content of total nitrogen is increased by 1.8 times, and a foundation is laid for subsequent vegetation recovery.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology of biological materials constructing artificial biological crust, and in particular to a novel artificial algal crust construction method based on community characteristics and application. BACKGROUND

[0002] As the "third pole of the world" and the ecological security barrier of Asia, the Qinghai-Tibet Plateau plays a key role in East Asian monsoon and global water cycle. However, in recent years, due to overgrazing and climate change, about 15.1% of the region is severely desertified and the area is expanding, resulting in a reduction of about 13.8% of farmland, causing huge economic losses, so it is of great significance to improve the vegetation coverage and restore biodiversity in this region. Inoculation of microbial communities in extreme environments is a new technology for ecological restoration, and algae in biological soil crusts are particularly important. Biological soil crusts cover 37% of the desertified land in the Qinghai-Tibet Plateau and are widely distributed in arid, semi-arid and alpine regions. They are pioneers of the ecosystem and have many key roles such as wind prevention and sand fixation. Among them, cyanobacteria, as the "engineer" of the ecosystem, can grow rapidly in harsh soil environments and promote the formation of early biological soil crusts.

[0003] Currently, the growth rate of artificial algal crusts in the wild is slow, which is still a "neck-breaking" problem that needs to be broken through. It is generally believed that the factors affecting the inoculation of artificial algal crusts mainly include the following aspects: soil salinity, soil texture, pH, water availability, inoculation amount, temperature and light intensity, but the interspecific competitive effect cannot be ignored. Although interspecific competition does not lead to the complete extinction of a species, it can inhibit its growth due to competition for nutrients and water. For example, after the formation of stable algal crusts, if the algae cannot utilize secondary metabolites to each other in limited environmental resources, it will increase the probability of resource competition between algae. In addition, ignoring the niche and network centrality of functional algae strains will weaken the ecological function of artificial algal crusts. At the same time, the importance of algal crust community characteristics is ignored in the existing technology, which still has defects to some extent,

[0004] Therefore, a novel artificial algal crust construction method based on community characteristics and application is needed to construct a novel artificial algal crust to solve the above technical problems. SUMMARY

[0005] In order to solve the above technical problems, the present application discloses a novel artificial algal crust construction method based on community characteristics and application. The algal crust constructed by the method has a faster growth rate in the wild. At the same time, the method is based on community characteristics and will not inhibit each other's growth. After the formation of stable algal crusts, algae can utilize secondary metabolites to each other in limited environmental resources, reducing resource competition and ensuring rapid growth in the wild.

[0006] In order to achieve the above technical effects, the application designs a new artificial algal crust construction method based on community characteristics, comprising the following steps:

[0007] S1: collecting mature natural algal crust samples in the target area;

[0008] S2: performing algal species separation, purification and molecular identification on the natural algal crust samples collected in S1, and obtaining the gene sequence of the purified algal strain;

[0009] S3: performing amplicon sequencing on the natural algal crust, constructing an OTU co-occurrence network and analyzing the topological properties, and screening algal species meeting the following conditions as breeding algal species:

[0010] modular hubs (Zi≥2.5 and Pi<0.62), connecting hubs (Zi<2.5 and Pi≥0.62), or network hubs (Zi≥2.5 and Pi≥0.62); there is a positive interaction between each other and no competitive relationship with the basic algal species;

[0011] S4: combining the basic algal species and the breeding algal species into an algal species combination, inoculating into the surface of the target soil, monitoring the crust growth index and screening the optimal combination.

[0012] Further, the basic algal species in S1 is selected from the most abundant algal strains in Nostoc, Microcoleus and Scytonema.

[0013] Further, the topological analysis in S3 comprises:

[0014] S3.1: using the Spearman correlation algorithm to construct a network, and setting the minimum abundance filtering threshold to 0.001;

[0015] S3.2: calculating the correlation between OTUs, and retaining significant correlations with P value≤0.01 and correlation coefficient absolute value≥0.8;

[0016] S3.3: calculating the intra-module connectivity (Zi) and inter-module connectivity (Pi) to divide the ecological roles.

