TWI424979B - Thermo-tolerant multi-functional phosphate- and potassium-solubilizing microbes and its biofertilizer preparations - Google Patents

Description

High temperature resistant multifunctional phosphorus and potassium microbe and its biological fertilizer production

The present invention relates to a high temperature resistant potassium phosphate microorganism Bacillus lichenifromis A3 BCRC 910522 and Bacillus subtilis H8 BCRC 910523 which can be grown at 25 and 50 ° C and has dissolved calcium phosphate, iron phosphate, aluminum phosphate, and hydroxyapatide. And four kinds of inorganic phosphorus activities of mineral phosphorus, can also dissolve three kinds of inorganic potassium activities of feldspar, illite and kaolinite, and have starchy, cellulose, chitin, pectin, protein, lipid, poly-xylose and Keratinolytic enzyme activity. When it is inoculated into raw material matrices such as agricultural waste and livestock manure waste, it can accelerate the decomposition of organic matter, promote decomposing, increase the content of soluble phosphorus and soluble potassium, improve the quality, and improve the medium temperature and high temperature resistance. Phosphorus and dissolved potassium microorganisms grow. Improve the ratio of medium temperature and high temperature resistant phosphorus and potassium dissolved microbes and their ratio to medium temperature and high temperature resistant bacteria. This kind of preparation of multifunctional biological fertilizer has deep sustainable application such as machine agriculture, resource recycling and environmental protection.

Phosphorus and potassium are one of the main nutrient elements of plant growth. They provide the source of biochemical metabolic energy during plant growth and the combination of intracellular genetic material, various organelles, macromolecules and chemical substances, as well as the storage of intracellular energy and the response of external environment. And the delivery of messages. Phosphorus and potassium have important functions for plant growth and reproduction. However, in nature, although the soil is rich in phosphorus and potassium sources, there are very few soluble forms of phosphorus and potassium that can be supplied to plants. For example, the average phosphorus content in the soil is 0.05% (w/w), of which only 0.1% can be used as a plant utilization type, and the remaining phosphorus source is fixed by the soil to form a fixed phosphorus which is not easily absorbed by the plant. In soil, the total potassium content is between <0.01-4% (Wild, 1988). Most soils have a total potassium content of about 1%, but soluble potassium is only a small fraction of the plant's absorption and utilization, and the direction of potassium movement in the soil is closely related to soil properties (Blake et al., 1999). In order to increase harvest, farmers use a large amount of chemical phosphate fertilizer and potash fertilizer to increase the availability of phosphorus and potassium in the soil and provide absorption and utilization of crops. However, the use of a large amount of phosphate fertilizer and potash fertilizer will cause environmental pollution problems such as acidification of the soil, pollution of groundwater sources and rivers, and chemical fertilizers and potash fertilizers will not form with the elements in the soil in a short time. The use of a type of composite reduces its effectiveness and causes environmental pollution and increases agricultural production costs. Therefore, how to use the insoluble phosphorus and potassium sources rich in soil to dissolve it to increase the content of soluble phosphorus and soluble potassium in the soil, not only can reduce the use of chemical fertilizers, but also the concept of sustainable management, which is now an important topic of agriculture. .

Microorganisms play an important role in the soil phosphorus and potassium cycle, such as: phosphate solubilizing bacteria and potassium solubilizing bacteria. Phosphorus solubilizing bacteria have the activity of dissolving inorganic phosphorus sources such as calcium phosphate, iron phosphate, aluminum phosphate, hydroxyapatite and mineral phosphorus (Chang and Yang, 2009; Ogbo, 2010), while potassium lysates are soluble. Inorganic potassium source activity such as feldspar, kaolinite, illite, mica and Montestone (Hutchens, 2009; Koele et al., 2009; Uroz et al., 2009, 2011). Therefore, direct inoculation of phosphorus-dissolving or potassium-dissolving microorganisms on crop seeds, plant roots and adjacent root-loop soils and the use of them to prepare high-soluble phosphorus and soluble potassium bio-fertilizers are currently commonly used in agriculture to enhance soluble phosphorus and potassium in soil. Content, increased agricultural production and sustainable agricultural operations (Badr et al., 2006; Nishanth and Biswas, 2008; Kumar et al, 2009; Aria et al, 2010; Behbahani, 2010; Singh and Reddy, 2011).

At present, the concept of organic agriculture has been promoted, and the use of biological fertilizer is one of them. Inoculation of nitrogen, phosphorus, potassium, organic matter decomposition, antibacterial and mycorrhizal microorganisms in biological fertilizers, in order to increase the speed of biological fertilizers, the concentration of nitrogen, phosphorus and potassium, improve soil physical properties and protect crops against disease. When the biological fertilizer is applied to the field, the phosphorus-dissolving or potassium-dissolving microorganisms contained therein can maintain the concentration of soluble phosphorus and soluble potassium in the fertilizer, and dissolve the insoluble phosphorus and potassium sources in the soil for the growth and metabolism of crops and soil microorganisms, increasing Agricultural land production (Nishanth and Biswas, 2008; Chang and Yang, 2009; Singh and Reddy, 2011). In addition, it also reduces the use of chemical phosphate fertilizers and potash fertilizers, reduces agricultural production costs and reduces environmental and ecological pollution caused by the extensive use of chemical fertilizers. Therefore, inoculation of suitable and effective potassium phosphate-dissolving microorganisms in various organic wastes to prepare bio-fertilizers rich in soluble phosphorus and soluble potassium can not only reduce the use of chemical phosphate fertilizers and potash fertilizers, but also utilize organic wastes to promote resource reuse. Protecting the environment and increasing production are worth promoting (Nishanth and Biswas, 2008; Chang and Yang, 2009; Martinez et al., 2009; Ogbo, 2010).

Due to the high temperature above 45-80 °C during the production of biological fertilizer. Most of the phosphorus-dissolving or potassium-dissolving microorganisms currently used are medium-temperature bacteria, which can only grow at moderate temperatures, and therefore are not suitable for initial inoculation of biological fertilizers. Because the bio-fertilizer is produced in the initial stage, it will produce a high temperature of 45-80 ° C to inhibit the growth of medium-temperature soluble phosphorus-potassium microorganisms. Therefore, the present invention intends to produce a biological fertilizer containing high-fertilization and high-concentration soluble phosphorus and soluble potassium, inoculated at a medium temperature and a high temperature, and has a high-temperature-soluble phosphorus-potassium microorganism which dissolves a plurality of phosphate ore activities and dissolves various potassium ore activities. Because of its high temperature resistance, it can be applied to the initial inoculation of bio-fertilizers, participate in bio-fertilizer decomposing, exhibit high-temperature phosphorus-dissolving and potassium-dissolving activities, shorten the time of bio-fertilizer decomposing, and it can also be grown at medium temperature for field application. Performance of moderately soluble phosphorus and potassium solubilizing helps plant growth. Therefore, the present invention separates several high temperature resistant potassium phosphate microorganisms from composting and biological fertilizer, and tests the dissolved calcium phosphate, iron phosphate, aluminum phosphate, and hydroxyapatite when cultured at 25 and 50 °C. , mineral phosphorus, feldspar, illite and kaolinite activity. Further selecting appropriate isolates for inoculation of agricultural waste and livestock manure waste for bio-fertilizer preparation, and studying the effects of bio-fertilizer maturity, quality, phosphorus and potassium movement and microbial phase change during the accumulation process for agricultural production increase , resource recovery, environmental protection and sustainable agricultural applications.