[0017] Further, the algal species combination in S4 includes one of the following groups:

[0018] basic algal species; basic algal species+connecting hub algal species (Nodosilinea); basic algal species+peripheral node algal species (Phormidesmis); basic algal species+Nodosilinea+Phormidesmis.

[0019] Further, the molecular identification in S2 comprises: extracting the algal strain genome, and amplifying the 16S rRNA sequence by PCR; then, Sanger sequencing is performed on the PCR product obtained by amplification, the sequencing result is spliced by using Geneious software, and then the 16S rRNA sequence is submitted to the GenBank database for BLAST comparison, and the classification information of the algal species is determined according to the comparison result.

[0020] Further, the algal species screened out in S3 is expanded, and the culture conditions for expansion are: temperature 25 DEG C, light intensity 15000 lx, light-dark ratio 12 h:12 h; when the algal liquid grows to a concentration meeting the experimental dosage, thick polyester cotton cloth bags are used for filtering or a high-speed refrigerated centrifuge is used for centrifugation at 8500 rpm at 4 DEG C for 10 min, and then the collected algal precipitate is placed in a freeze dryer for vacuum freeze-drying; the freeze-dried algal precipitate is ground into algal powder at low speed by using a multifunctional grinder, and is stored in a refrigerator at-20 DEG C for use as needed.

[0021] Further, the inoculation conditions in S3 are: the corresponding freeze-dried algal powder is mixed in a ratio of 1:1 according to each algal species, and the mixed algal liquid is sprayed to the soil surface with an inoculation amount of 8 g dry weight / m 2 The growth rate of each group of algal species combination is compared, and the algal species combination with the fastest growth rate is selected as the optimal algal species combination.

[0022] Further, in S1, the target area is the alpine desertification area of the Qinghai-Tibet Plateau.

[0023] The beneficial effects of the present application are:

[0024] The present application designs a new artificial algal crust construction method based on community characteristics and application, which at least has the following beneficial effects:

[0025] 1. Through OTU co-occurrence network topology analysis (Zi / Pi value screening module hub, connection hub and network hub), the hub algal species and peripheral nodes (such as Nodosilinea OTU160 and Phormidesmis OTU53) with no competitive relationship with the basic algal species (Nostoc, Microcoleus, Scytonema) and positive effects on each other are accurately screened, the inter-species resource competition is avoided, and the crust anti-interference ability and formation speed are significantly improved. In the pot experiment, the artificial algal crust obtained has a crust coverage of 75% within 30 days, which is increased by 40% compared with a single algal species, and the current slow growth of artificial algal crust is solved.

[0026] 2. The method finally obtains an algal crust water-holding soil layer depth of 7.2 cm, which can effectively alleviate water evaporation in the alpine region; the soil fertility is significantly improved: the organic carbon content is increased by 1.4 times, and the total nitrogen content is increased by 1.25 times, laying a foundation for subsequent vegetation restoration;

[0027] 3. The algal crust obtained by the application not only can colonize efficiently in the field test, but also can quickly build a surface barrier, and the coverage degree is as high as about 40% in 30 days, and the sand soil in the covered area can effectively resist wind and sand erosion under the condition of strong wind of 10 m / s, which provides key technical support for desertification control. BRIEF DESCRIPTION OF DRAWINGS

[0028] In order to more clearly illustrate the technical solutions of the embodiments of the application, the drawings needed for the embodiment description will be briefly introduced as follows.

[0029] Figure 1 is the OTU interaction type and intensity in the application;

[0030] Figure 2 is the thickness of each combined crust in the application;

[0031] Figure 3 is the coverage of each combined crust in the application;

[0032] Figure 4 is the comparison of the organic carbon content of the best algal species combination and bare soil in the application;

[0033] Figure 5 is the comparison of the total nitrogen content of the optimal algal species combination and bare soil in the application;

[0034] Figure 6 is the coverage of the crust formed by the optimal algal species combination in the field in the application;

[0035] Figure 7 is the growth of the crust formed by the optimal algal species combination in the field in the application;

[0036] Figure 8 is the condition of the crust covered by the optimal algal species combination under strong wind. DETAILED DESCRIPTION

[0037] Embodiment 1

[0038] A new artificial algal crust construction method based on community characteristics and application, comprising the following steps:

[0039] S1: Collecting mature natural algal crust samples developed in the alpine region of the Qinghai-Tibet Plateau;

[0040] S2: performing algal species separation, purification and molecular identification on the natural algal crust sample collected in S1 to obtain the gene sequence of the purified algal strain;

[0041] The separation and purification process is as follows: the collected algal crust is placed in an oven for low-temperature drying, ground with a mortar, and the crust particles are collected in a serum bottle, 2 mL of pure water is added for hydration for 24 h. 50 μL of the suspension is taken and evenly coated on the BG11 solid culture medium, and cultured in an artificial climate chamber for 7-10 days (culture conditions: temperature 25 ℃, light intensity 15000 lx, light-dark ratio 12h:12h). After the algal filaments grow, a single algal filament is picked up with tweezers and inoculated on a new BG11 solid culture medium for streaking, and after several rounds of purification, relatively clean algal cells are obtained;

[0042] Algal species propagation: a single algal filament is picked up with tweezers and inoculated in a 12-hole cell culture plate containing BG11 liquid medium for the first stage of propagation culture (2-3 ml of medium per hole). After culturing to the logarithmic growth phase, the algal liquid is transferred to a 100 ml conical flask containing BG11 liquid medium and connected to an oxygen supply pump for the second stage of propagation. Similarly, after the algal cells in the conical flask grow to the logarithmic growth phase, the algal liquid is transferred to a 500 ml conical flask containing BG11 liquid medium and connected to an oxygen supply pump for the third stage of propagation culture.

[0043] The molecular identification process in the above process is as follows: 300 mL of algal liquid cultured to the logarithmic growth phase in step (3) above is collected, centrifuged at 8500 rpm for 10 min, the supernatant is discarded, and then the genome is extracted using a plant DNA extraction kit (Novozyme, China), and the quality of the extracted genome is evaluated by agarose gel electrophoresis and Nanodrop. The qualified genome can be used for subsequent experiments. Next, the 16S rRNA sequence of cyanobacteria is amplified by PCR, and the amplification primers and procedures are shown in Table 1. The reaction system of PCR is 50 μL: 25 μL Taq enzyme PCR premix; 1 μM primer; 1 μg genomic DNA; the remaining volume is supplemented with ddH2O, then the PCR product obtained by amplification is subjected to Sanger sequencing, the sequencing results are spliced using Geneious software, and then the 16S rRNA sequence is submitted to the GenBank database for BLAST comparison, and the classification information of the algal species is preliminarily determined according to the comparison results. The specific primers used are shown in Table 1 below:

[0044] Table Primer used for amplification

[0045]

[0046] S3: The natural algal crust is subjected to amplicon sequencing, and an OTU co-occurrence network is constructed and the topological properties are analyzed to screen algae species meeting the conditions as breeding algae species, and the specific process is as follows:

[0047] In order to screen out potential high-efficiency blue-green algae community, the abundance of each algae species in the wild algal crust community and the interaction strength between each other are obtained by amplicon sequencing. According to the records in the prior art, Nostoc, Microcoleus and Scytonema are the blue-green algae that first colonize the surface layer of soil and generate crust, and are also the most commonly used blue-green algae for preparing artificial crust; based on the guidance in the prior art, the three algae species of Nostoc, Microcoleus and Scytonema are used as basic algae species in the present application.