The invention adopts composting and biological fertilizer as a separation source of high temperature resistant multifunctional phosphorus-potassium microorganisms. Because of its complex organic waste composition and high temperature characteristics, microorganisms participating in the composting process should have multifunctional enzyme activity and high temperature physiological characteristics. . A total of 22 composting field compost samples and 4 bio-fertilizer samples were collected in different regions, substrates and adjustment materials in Taiwan (eg rice straw, rice husk, wood chips, abandoned space bags, coffee grounds, distiller's grains, Chinese medicine residues, food waste) And the residue of the food processing, and the composting field of the livestock manure (such as pig, chicken and cow dung) in the stacking mode to collect compost samples of different accumulation time. The pig manure composting field was sampled 10 times, mainly prepared by orbital stacking method, with a stacking time of 8-10 weeks and a temperature of 19-78 °C. The chicken manure composting field was sampled 10 times, mainly prepared by orbital stacking method, with a stacking time of 6-9 weeks and a temperature of 19-68 °C. The cow manure composting field was sampled twice and prepared by a strip-type stacking method with a stacking time of 5-13 weeks and a temperature of 31-70 °C. In order to increase the diversity of the isolated microorganisms, the present invention also collects four kinds of biological fertilizer raw materials, semi-finished products, finished products and biological fertilizer inoculated soils provided by the biotechnology industry, and the temperature is between 26-62 °C.

The microbial population was detected and screened by serial dilution of the pour culture medium. The medium temperature microorganisms are colonies grown at 25 ° C, while the heat resistant bacteria are cultured at 50 ° C. The number of phosphorus-dissolving microbial bacteria was detected and screened by using three different nutrients of calcium phosphate as the sole phosphorus source medium. Respectively NBRIP (National Botanical Research Institute's phosphate ) medium, containing per liter _{glucose 10 g, Ca 3 (PO} 4) 2 5 g, MgCl 2 ‧6H 2 O 5 g, MgSO 4 ‧7H 2 O 0.25 g, KCl 0.2 g (NH ₄ ) ₂ SO ₄ 0.1 g and agar 15 g, pH 6.5 ± 0.1. SCP (Sucrose calcium phosphate) medium, containing per liter, _{sucrose 10 g, Ca 3 (PO} 4) 2 5 g, NH 4 NO 3 0.27 g, MgSO 4 ‧7H 2 O 0.1 g, KCl 0.2 g, yeast extract 0.1 g, MnSO ₄ ‧H ₂ O 0.001 g, FeSO ₄ ‧7H ₂ O 0.001 g and agar 15 g, pH 6.5±0.1. And PVK (Pikovskaya's) medium, containing per liter _{glucose 10 g, Ca 3 (PO} 4) 2 5 g, (NH 4) 2 SO 4 0.5 g, NaCl 0.2 g, MgSO 4 ‧7H 2 O 0.1 g, KCl 0.2 g Yeast extract 0.5 g, MnSO ₄ ‧H ₂ O 0.002 g, FeSO ₄ ‧7H ₂ O 0.002 g and agar, 15 g, pH 6.5 ± 0.1.

A total of 977 phosphate-dissolving microorganisms were isolated from the above-mentioned pig, chicken and cow manure compost and biological fertilizer, of which 694 were high temperature resistant microorganisms and 283 were medium temperature microorganisms. It contains 621 high temperature resistant phosphorus solubilizing bacteria, 202 medium temperature soluble phosphorus bacteria, 50 high temperature resistant phosphorus actinomycetes, 40 medium temperature soluble phosphorus actinomycetes, 23 high temperature resistant phosphorus solubilizing fungi and 41 medium temperature soluble phosphorus fungi. . The isolates were cultured at 25 and 50 °C for 5 days using NBRIP, SCP and PVK plate assays respectively. The diameters of colonies (CS) and dissolved phosphorus rings (CZ) were measured, and the CZ/CS ratio was calculated as the activity parameters of calcium phosphate phosphate ( TCPSAI) for screening.

694 high-temperature-soluble phosphorus-dissolving microorganisms can grow at 25 and 50 °C, and further screening colonies at these two temperatures are larger (representing rapid growth), larger dissolved phosphorus rings (representing high phosphorus-dissolving activity), and dissolved phosphoric acid. An isolate with a higher calcium activity parameter (representing a higher phosphorus solubilizing efficiency). A total of 59 strains of high-temperature phosphate-dissolving bacteria, 7 strains of high-temperature resistant actinomycetes and 5 strains of high-temperature phosphate-dissolving fungi were selected, and the calcium phosphate activity parameters were tested at 25 and 50 °C in NBRIP, SCP and PVK. .

In the study of microbial elution minerals, it was found that the mechanisms of phosphorus solubilization and potassium solubilization were similar, all of which were acidification, enzymolysis, organic acid production, and capsule absorption. And polymer substance formation, the phosphorus-dissolving microorganism may also have a mechanism of potassium dissolution. Therefore, the present invention detects the growth of the high-temperature-soluble phosphorus-dissolving microorganism and the potassium-dissolving activity after 5 days of culture at 25 and 50 ° C using the modified Bromfield medium plate assay using potassium ore as the sole potassium source. The modified Bromfield medium with potassium ore as the sole potassium source contains 5 g of glucose, 0.5 g of NaH ₂ PO _{4 ,} 0.5 g of MgSO ₄ ‧7H ₂ O, 1.0 g of (NH ₄ ) ₂ SO ₄ , and 0.2 g of NaCl per liter. Test potassium ore 50 g and agar 20 g, pH 6.5 ± 0.1. The potassium ore powders tested were feldspar (total nitrogen, total phosphorus and total potassium content of 0.0025±0.0008, 0.034±0.004 and 42.2±5.8 g kg ^-1 feldspar, respectively) and illite (total nitrogen, total phosphorus and total potassium). They were 0.0012±0.0003, 0.024±0.006 and 3.2±0.8 g kg ^-1 illite) and kaolinite (total nitrogen, total phosphorus and total potassium were 0.0028±0.0009, 0.023±0.006 and 4.1±0.3 g kg ^-1 kaolinite respectively). ). The modified Bromfield medium with feldspar, illite and kaolinite as the sole potassium source is referred to as feldspar, illite and kaolinite medium, respectively, in the present invention.

The colonies with high temperature-soluble phosphorus-dissolving bacteria were cultured at 25 and 50 °C with colony diameters of 2.0 ± 0.2-13.0 ± 2.3 mm and 2.5 ± 0.0-23.2 ± 1.3 mm, respectively. The colony diameters were 1.9 ± 0.2-12.4 ± 1.2 mm and 2.1 ± 0.2 - 21.6 ± 2.9 mm when cultured at 25 and 50 ° C in illite medium. When the kaolinite medium was cultured at 25 and 50 ° C, the colony diameters were 1.5 ± 0.2-10.6 ± 0.1 mm and 1.6 ± 0.0-14.7 ± 2.6 mm, respectively. The isolates Bacillus lichenifromis A3 BCRC 910522 and B. subtilis H8 BCRC 910523 all formed larger colonies when cultured at 50 ° C, so the two isolates were selected for further study.

The high temperature resistant phosphorus-dissolving microorganisms have potassium-dissolving activity when grown at 25 and 50 ° C, and can be called high temperature resistant phosphorus-potassium microorganisms. The invention screens the high temperature resistant potassium phosphate bacteria B. lichenifromis A3 BCRC 910522 and B. subtilis H8 BCRC 910523 respectively, and has large colonies when cultured at 50 ° C in feldspar, illite and kaolinite medium, respectively, which is known to be high temperature and In the potassium ore environment, it can rapidly grow and express the activity of potassium solubilization. It is suitable for inoculation in compost preparation, improve the soluble potassium concentration of compost and improve the quality. Colonies can also be formed in three potassium ore cultures at 25 ° C. It can be seen that when the compost is applied to the field, potassium can be grown and dissolved in the medium temperature soil for the crops to increase the yield. These two microorganisms have both medium-temperature and high-temperature-soluble phosphorus and potassium-dissolving activities, which can be used in composting and agricultural production, and improve the content of soluble phosphorus and soluble potassium in compost and soil, which is worthy of resource recovery and sustainable agriculture.