[0048] According to the amplicon sequencing results, the algal strains with the highest abundance in Nostoc, Microcoleus and Scytonema are selected as basic algae species; at the same time, the network central algae species in the algal crust community are used as breeding algae species, and the specific steps are as follows:

[0049] a. First, the microeco package is used to construct the OTU co-occurrence network and perform topological analysis, the network is constructed by using the Spearman correlation algorithm, and the minimum abundance filtering threshold is set to 0.001;

[0050] b. The correlation between OTUs is calculated, and the significant correlation with P value ≤0.01 and absolute value of correlation coefficient ≥0.8 is retained;

[0051] c. Finally, the network topological properties are calculated and the modules are divided, and the node topological property table is extracted, including the intra-module connectivity (Zi), the inter-module connectivity (Pi) and the ecological role classification, wherein the ecological role is determined according to the values of Zi and Pi, and the nodes are divided into: ① peripheral node (Zi<2.5 and Pi<0.62): connected to a few nodes within the module; ② module hub (Zi≥2.5 and Pi<0.62): core node within the module; ③ connecting hub (Zi<2.5 and Pi≥0.62): bridge node connecting different modules; ④ network hub (Zi≥2.5 and Pi≥0.62): super core node across modules. Preferably, the OTUs that do not have competitive relationship with the basic algae species and have positive interaction with each other are selected as breeding algae species candidates from the module hub, the connecting hub and the network hub, and if there is a peripheral node having positive interaction with the breeding algae species pioneer and not having competitive relationship with the basic algae species, the peripheral node is added as the breeding algae species. (In the present application, the connecting hub OTU160 is selected as the breeding algae species pioneer, and the peripheral node OTU53 is selected as the other breeding algae species, and the two have positive interaction and do not have competitive relationship with the basic algae species).

[0052] The 16S rRNA sequences of the laboratory-isolated algal species are taken as the sequence data to be aligned, and the OTU representative sequences of amplicon sequencing are taken as the database to be aligned. First, use the cat command (cat *.fasta>combined_result.fasta) to combine all the 16S rRNA sequence files of the laboratory-isolated algal species into a complete FASTA file, and use the awk command (awk'NR % 4 == 1 {print ">" substr($0,2)} NR % 4 == 2 {print $0}' OUT_seq.fastq >database_pre.fasta) to convert the OTU representative sequences from the FASTQ format file to the FASTA format file, to obtain a file suitable for the BLAST database construction format. Then use makeblastdb to construct a nucleotide sequence database (makeblastdb -in database_pre.fasta -dbtype nucl -out database_NR). Finally, use blastn to perform sequence alignment (blastn-query combined_result.fasta -db database_NR -outresult.txt -outfmt "6 qseqid sseqid pident length evalue bitscore" -evalue1e-5 -num_threads 5), with the following parameter settings: the threshold of the expected value (E-value) of sequence similarity evaluation is 1e-5, and 5 computing threads are enabled to speed up the analysis process. The output result is in the format 6 separated by tabs, containing key information such as the name of the sequence to be aligned, the name of the database sequence, the percentage of sequence identity, the length of alignment, the expected value, and the bit score.

[0053] Take 99% as the threshold of the percentage of sequence identity, and prefer the alignment results with a percentage of sequence identity of 100%, to confirm the correspondence between the laboratory-isolated algal species and the OTUs obtained by amplicon sequencing. Then submit the obtained OTU representative sequences to the GenBank database for BLAST alignment, and compare the classification results of the OTUs and the classification alignment results of the 16S rRNA, to confirm the classification information of the algal species;

[0054] S4: The algae species screened in the above step were expanded (the culture conditions were as follows: temperature 25 ℃, light intensity 15000 lx, light / dark ratio 12 h:12 h). When the algal liquid grew to a concentration meeting the experimental dosage, it was filtered using a thick polyester cotton bag or centrifuged at 8500 rpm for 10 min at 4 ℃ using a high-speed refrigerated centrifuge, and then the collected algal precipitate was placed in a freeze dryer for vacuum freeze-drying. The freeze-dried algal precipitate was ground into algal powder at low speed using a multifunctional grinder, and was stored in a refrigerator at-20 ℃ for use as needed. Then the basic algae species and the selected algae species were randomly combined in the following ways: ① basic algae species; ② Nodosilinea; ③ Phormidesmis; ④ basic algae species+Nodosilinea; ⑤ basic algae species+Phormidesmis; ⑥ basic algae species+Nodosilinea+Phormidesmis, and the corresponding freeze-dried algal powder was mixed in a ratio of 1:1 of each algae species, and 8 g of dry weight / m2 of inoculum was sprayed to the surface of the potting soil, and the water amount was 10 mL per day, and the drought was 2 days every 5 days, and the crust growth rate such as crust coverage and crust thickness was detected every 7 days, the growth rate of each group of algae species combination was compared, the fastest growth rate of algae species combination was selected as the optimal algae species combination, and the improvement effect of the optimal algae species combination on the soil physical and chemical properties was confirmed. The results of the above process screening are shown in Table 2 below: 2