Since the livestock manure composting is produced, a high temperature of 60-80 ° C is generated at the initial stage of fermentation, so that the high temperature resistant potassium phosphate microorganism which can grow at this high temperature is a preferable choice for preparing the biological fertilizer. For example, these microorganisms have a variety of enzyme activities at this high temperature, which can accelerate compost maturity, shorten preparation time and reduce cost. For example, when these activities are simultaneously exhibited at medium temperature, the application value is higher, which can be called multifunctional High temperature resistant phosphorus and potassium microorganisms. The high temperature resistant potassium phosphate bacterial isolates B. lichenifromis A3 BCRC 910522 and B. subtilis H8 BCRC 910523 have growth temperatures ranging from 10 to 75 °C. They all have the activity of dissolving a variety of inorganic phosphate ore and inorganic potassium ore. Therefore, the present invention is selected as a patented strain, which can accelerate the conversion of livestock manure compost into biological fertilizer and increase the content of soluble phosphorus and soluble potassium in the fertilizer. Examples of the phosphorus and potassium solubilizing activities, enzyme activities, and biological fertilizers of these microorganisms are shown below to further understand the present invention, but there is no limitation on the attached patent portion.

Example 1: Identification of high temperature resistant multi-functional phosphorus and potassium bacteria

The types of the high temperature resistant multifunctional phosphorus-dissolving potassium bacteria isolate A3 and the isolate H8 are shown in Figs. 1 and 2. Both isolates are bacilli with endospores. Its biochemical characteristics are shown in Table 1. Both isolates can hydrolyze starch and casein, all of which can be grown at 10-75 °C. The results of identification of 16S ribosomal DNA molecules of isolate A3 and isolate H8 are shown in Table 2. The isolate A3 and Bacillus licheniformis ATCC 14580 have a similarity of 99.9%, hence the name Bacillus licheniformis A3. The isolate H8 and Bacillus subtilis strain BJ-1 have a similarity of 100%, hence the name Bacillus subtilis H8. Bacillus licheniformis A3 and Bacillus subtilis H8 were deposited in the Bioresource Conservation and Research Center of the Food Industry Development Research Institute, numbered Bacillus licheniformis A3 BCRC 910522 and Bacillus subtilis H8 BCRC 910523, respectively.

Example 2: High temperature resistant multi-functional phosphorus-dissolving potassium bacteria, calcium phosphate, aluminum phosphate, hydroxyl apatite and phosphate rock activity

The activity of soluble inorganic phosphorus of B. lichenifromis A3 BCRC 910522 and B. subtilis H8 BCRC 910523 is shown in Table 3 and Figure 3-7. Its calcium phosphate activity is measured by NBRIP, PVK and SCP. Aluminum phosphate (AlPO ₄ ), iron phosphate (FePO ₄ ), hydroxyapatite and phosphate rock (Isra Phosphate ore, containing P 285 grams per kilogram, N < 0.2 grams, K 0.9 grams, Na 40 The activity of gram, Ca 13 g, Mg 38 g and C 341 g) were determined by PVK medium with the same amount of aluminum phosphate, iron phosphate, hydroxyapatite and phosphate rock instead of calcium phosphate as the sole phosphorus source (abbreviated as Aluminum phosphate, iron phosphate, hydroxyapatite and phosphate rock media).

As shown in Table 3, both strains were able to grow and express calcium phosphate activity on NBRIP, PVK and SCP at 25 and 50 °C. B. subtilis H8 BCRC 910523 has aluminophosphate activity at 25 and 50 °C. B. licheniformis A3 BCRC 910522 and B. subtilis H8 BCRC 910523 also have hydrogen oxyapatite and mineral phosphorus activity at 25 and 50 °C. The acidification medium and dissolved inorganic phosphorus activity of the two strains were continuously cultured for 10 days at 25 and 50 ° C by liquid detection methods of NBRIP, PVK, SCP, aluminum phosphate, iron phosphate, hydroxyapatite and phosphate rock medium, respectively. Culture medium pH and soluble phosphorus content.

The changes of the acidification medium and calcium phosphate activity of the two strains tested in NBRIP, PVK and SCP liquid medium at 25 and 50 ° C for 10 days are shown in Fig. 3. There was a positive correlation between the activity of the isolate calcium phosphate and the acidified medium. When cultured in NBRIP liquid medium at 25 ° C, the pH and soluble phosphorus content of the uninoculated microbial control group were kept constant. B. licheniformis A3 BCRC 910522 and B. subtilis H8 BCRC 910523 acidification medium and calcium phosphate phosphate activity increased with incubation time, and had the lowest pH and the highest soluble phosphorus content on the 10th day of culture (Fig. 3a and 3b).

There was no significant change in pH and soluble phosphorus content of the uninoculated microbial control group when NBRIP was cultured at 50 °C. The acidification medium and calcium phosphate phosphate inoculated with B. licheniformis A3 BCRC 910522 increased with the culture time, and the lowest pH value and the highest soluble phosphorus content of the medium on the 10th day of culture. There was no significant change in pH and soluble phosphorus content of B. subtilis H8 BCRC 910523 (Figures 3c and 3d).

There was no significant change in the pH and soluble phosphorus content of the uninoculated microbial control medium when cultured in PVK liquid medium at 25 °C. B. subtilis H8 BCRC 910523 had the highest acidification medium activity and calcium phosphate activity on the second day of culture, and then gradually decreased with the culture time. Inoculation of B. licheniformis A3 BCRC 910522 showed the highest acidification medium activity and calcium phosphate activity on the second day of culture, after which the activity of the acidified medium continued to decrease, and the activity of calcium phosphate phosphate appeared the lowest value on the sixth day of culture, followed by a slight increase (Fig. 3e and 3f).

When the PVK was cultured at 50 ° C, the pH and soluble phosphorus content of the uninoculated microbial control medium remained stable without significant change. B. licheniformis A3 BCRC 910522 and B. subtilis H8 BCRC 910523 showed the highest acidification medium activity on the second day of culture, and the calcium phosphate activity also increased rapidly on the second day, then decreased slightly, and then rose to the highest on the 10th day ( Figures 3g and 3h).

The acidified medium and calcium phosphate dissolved activity of the test strain cultured at 25 ° C in SCP liquid medium are shown in Figures 3i and 3j. The pH and soluble phosphorus content of the uninoculated control medium were maintained. Inoculation B. licheniformis A3 BCRC 910522 The lowest pH on day 2 of culture, then gradually increased. The activity of the calcium phosphate phosphate was also highest on the second day of culture, then decreased slightly, and the lowest value on the 10th day of culture, and less than the uninoculated control group, the activity of the dissolved calcium phosphate was lower than that converted into biomass activity. The inoculum B. subtilis H8 BCRC 910523 acidification medium activity reached the highest on the fourth day of culture, then gradually increased with the culture time, and the pH value was highest on the tenth day of culture. The content of soluble phosphorus in the culture process was lower than that in the non-inoculated control group, and the activity of dissolved calcium phosphate was lower than that of conversion to biomass.

There was no significant change in the pH and soluble phosphorus content of the SCP liquid medium without inoculation of microorganisms at 50 ° C for 10 days. Seeded B. licheniformis A3 BCRC 910522 2nd day the culture medium was acidified and the highest activity of soluble calcium, pH of the medium after rising to 10 days. Calcium phosphate activity is lower than the absorption of soluble phosphorus by microorganisms and converted into biomass. The activity of the acidified medium inoculated with B. subtilis H8 BCRC 910523 continued to rise from day 0 to day 6, and then slowly decreased to day 10. Calcium phosphate activity increased slowly from day 0 to day 6, then gradually decreased to day 10 (Figures 3k and 3l).