[0055] Table 2 16S rRNA sequence and OTU sequence alignment results

[0056]

[0057] Example 2

[0058] In this example, the water holding depth of the algae obtained in the above example 1 under different combinations of algae, the crust thickness and the crust coverage of different algae combinations were explored; the specific water holding depth is shown in Table 3 below:

[0059] Table Water holding depth of crust under different combinations

[0060]

[0061] ​From Table 3, it can be seen that, under the blank control of different combinations of algae and bare soil, the best water holding depth is the basic algae + Nodosilinea + Phormidesmis, and the water holding depth is 7.2 cm, which is about 67% higher than that of bare soil (4.3 cm), which can significantly improve the water supply of the surface soil; at the same time, it is 1.1 cm higher than the basic algae + Nodosilinea, and 0.7 cm higher than the basic algae + Phormidesmis, which shows that the water retention capacity of the basic algae + Nodosilinea + Phormidesmis is good, and it has better water retention capacity than other algae combinations.

[0062] In addition, the crust thickness and coverage of different algae combinations are as shown in Figure 2 and Figure 3 On the 14th day, the thickness of the combination containing the basic algae is thicker than that of the combination not containing the basic algae, and the coverage of the basic algae + Nodosilinea + Phormidesmis is as high as 75%, which is significantly higher than that of other algae combinations. This result fully proves that the growth rate of the basic algae + Nodosilinea + Phormidesmis is the fastest among these algae combinations, and it is proved that the algae crust prepared by the method has high coverage and good application prospect.

[0063] Example 3

[0064] In this embodiment, it is known from the above examples 1 and 2 that the basic algae + Nodosilinea + Phormidesmis is the preferred combination, and the preferred combination is now explored. The specific process is that the algae crust formed by the optimal combination is cultivated in a pot for 30 days, and then the soil under the algae crust and the bare soil are compared in terms of organic carbon content and total nitrogen content, as shown in Figure 4 and Figure 5 The organic carbon content and total nitrogen content of the soil under the algae crust are significantly higher than those of the bare soil, and the organic carbon content is 43% higher and the total nitrogen content is 25% higher. These results show that the optimal algae combination not only can significantly improve the growth rate of the crust, but also can promote nutrient cycling by increasing the nutrient content of the soil, and provide a good environment for the planting of plants.

[0065] Example 4

[0066] In this embodiment, the preferred combination in the above examples is sprayed in the Tengger Desert for field experiment to explore its survival in actual desert application. The specific process is: 6 g of dry weight / m 2The spray amount of the freeze-dried algal powder of the algal species combination and the BG11 culture medium is sprayed to the soil surface, and the water amount sprayed per day is 3 L / m 2 , and the watering is stopped every 5 days to make it dry for 2 days. The results are shown in Figure 6 , Figure 7 and Figure 8 , the optimal algal species combination shows superior adaptability in the field, and the coverage is up to about 40% in only 30 days, and in practical application, it shows a strong wind-preventing and sand-fixing effect: under strong wind of 10 m / s, the sand soil in the covered area can effectively resist wind and sand erosion, forming a stable surface protection, while the sand soil in the uncovered area is loose and easily eroded. This clear contrast fully verifies that the algal species cultivated by the method can not only survive in the field but also achieve a high coverage.

[0067] The preferred embodiments of the present application disclosed above are only used to help illustrate the present application, and the preferred embodiments do not describe all the details and limit the present application to the specific embodiments described.