B. licheniformis A3 BCRC 910522 was cultured in PVK liquid medium at 25 ° C for 2 days with the highest acidification medium activity, cultured for 4 days, and cultured at 50 ° C for 10 days with SCP liquid medium. The highest calcium phosphate activity was observed in PVK liquid medium at 50 ° C for 10 days, and cultured at 25 ° C for 2 days, and cultured in SCP liquid medium at 50 ° C for 10 days. B. subtilis H8 BCRC 910523 was cultured in PVK liquid medium at 50 ° C for 10 days with the highest acidification medium activity, cultured at 25 ° C for 2 days, and cultured in SCP liquid medium at 25 ° C for 10 days with the lowest value. However, it was cultured in PVK liquid medium at 25 ° C for 2 days and had the highest calcium phosphate activity, cultured at 50 ° C for 10 days, and NBRIP liquid medium at 50 ° C for 10 days.

Therefore, the high-temperature-soluble potassium-potassium bacteria have the highest acidification medium and calcium phosphate activity when cultured in PVK medium, followed by NBRIP, and the lowest with SCP. PVK is a high-temperature potassium phosphate-tolerant bacteria showing acidification medium and calcium phosphate dissolution activity, and SCP is poor. When cultured in NBRIP, B. subtilis H8 BCRC 910523 had the highest calcium phosphate activity when cultured at 25 °C.

When the test strain was cultured in a liquid aluminum phosphate medium, the acidification medium and the aluminum phosphate solution activity are shown in Fig. 4. When cultured at 25 ° C, the pH and soluble phosphorus content of the uninoculated microbial control group were kept constant. When inoculatedB .Licheniformis At A3 BCRC 910522, the pH of the medium remained stable without significant change, and there was no significant difference from the uninoculated microbial control group. However, the soluble phosphorus content in the culture process was lower than that of the uninoculated microbial control group, and gradually decreased to the lowest value on the fourth day, and then slowly rose to the 10th day. Four days before the culture, the inoculated microorganisms were subjected to growth and metabolism using the soluble phosphorus contained in the medium to reduce the soluble phosphorus content. After that, the growth of the microorganisms was hindered by the lack of soluble phosphorus, and some of the broken cells released soluble phosphorus, which led to a slow increase in soluble phosphorus content. VaccinationB .Subtilis H8 The acidification medium and the dissolved aluminum phosphate of BCRC 910523 were the highest on the second day of culture, then gradually decreased, and the lowest value was observed for 10 days and was not significantly different from the non-inoculated control group (Fig. 4a and 4b).

When cultured at 50 ° C, the pH and soluble phosphorus content of the uninoculated microbial control group remained stable. When the activity of acidified medium and aluminum phosphate dissolved in B. subtilis H8 BCRC 910523 gradually increased with the culture time, the maximum activity appeared on the 10th day of culture, and the medium had the lowest pH and the highest soluble phosphorus content. When B. licheniformis A3 BCRC 910522 was inoculated with acidification medium and aluminum phosphate dissolving activity, the pH of the medium remained stable and was not significantly different from the control group. The soluble phosphorus content was lower than that of the uninoculated microorganisms, and appeared on the second day. The lowest value, then slowly rose to the 10th day (Figures 4c and 4d). It can be seen that B. licheniformis A3 BCRC 910522 grows with soluble phosphorus in the medium 2 days before culture, but after 2 days, it gradually dies due to low soluble phosphorus content, and cell rupture releases soluble phosphorus, which leads to a slight increase in soluble phosphorus in the medium.

The acidification medium and iron phosphate activity of the test strain in the liquid iron phosphate medium are shown in Fig. 5. There was no significant difference in the pH and soluble phosphorus content of the uninoculated microbial control medium at 10 and 50 °C for 10 days. The content of soluble phosphorus in the inoculated test strains was lower than that in the uninoculated microbial control group, and the lowest value appeared on the first 2-4 days of culture, and then increased slowly. When the test strain was cultured at 50 °C, the minimum soluble phosphorus content was earlier than 25 °C. Because the test strain was high temperature resistant bacteria, it grew faster at 50 °C than 25 °C, resulting in absorption of soluble phosphorus content at 50 °C. °C high.

Figure 6 is a graph showing changes in pH and hydrogen oxyapatite activity of a test strain in a liquid hydroxyapatite medium at 25 and 50 ° C for 10 days. The pH and soluble phosphorus content were maintained constant at 25 and 50 ° C in the uninoculated microbial control group. When B. subtilis H8 BCRC 910523 was inoculated at 25 ° C, the acidification medium and the hydrogen oxyapatite activity were highest on the second day of culture, and then slowly decreased to the lowest on the 10th day. When cultured at 50 ° C, the activity of the inoculum B. subtilis H8 BCRC 910523 acidified medium gradually increased with time, and the highest dissolved hydrogen oxyapatite activity on the 10th day. Inoculation B. licheniformis A3 BCRC 910522 exhibited the highest acidification medium activity on day 2 when cultured at 25 ° C, after which the pH slowly rose to day 10. However, when cultured at 50 ° C, the pH of the medium rapidly decreased from day 0 to day 2, after which no significant difference remained constant until day 10. The hydrogen oxyapatite activity was also highest on day 2 and then decreased slightly.

The acidification medium and phosphate rock ore activity of the two test strains cultured in a liquid phosphate rock medium are shown in Fig. 7. When the non-inoculated control medium was cultured at 25 and 50 ° C, its pH and soluble phosphorus content were kept constant. After incubation at 25 ° C for 10 days, B. licheniformis A3 BCRC 910522 and B. subtilis H8 BCRC 910523 showed the highest acidification medium and phosphate rock ore activity on the second day, the medium had the lowest pH and the highest soluble phosphorus content, then gradually decreased, the 10th The medium has the highest pH and the lowest soluble phosphorus content. When cultured at 50 °C, the activity of acidified medium and phosphate rock ore of B. licheniformis A3 BCRC 910522 increased with the incubation time, and the maximum appeared on the 10th day. The medium had the lowest pH and the highest soluble phosphorus content. The acidification medium and phosphate rock ore activity of B. subtilis H8 BCRC 910523 increased rapidly from day 0 and then slowly rose to maintain stable pH and soluble phosphorus content after day 4.

Therefore, the activity of the high-temperature potassium-dissolving bacteria to dissolve aluminum phosphate, hydroxide apatite and phosphate rock has a positive correlation with the activity of the acidified medium. B. subtilis H8 BCRC 910523 had the highest aluminum phosphate activity on day 2 of culture at 25 °C. B. Licheniformis A3 BCRC 910522 cultured at 25 deg.] C for 2 days showed the highest activity of hydroxyapatite solution, but at 50 ℃, the maximum activity appeared insoluble hydroxyapatite by side for 10 days.

It is cultured in liquid PVK, aluminum phosphate, iron phosphate, hydroxyapatite and phosphate rock medium at 25 °C. B. licheniformis A3 BCRC 910522 exhibits three kinds of soluble inorganic phosphorus activity, with the highest activity of dissolved calcium phosphate, hydrogen oxylate Apatite activity is second, while phosphate rock ore is the least active. B. subtilis H8 BCRC 910523 exhibits four kinds of dissolved inorganic phosphorus activities (calcium phosphate, aluminum phosphate, hydroxyapatite and phosphate rock), with the highest activity of dissolved calcium phosphate, followed by phosphate rock ore activity, and aluminum phosphate soluble activity. lowest. When cultured at 50 °C, B. licheniformis A3 BCRC 910522 has three kinds of soluble inorganic phosphorus activity, the highest activity of dissolved calcium phosphate, followed by the activity of dissolved hydrogen oxyapatite, and the lowest activity of dissolved phosphate ore. B subtilis H8 BCRC 910523 exhibits four kinds of soluble inorganic phosphorus activities (calcium phosphate, aluminum phosphate, hydroxyapatite and phosphate rock) when cultured at 50 °C, with the highest activity of dissolved calcium phosphate and the activity of phosphate rock ore. The aluminum phosphate solution has the lowest activity. Therefore, the two strains of bacteria use calcium phosphate as the best phosphorus source, which can be used to make biological fertilizers and improve the soluble phosphorus content of the strain.

Example 3: Activity of feldspar, illite and kaolinite in high temperature resistant multi-functional phosphorus-potassium bacteria isolates

Since feldspar, illite and kaolinite are potassium minerals rich in natural matter, the potassium dissolution activity of B. licheniformis A3 BCRC 910522 and B. subtilis H8 BCRC 910523 is to detect the soluble potassium of feldspar, illite and kaolinite. The ionic activity is representative, and its acidified medium activity is simultaneously measured (Han and Lee, 2005; Badr et al, 2006; Uroz et al, 2009). The two microorganisms were cultured in liquid feldspar, illite and kaolinite at 25 and 50 ° C for 20 days, and the pH of the medium and the atomic absorption spectrometry and ICP were measured by an automated pH meter. The results are shown in Fig. 8. Both strains were able to grow and express acidification medium and feldspar, illite and kaolinite activity in feldspar, illite and kaolinite medium, and exhibited higher potassium dissolution activity at 25 °C than cultured at 50 °C. The feldspar is the most suitable source of potassium, followed by the illy stone, and the kaolinite is poor. In the study of microbial elution of minerals, the mechanism of potassium dissolution by microorganisms has acidification, enzyme decomposition, organic acid secretion, membrane adsorption and polymer formation. Therefore, the two test strains of the present invention may utilize two or more mechanisms for potassium ore dissolution to release soluble potassium.

The pH and soluble potassium content of the uninoculated microbial control medium were maintained at 25 and 50 ° C, and there was no significant change (Fig. 8). The pH and soluble potassium content of the non-microbial feldspar medium cultured at 25 and 50 ° C for 20 days were between 6.42 ± 0.10 - 6.52 ± 0.07 and 0.13 ± 0.02 - 0.14 ± 0.02 μg ml ^-1 . The illite medium not inoculated with microorganisms was between 6.48 ± 0.02 - 6.51 ± 0.01 and 0.09 ± 0.00 - 0.10 ± 0.01 μg ml ^-1 . The kaolinite medium not inoculated with microorganisms is between 6.49 ± 0.00 - 6.51 ± 0.01 and 0.11 ± 0.01 - 0.12 ± 0.01 μg ml ^-1 . The activity of the acidified medium in the two strains tested by liquid feldspar, illite and kaolinite culture at 25 and 50 °C had the maximum on the fourth day of culture, and then decreased with the culture time, due to the use of carbon sources by microorganisms. Later, it metabolizes nitrogen-containing substances such as proteins. The acidification medium activity was higher at 50 ° C than at 25 ° C 10 days before the culture, because the culture strain of the test strain culture at 50 ° C was faster than 25 ° C. However, after 10 days of culture, the acidification medium activity at 25 ° C was higher than that at 50 ° C, and the soluble potassium content continued to rise in a cumulative manner until the maximum appeared on the 20th day.

B. licheniformis A3 BCRC 910522 and B. subtilis H8 BCRC 910523 have the highest acidification medium and potassium lysate activity in liquid feldspar, illite and kaolinite medium assays at 25 and 50 °C for 20 days, so further biofertilizers Inoculated strains that produce and enhance their soluble potassium content.

Example 4: Starch, chitin, cellulose, keratin, lipid, protein, pectin and polyxylose decomposing enzyme activities of high temperature resistant multi-functional phosphorus-potassium bacteria isolates

Because of the wide variety of organic wastes and the complex composition of biological fertilizers, including agriculture, animal husbandry, poultry manure, slaughterhouses, fruit and vegetable markets, food processing industry, aquaculture fisheries and urban kitchen waste, it is used in bio-fertilizer production. The inoculated strains need to have a variety of enzyme activities in order to grow and metabolize organic matter, enhance composting activity, reduce production costs and improve compost quality during the production process. The activities of starch, chitin, cellulose, hemicellulose, keratin, lipid, protein, pectin and polyxylase in compost are related to compost maturity, quality and microbial population. Therefore, the B. licheniformis A3 BCRC 910522 and B. subtilis H8 BCRC 910523 test strains of the present invention were cultured at 25 and 50 ° C for detection of starch, chitin, cellulose, lipid, and pectin by plate and liquid assay, respectively. , Xylose, keratin and proteolytic activity, the results are shown in Figures 9 and 10. Amyloid, chitin, cellulose, lipid, pectin, poly-xylose and proteolytic enzyme activities were respectively soluble starch-yeast extract medium (yeast extract 10 g, soluble starch 10 g and agar 15 g per liter) , pH 7.0 ± 0.1), chitin decomposing enzyme assay medium (colloidal chitin 10 g per liter, urea 0.3 g, (NH ₄ ) ₂ SO ₄ 1.4 g, KH ₂ PO ₄ 2 g, CaCl ₂ ‧6H ₂ O 0.4 g, MgSO ₄ ‧7H ₂ O 0.3 g, FeSO ₄ ‧7H ₂ O 0.005 g, ZnSO ₄ ‧7H ₂ O 0.014 g, MnSO ₄ ‧4H ₂ O 0.016 g, CoCl ₂ ‧6H ₂ O 0.002 g and agar 15 g, pH 7.0 ± 0.1), Mandels-Reese medium (10 g per liter of carboxymethylcellulose (CMC), 1 g of peptone, 0.3 g of ureaa, 1.4 g of (NH ₄ ) ₂ SO ₄ , ₂ g of KH ₂ PO ₄ 2 g, CaCl ₂ ‧6H ₂ O 0.4 g, MgSO ₄ ‧7H ₂ O 0.3 g, FeSO ₄ ‧7H ₂ O 0.005 g, ZnSO ₄ ‧7H ₂ O 0.014 g, MnSO ₄ ‧4H ₂ O 0.016 g, CoCl ₂ ‧6H ₂ O 0.002 g and agar 15 g, pH 7.0 ± 0.1), butyrate glyceride medium (peptone from meat 2.5 g per liter, peptone from casein 2.5 g, yeast extract 3 g, tributyrin 10 ml and agar 15 g, pH 7.0±0.1), pectin decomposing enzyme assay medium (containing 10 g of pectin, 0.3 g of urea, 3.5 g of (NH ₄ ) ₂ SO ₄ , KH ₂ PO ₄ 2 g, CaCl ₂ ‧6H ₂ O 0.4 g per liter, MgSO ₄ ‧7H ₂ O 0.3 g, FeSO ₄ ‧7H ₂ O 0.005 g, ZnSO ₄ ‧7H ₂ O 0.014 g, MnSO ₄ ‧4H ₂ O 0.016 g, CoCl ₂ ‧6H ₂ O 0.002 g and agar 15 g, pH 7.0±0.1), polyxylase enzyme assay medium (10 g per liter oat xylan, urea 0.3 g, (NH ₄ ) ₂ SO ₄ 1.4 g, KH ₂ PO ₄ 2 g, CaCl ₂ ‧6H ₂ O 0.4 g, MgSO ₄ ‧7H ₂ O 0.3 g, FeSO ₄ ‧7H ₂ O 0.005 g, ZnSO ₄ ‧7H ₂ O 0.014 g, MnSO ₄ ‧4H ₂ O 0.016 g, CoCl ₂ ‧6H ₂ O 0.002 g and agar 15 g , pH 7.0 ± 0.1) and skim milk powder medium (skim milk 5 g per liter, glucose 0.005 g, nutrientbroth 1 g, KH ₂ PO ₄ 0.5 and agar 15 g, pH 7.0 ± 0.1); keratinolytic activity Feather decomposing medium (per liter of chicken feather waste 10 g, NaCl 0.5 g, K ₂ HPO ₄ 0.3 g, KH ₂ PO ₄ 0.4 g and agar 20 g, pH 7.0 ± 0.1) and keratinase production Medium (chicken feather waste 10 per liter) g, starch 3 g, NH ₄ Cl 5 g, K ₂ HPO ₄ 4 g, Na ₂ SO ₄ 1 g and agar 20 g, pH 7.5 ± 0.1).