Claims

1. A novel method for constructing artificial algae crusts based on community characteristics, characterized in that: The following steps are involved: S1: Collect mature natural algal crust samples from the target area; S2: Isolate, purify and molecularly identify the natural algal crust samples collected in S1 to obtain the gene sequence of the purified algal strain; S3: Perform amplicon sequencing on natural algal crusts, construct an OTU co-occurrence network and analyze its topological properties, and select algae species that meet the following conditions for breeding: Module hubs (Zi ≥ 2.5 and Pi < 0.62), connection hubs (Zi < 2.5 and Pi ≥ 0.62), or network hubs (Zi ≥ 2.5 and Pi ≥ 0.62); there are positive interactions between them and no competition with the basic algae species; S4: Combine the basic algae species and the selected algae species into an algae species combination, inoculate it onto the target soil surface, monitor the crust growth indicators and screen for the optimal combination.

2. A novel method for constructing artificial algae crusts based on community characteristics according to claim 1, characterized in that: The basic algae species in S1 are selected from the most abundant algae strains in the genera Nostoc, Microcoleus and Pseudoclonis.

3. The method for constructing a novel artificial algae crust based on community characteristics according to claim 1, wherein: The topology analysis in S3 includes: S3.1: The network was constructed using the Spearman correlation algorithm, with the minimum abundance filtering threshold set to 0.001; S3.2: Calculate the correlation between OTUs and retain significant associations with a P value ≤ 0.01 and an absolute value of the correlation coefficient ≥ 0.8; S3.3: Calculate intra-module connectivity (Zi) and inter-module connectivity (Pi) to divide ecological roles.

4. The method for constructing a novel artificial algae crust based on community characteristics according to claim 1, wherein: The algae species combination in S4 includes one of the following groups: Basic algae species; basic algae species + connecting hub algae species (Nodosilinea); basic algae species + peripheral node algae species (Phormidesmis); basic algae species + Nodosilinea + Phormidesmis.

5. The method for constructing a novel artificial algae crust based on community characteristics according to claim 1, characterized in that: The molecular identification in S2 includes: extracting the algae strain genome and amplifying the 16S rRNA sequence by PCR; then performing Sanger sequencing on the amplified PCR product, splicing the sequencing results using Geneious software, and then submitting the 16S rRNA sequence to the GenBank database for BLAST comparison, and determining the classification information of the algae species based on the comparison results.

6. The method for constructing a novel artificial algae crust based on community characteristics according to claim 1, characterized in that: The algae strains screened in S3 were propagated under the following culture conditions: temperature 25°C, light intensity 15,000 lx, and light-dark ratio 12 h:12 h. When the algae solution grew to a concentration sufficient for the experimental dosage, it was filtered using a thick polyester-cotton bag or centrifuged using a high-speed refrigerated centrifuge at 8,500 rpm for 10 min at 4°C. The collected algae pellet was then placed in a freeze dryer and vacuum-dried. Use a multifunctional grinder to grind the freeze-dried algae precipitate into algae powder at a low speed, and store it in a refrigerator at -20°C for immediate use.

7. The method for constructing a novel artificial algae crust based on community characteristics according to claim 1, characterized in that: The inoculation conditions in S3 are as follows: the corresponding freeze-dried algae powder is mixed at a ratio of 1:1 for each algae species, and 8g dry weight / m 2 The inoculation amount was sprayed on the soil surface with the mixed algae solution, and 10 mL of water was applied every day. At the same time, the growth rates of the algae species combinations in each group were compared, and the algae species combination with the fastest growth rate was selected as the optimal algae species combination.

8. The method for constructing a novel artificial algae crust based on community characteristics according to claim 1, characterized in that: In S1, the target area is the high-altitude cold desertified area of ​​the Qinghai-Tibet Plateau.

9. Application of a novel artificial algal crust construction method based on community characteristics according to any one of claims 1 to 8 in the treatment of alpine desertification areas on the Qinghai-Tibet Plateau.

10. The use according to claim 9, characterized in that Based on 6 g dry weight / m 2 The spraying amount is to mix the freeze-dried algae powder of the algae species combination with the BG11 culture medium and spray it on the soil surface. The spraying amount of water is 3 L / m per day. 2 , stop watering every 5 days and let it dry for 2 days.

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