The enzyme activity of the test strains cultured at 25 and 50 °C by liquid assay was positively correlated with the enzyme activity parameters of the plate assay. B. licheniformis A3 BCRC 910522 and B. subtilis H8 BCRC 910523 can express 8 enzyme activities at 25 and 50 °C, including amylose, chitin, cellulose, keratin, lipid, protein, pectin and poly Xylose decomposing enzyme activity. No enzyme activity was detected in the uninoculated microbial control group, and the microbial enzyme activity showed a pyramidal change, and the maximum value appeared during the culture. The maximum enzyme activity of the test strains which were continuously cultured for 7 days at 50 °C was higher than 25 °C, because the growth metabolism rate at 50 °C was higher than that at 25 °C. The amylose, lipid, pectin, polyxylose, protein and keratinolytic enzyme activities all reached a maximum after 3 days of culture at 25 and 50 °C. The chitinolytic enzyme was the highest on the 5th day of culture at 25 ° C, but the 4th day when cultured at 50 ° C. The cellulolytic enzyme activity was highest on the fourth day of culture at 25 ° C, but on day 3 when cultured at 50 ° C.

B. licheniformis A3 BCRC 910522 exhibits four highest enzyme activities such as starch, cellulose, lipid and protein degradation when cultured at 25 and 50 °C. B. subtilis H8 BCRC 910523 exhibit the highest enzyme activity four kinds of chitin, pectin, poly-xylose and keratin decomposition (FIGS. 9 and 10) and to the culture 25 50 ℃. Therefore, the two tested strains can be used as inoculated strains for accelerating composting.

Example 5: High temperature resistant multi-functional phosphorus-potassium bacteria isolate for bio-fertilizer production

B. licheniformis A3 BCRC 910522 and B. subtilis H8 BCRC 910523 were inoculated in small fermenters (30-31 kg) to produce biofertilizers to assess their effects on biofertilage maturity, quality, soluble phosphorus and potassium content and microbial populations. The odor, color, temperature, pH, moisture content, organic matter content, total carbon content, total nitrogen content, C/N ratio and germination rate of bio-fertilizer were used as indicators of maturity, and the effects of inoculated microorganisms on composting speed and quality were evaluated. (Figures 11 and 12).

The raw materials for biological fertilizers are 10% chicken manure, 30% wood chips and waste rice bran, and 60% sludge in food processing industry. The raw material is grayish brown, sticky, the particle diameter is between 1-30 mm, and the mixed food is fermented and smelly [determined as grade 2 (2-) malodor], temperature is 34.3±0.9 °C, pH is 6.4±0.3, moisture content is 45.3. ±1.8%, organic matter content 71.4±3.3%, total organic carbon content 61.1±0.7%, total nitrogen content 1.35±0.05%, and germination rate 73.3±3.1%. When the gram of dry weight of the raw material was inoculated with the test strain 1 × 10 ⁵ CFU, and the uninoculated microorganism compost was used as the control group. The shovel is evenly mixed so that the raw material particles are less than 40 mm in diameter, the moisture content is adjusted to be about 65%, and the carbon to nitrogen ratio is between 20 and 25. The mixed raw materials were placed on the three-seat 35-liter plastic fermenter (the upper inner ring diameter was 37 cm, the lower inner ring diameter was 28 cm, and the height was 45 cm). The compost height was about 40 cm, and the weight of each fermenter was Between 31-32 kg. During the accumulation process, it was turned over once every 3-4 days. The upper, middle and lower layers of bio-fertilizer were mixed with a shovel, and the large particles were smashed with a small shovel and piled up for 56 days to observe the color and smell. Every 7 days, the temperature of the biological fertilizer center is about 100-120 cm from the surface, and the temperature is atmospheric temperature. The sample is about 10-20 cm deep from the surface of the bio-fertilizer, and the bio-fertilizer maturity index and microbial population analysis are performed. The temperature of the biofertilizer increased rapidly after the accumulation of the temperature, reached the highest on the 7th day, and decreased slightly by 2.19-3.75 °C on the 14th day, and then decreased rapidly thereafter, and there was no significant difference with the atmospheric temperature on the 56th day (Fig. 11a). The three different microbial treatment bio-fertilizers had higher microbial activity 14 days before the initial stage of accumulation, and then the composting slowed down rapidly, and reached a stable and maturing state after 56 days of accumulation. The biofertilizer inoculated with B. subtilis H8 BCRC 910523 was the highest temperature on the 7th day of accumulation (53.6±0.5°C), followed by the inoculation B. licheniformis A3 BCRC 910522, and the lowest in the non-inoculated microbial control group (46.5±1.6°C) .

The pH, total nitrogen content and germination rate increased rapidly during the 0-1-4 days of accumulation, and then increased slowly with the accumulation time due to decomposition of organic matter and degradation of organic acids or toxic substances to plants. B. subtilis H8 BCRC 910523 had higher pH, total nitrogen content and germination rate during the accumulation process, followed by B. licheniformis A3 BCRC 910522, while the uninoculated microbial control group was lower. The bio-fertilizer inoculated with B. subtilis H8 BCRC 910523 had lower moisture content, organic matter content, total organic carbon content and C/N ratio during the 56-day accumulation process, and was inoculated with B. licheniformis A3 BCRC 910522, but not inoculated. The microbial control group was higher.

The color and odor changes of the composting of the two groups of biofertilizers and non-inoculated microorganisms during the accumulation process are shown in Table 4. At the beginning of the accumulation, the biological fertilizer and the uninoculated microbial composting materials were all gray-brown, mixed with ammonia gas, chicken feces and odor, and food spoiled sour taste, which was judged to be Grade 2 malodor (2-). After the 21st day of accumulation, the color began to turn into dark grayish brown. On the 42nd day of accumulation, the bio-fertilizers inoculated with microorganisms were all turned into gray, and converted to dark gray on the 49th day of accumulation. However, the compost of the uninoculated microbial control group changed from dark gray to gray on the 49th day of accumulation, and dark gray appeared on the 56th day of accumulation. After the three kinds of treated biological fertilizers were accumulated for 7 days, the odor was mixed with the odor of microbial fermentation and food spoilage, and it was judged to be a grade 3 malodor, and then the malodor odor gradually disappeared. Inoculation of B. subtilis H8 BCRC 910523 bio-fertilizer in the accumulation of the 42nd weather smell is the smell of aromatic soil, which makes people feel happy, and is judged as a grade 4 aroma (4+). The bio-fertilizer inoculated with B. licheniformis A3 BCRC 910522 showed a grade 4 aroma on the 49th day of accumulation. The uninoculated microbial control group composted on the 56th day of accumulation and a grade 4 aroma appeared. Therefore, inoculation of microorganisms in the composting material can reduce malodor and promote decomposing during the accumulation process, deepen the color and reduce the generation of odor.

In the preparation process of bio-fertilizer, there is higher microbial activity in the first 14 days of the initial stage of accumulation, and inoculation of high-temperature-soluble phosphorus-potassium microorganisms can indeed raise the pH, shorten the preparation time, deepen the color and reduce the malodor. The biofertilizer inoculated with B. subtilis H8 BCRC 910523 had the highest microbial activity, followed by B. licheniformis A3 BCRC 910522.

Example 6: Effect of Inoculation of High Temperature and Multi-functional Phosphorus-Phosphorus-Bacterial Isolates on the Content of Soluble Phosphorus and Potassium

The total phosphorus, total potassium, soluble phosphorus and soluble potassium content, phosphorus solubilization efficiency and potassium dissolution efficiency during the production of biological fertilizer are shown in Fig. 12. The total phosphorus, total potassium and soluble potassium content gradually increased with the accumulation time, and the highest value appeared on the 56th day of accumulation. The bio-fertilizer inoculated with B. subtilis H8 BCRC 910523 had higher total phosphorus, total potassium and soluble potassium content during the accumulation period, and B. licheniformis A3 BCRC 910522 was inoculated, while the uninoculated microbial control group had lower compost.

The dissolved potassium efficiency is the ratio of soluble potassium content to total potassium content, which indicates that the movement of potassium in the process of bio-fertilizer accumulation is affected by the activity of microbial dissolved potassium and the bio-fertilization of bio-fertilizer. The potassium solubilization efficiency of the three different treated biological fertilizers decreased with the accumulation time. The biofertilizer inoculated with B. subtilis H8 BCRC 910523 accumulated the highest potassium dissolution efficiency (12.1±0.1%) on the 56th day, and the B. licheniformis A3 BCRC 910522 was inoculated, and the uninoculated microorganisms were significantly lower. Thus B. subtilis H8 BCRC 910523 is the preferred strain for the production of biological fertilizers to increase the soluble potassium content, B. licheniformis A3 BCRC 910522.

The changes in soluble phosphorus content and phosphorus solubilization efficiency were similar. After the 7th day of accumulation, all treated biological fertilizers continued to decline, which was the lowest value on the 56th day of accumulation (Fig. 12c and 12e). As the pH gradually increases, the phosphorus solubilizing activity of the phosphorus-dissolving microorganism decreases. On the 7th day of accumulation, the soluble phosphorus content of the biofertilizer inoculated with B. licheniformis A3 BCRC 910522 and B. subtilis H8 BCRC 910523 increased to a maximum. It can be seen that B. licheniformis A3 BCRC 910522 and B. subtilis H8 BCRC 910523 can effectively exert their phosphorus solubilizing activity in the initial stage of biological fertilizer production, and provide soluble phosphorus for microbial metabolism. Phosphorus solubilization efficiency is the ratio of soluble phosphorus content to total phosphorus content, which represents the release efficiency of insoluble phosphorus due to the physical and chemical action of microbial phosphorus solubilizing activity and composting in the composting environment. Only the inoculated B. licheniformis A3 BCRC 910522 bio-fertilizer rose to the maximum on the 7th day of accumulation, while the composting of the bio-fertilizer inoculated with B. subtilis H8 BCRC 910523 and the non-inoculated microorganisms was less obvious. It can be seen that inoculation B. licheniformis A3 BCRC 910522 can show better phosphorus solubilizing activity in the early stage of converting agricultural and food processing waste into biological fertilizer preparation, and B. subtilis H8 BCRC 910523.

The bio-fertilizer inoculated with B. licheniformis A3 BCRC 910522 had significantly higher soluble phosphorus content and phosphorus solubilization efficiency during the accumulation process, and was inoculated with B. subtilis H8 BCRC 910523, but the unfertilized microbial control group had lower compost. After 56 days of accumulation, the biological fertilizer inoculated with B. licheniformis A3 BCRC 910522 had the highest soluble phosphorus content and phosphorus solubilization efficiency (1.6±0.0 g kg ^-1 and 4.7±0.1%, respectively), and inoculated with B. subtilis H8 BCRC 910523 However, the compost of the uninoculated microbial control group was significantly lower. It can be seen that B. licheniformis A3 BCRC 910522 is the best inoculated strain for improving the soluble phosphorus content of biological fertilizer, and B. subtilis H8 BCRC 910523 is the second.

Example 7: Effect of high temperature resistant multi-functional phosphorus-potassium bacteria isolates on phosphorus and potassium microbial populations of biological fertilizers

The microbial population changes in the three differently treated biological fertilizers are shown in Figure 13. The microbial population in the biofertilizer was highest at 7-14 days after accumulation, and then decreased with accumulation time. The population of phosphate-dissolving bacteria is significantly higher than the number of potassium-dissolving bacteria. Therefore, it is feasible to add potassium-soluble bacteria suitable for high temperature to enhance the potassium-dissolving bacteria population and soluble potassium content. The bio-fertilizer inoculated with B. subtilis H8 BCRC 910523 had significantly higher total number of bacteria in the high temperature resistant microbial population, the number of high temperature resistant phosphate bacteria and the number of high temperature resistant potassium bacteria in the accumulation process. B. licheniformis A3 BCRC 910522 was inoculated, while compost was significantly lower in the uninoculated microbial control group. In addition, after inoculation of B. subtilis H8 BCRC 910523 bio-fertilizer for 56 days, there were also higher number of medium-temperature soluble phosphate bacteria and medium-temperature potassium-dissolving bacteria. B. licheniformis A3 BCRC 910522 was inoculated, and the uninoculated microbial control group was significantly lower. Therefore, inoculation of high-temperature-soluble phosphorus-potassium bacteria can increase the total number of mild and high-temperature resistant bacteria in the biological fertilizer, the number of medium-temperature and high-temperature-soluble phosphorus-dissolving bacteria, and the number of medium-temperature and high-temperature-soluble potassium-dissolving bacteria. B. subtilis H8 BCRC 910523 and B. licheniformis A3 BCRC 910522 were selected as preferred inoculum strains.

The proportion of the total temperature-to-temperature ratio of the medium-temperature soluble phosphate bacteria and the medium-temperature potassium-dissolving bacteria in the medium temperature and the ratio of the high-temperature-soluble phosphate-dissolving bacteria and the high-temperature-soluble potassium-dissolving bacteria in the high-temperature-tolerant bacteria are shown in Fig. 14. The proportion of total dissolved bacteria in the middle temperature of the three kinds of different treatment biological fertilizers and the ratio of the total number of bacteria in the middle temperature and the high temperature-resistant potassium-dissolving bacteria in the high temperature-resistant bacteria were 14 days before the initial accumulation. It continued to decline, and the lowest value appeared on the 14th day of accumulation, because the non-phosphorus-potassium microorganisms multiplied at the beginning of the accumulation and the highest microbial population on the 14th day. Then the proportion slowly rose to the 56th day. Because soluble phosphorus is one of the microbial growth factors, solubilized bacteria have higher ability to resist hunger than non-phosphorus microorganisms.

Inoculation B. licheniformis A3 BCRC 910522 biological fertilizer in the accumulation process have the highest proportion of medium-temperature dissolved phosphorus bacteria in the total temperature of the total number of bacteria and the proportion of high-temperature resistant bacteria in the high temperature-tolerant bacteria, inoculated B. subtilis H8 BCRC 910,523 times However, the uninoculated microbial control group was significantly lower. On the 56th day of accumulation, the bio-fertilizer inoculated with B. licheniformis A3 BCRC 910522 has the highest ratio of the highest temperature-dissolved phosphorus bacteria and the medium-temperature soluble phosphate bacteria to the total temperature in the middle temperature and the high-temperature-soluble phosphate-dissolving bacteria and high-temperature-soluble phosphate-dissolving bacteria. The proportion of total bacteria in high temperature was inoculated with B. subtilis H8 BCRC 910523, and the uninoculated microbial control group was significantly lower. Inoculation of B. subtilis H8 BCRC 910523 bio-fertilizer during the accumulation process, the highest proportion of medium-temperature potassium-dissolving bacteria in the middle temperature and the proportion of high-temperature resistant potassium bacteria in the high temperature-tolerant bacteria, inoculation B. subtilis A3 BCRC 910522 times However, the uninoculated microbial control group was significantly lower. Therefore, inoculation of high-temperature-soluble phosphorus-potassium bacteria can increase the proportion of warm-dissolved phosphorus bacteria and medium-temperature dissolved potassium bacteria in the total fertilizer to the moderate-temperature bacteria group, and the high temperature-resistant phosphate-dissolving bacteria and the high-temperature-soluble potassium-soluble bacteria group in the total heat-tolerant bacteria. Ethnicity ratio.

The bio-fertilizer inoculated with B. licheniformis A3 BCRC 910522 has the highest soluble phosphorus content, phosphorus solubilizing efficiency, the proportion of medium-temperature soluble phosphorus bacteria in the middle temperature, and the proportion of high-temperature resistant phosphorus-dissolving bacteria in the high temperature-tolerant bacteria. . The bio-fertilizer inoculated with B. subtilis H8 BCRC 910523 has the highest soluble potassium content, potassium dissolution efficiency, the ratio of medium-temperature potassium-dissolving bacteria to medium-temperature total bacteria and the ratio of high temperature-resistant potassium bacteria to high temperature-tolerant bacteria. . The uninoculated microbial control group was significantly lower.

The invention successfully separates two strains of high-temperature-soluble phosphorus-potassium bacteria isolates from livestock manure compost, and has the activities of dissolving calcium phosphate, aluminum phosphate, hydroxide apatite and mineral phosphorus and dissolving potassium dissolution of feldspar, illite and kaolinite. . These isolates were cultured at 25 and 50 ° C. B. licheniformis A3 BCRC 910522 had the highest dissolved hydrogen oxyapatite activity and sub-high dissolved albite, illite and kaolinite activity and B. subtilis H8 BCRC 910523 Phosphorus solubilizing activity and activity of the highest dissolved feldspar, illite and kaolinite were selected as inoculated strains prepared by biological fertilizer. Inoculation of the two strains in the agricultural and food processing industry waste bio-fertilizer can accelerate its maturity, increase the content of soluble phosphorus and potassium to improve the quality, and improve the growth of medium-temperature and high-temperature-soluble phosphate-dissolving bacteria and potassium-dissolving bacteria.

The rate of bio-fertilizer decomposing was positively correlated with the total number of bacteria, phosphorus-dissolving bacteria and potassium-dissolving bacteria groups, but the ratio of soluble phosphorus and soluble potassium to the ratio of the number of dissolved phosphorus bacteria to the total number of bacteria and the proportion of potassium-dissolving bacteria to the total number of bacteria. There is a positive correlation. Inoculation B. licheniformis A3 BCRC 910522 has the highest soluble phosphorus content, the efficiency of phosphorus solubilization and the ratio of the number of phosphate solubilizing bacteria to the total number of bacteria in the biological fertilizer. Inoculation of B. subtilis H8 BCRC 910523 showed the highest composting rate, soluble potassium content, potassium solubilization efficiency, potassium-dissolving bacteria number and potassium-dissolving bacteria number in total bacterial count. Mixed inoculation of B. licheniformis A3 BCRC 910522 and B. subtilis H8 BCRC 910523 and other high-temperature-soluble phosphorus-potassium microorganisms in agriculture, kitchen waste, fruit and vegetable market, food processing and livestock manure waste, can prepare multi-functional biological fertilizer, which can not only improve Its soluble phosphorus and soluble potassium content can accelerate the bio-fertilizer decomposing, improve the quality of bio-fertilizer and increase the proportion of medium-temperature and high-temperature resistant phosphorus and potassium-dissolving microbial populations and their ratios to medium-temperature and high-temperature resistant bacteria. Deep application of biological fertilizer production, resource recovery, agricultural production and sustainable agricultural management.

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Figure 1. Isolated strain B. licheniformis A3 BCRC 910522 strain under microscope.

Figure 2. Isolated strain B. subtilis H8 BCRC 910523 strain under microscope.

Figure 3. Calcium phosphate activity of the test strain. (●) uninoculated control group, (▼) B. licheniformis A3 BCRC 910522, (Δ) B. subtilis H8 BCRC 910523. The data is expressed as an average, and the superscript is the standard deviation (n 3).

Figure 4. Activity of the solution-dissolved aluminum phosphate. (●) unvaccinated control group, (▼) B. licheniformis A3 BCRC 910522, (Δ) B. subtilis H8 BCRC 910523. The data is expressed as an average, and the superscript is the standard deviation (n 3).

Figure 5. Activity of iron phosphate dissolved in the test strain. (●) uninoculated control group, (▼) B. licheniformis A3 BCRC 910522, (Δ) B. subtilis H8 BCRC 910523. The data is expressed as an average, and the superscript is the standard deviation (n 3).

Figure 6. Hydroxyapatite activity of the test strain. (●) uninoculated control group, (▼) B. licheniformis A3 BCRC 910522, (Δ) B. subtilis H8 BCRC 910523. The data is expressed as an average, and the superscript is the standard deviation (n 3).

Figure 7. Phosphate ore activity of the test strain. (●) uninoculated control group, (▼) B. licheniformis A3 BCRC 910522, (Δ) B subtilis H8 BCRC 910523. The data is expressed as an average, and the superscript is the standard deviation (n 3).

Figure 8. Potassium ore activity of the test strain. (●) uninoculated control group, (▼) B. licheniformis A3 BCRC 910522, (Δ) B. subtilis H8 BCRC 910523. The data is expressed as an average, and the superscript is the standard deviation (n 3).

Figure 9. The test strain decomposes carbon source enzyme activity. (●) uninoculated control group, (▼) B. licheniformis A3 BCRC 910522, (Δ) B. subtilis H8 BCRC 910523. The data is expressed as an average, and the superscript is the standard deviation (n 3).

Figure 10. The test strain decomposes the activity of nitrogen-derived enzymes. (●) uninoculated control group, (▼) B. licheniformis A3 BCRC 910522, (Δ) B. subtilis H8 BCRC 910523. The data is expressed as an average, and the superscript is the standard deviation (n 3).

Figure 11. Changes in the nature of the production of small amounts of bio-fertilizer. (●) uninoculated control group, (▼) inoculated B. licheniformis A3 BCRC 910522, (Δ) inoculated B. subtilis H8 BCRC 910523, (█) atmospheric temperature. The data is expressed as an average, and the superscript is the standard deviation (n 3).

Figure 12. Phosphorus and potassium movement of a small amount of bio-fertilizer. (●) uninoculated control group, (▼) inoculated B. licheniformis A3 BCRC 910522, (Δ) inoculated B. subtilis H8 BCRC 910523. The data is expressed as an average, and the superscript is the standard deviation (n 3).

Figure 13. Changes in microbial populations of small amounts of biofertilizers. (●) uninoculated control group, (▼) inoculated B. licheniformis A3 BCRC 910522, (Δ) inoculated B. subtilis H8 BCRC 910523. The data is expressed as an average, and the superscript is the standard deviation (n 3).

Figure 14. Proportion of microbial populations of phosphorus and potassium microbes in a small amount of bio-fertilizer. (●) uninoculated control group, (▼) inoculated B. licheniformis A3 BCRC 910522, (Δ) inoculated B. subtilis H8 BCRC 910523. The data is expressed as an average, and the superscript is the standard deviation (n 3).

Claims (4)

A method for producing multifunctional biological fertilizer is inoculated with high-temperature resistant potassium-dissolving bacteria Bacillus lichenifromis A3 BCRC 910522 or Bacillus subtilis H8 BCRC 910523, which can be characterized by converting raw materials such as agricultural waste and livestock manure waste into biological fertilizer. . As described in claim 1, the high temperature resistant potassium phosphate bacteria have dissolved calcium phosphate, iron phosphate, aluminum phosphate, hydroxyapatide, mineral phosphorus, feldspar, illite and kaolinite. Ability to grow at 15-75 °C. As described in the first paragraph of the patent application, the high temperature resistant potassium phosphate bacteria have amylolytic enzyme activity, cellulolytic enzyme activity, chitinolytic activity, pectin decomposing enzyme activity, proteolytic activity, lipid It decomposes enzyme activity and keratinase activity and can grow at 15-75 °C. For example, in the method described in claim 1, the inoculation of high temperature resistant potassium phosphate bacteria can shorten the time of biological fertilizer decomposing, and increase the total nitrogen content, ash content, soluble phosphorus content, soluble potassium content and seed germination rate of the produced biological fertilizer. Reduce the total organic carbon content, carbon to nitrogen ratio and crude fat content, and improve the quality of biological fertilizers.