CN104819951A - Soil degrading bacteria and experimental method of combinations of soil degrading bacteria for degrading humic acid (HA) in farmland - Google Patents

Abstract

The invention discloses soil degrading bacteria and an experimental method of combinations of the soil degrading bacteria for degrading humic acid (HA) in a farmland. Three soil HA degrading bacteria are selected, and the influences of three bacteria and different combinations of the three bacteria to the degrading degree of HA in the farmland and structural property are researched. The total colony count is T13>T3>T123>T23>T1>T2>T12 after HA is degraded for 14 days, the colony counts of T1 and T2 respectively account for 53.95% and 26.98% of the colony count of T3, the first bacterium has relatively strong capacity for degrading an aryl carbon structure after being treated, the humification degree of an HA culture medium is lower than that of a non-degraded HA culture medium, the structure tends to be simple, and the second bacterium is relatively low in capacity. The quantities of all treated fats and nitrogen-containing substances are respectively increased by 2.33-3.23 times and 1.48-1.74 times as comparison with the quantities of untreated fats and nitrogen-containing substances, thereby further proving the degrading capacity and tendency of the combination of the first bacterium and the third bacterium to complex organic carbon.

Description

Soil degrading bacteria and combination thereof are to the experimental technique of arable land humic acid degraded

Technical field

The present invention relates to the experimental technique of a kind of humic acids from different soils degraded feature, specifically, the experimental technique relating to a kind of soil degrading bacteria and combine the degraded of arable land humic acid.

Background technology

Humic acid (humic acid, HA) is that soil humic acid middle-molecular-weihydroxyethyl is medium, and structure is more complicated, the component that chemistry and biological property are comparatively enlivened.HA and soil microorganism in close relations, C, N in fatty C contained in its structure, Small molecular carbohydrates, aromatic rings side chain all can serve as C, N source for microbial metabolism.Be separated qualification aspect about degradation bacteria mainly concentrates on HA degradation bacteria with the research of HA mutual relationship in the past.This research is separated 5 strain HA degradation bacteria from five kinds of vegetation patterns (farmland, Chinese pine, locust tree, sea-buckthorn, mixed forest) soil, choose B.licheniformis, M.resistens, S.maltophilia 3 strain degradation bacteria further, inquire into this 3 strain bacterium and the palliating degradation degree of combination to arable land HA thereof.

Summary of the invention

The object of the invention is to the defect overcoming the existence of above-mentioned technology, there is provided a kind of soil degrading bacteria and combination thereof to the experimental technique of arable land humic acid degraded, first, using arable soil HA as unique C, N source of nutrient culture media, and research used medium in the past often selects one matter to supplement C source (glucose) and N source (yeast extract powder, ammonium tartrate, NH ₄nO ₃deng).The second, single bacterium colony and various combination process are carried out to 3 strain Soil HA degradation bacteria, the palliating degradation degree of degradation bacteria on C in HA and the impact of architectural feature in conjunction with infrared spectroscopic study.To fixedly providing foundation for architectural feature change and organic carbon in the HA degradation process of arable land.

Its concrete technical scheme is:

Soil degrading bacteria and combination thereof, to an experimental technique for arable land humic acid degraded, comprise the steps:

(1) respiratory capacity carbon measures: combined by 90mm double dish, and junction uses Arabic gum sealing, and outer tire layer sealed membrane is to ensure impermeability, and HA solid medium sticks to top, with 1.0molL ^-1naOH 10.0mL collect degradation bacteria breathe the CO discharged ₂, every 24h 0.5molL ^-1hCl titration once, the concrete concentration Na of HCl ₂b ₄o ₇10H ₂o demarcates, and cultivating 14d in 28 DEG C, is culture media supplemented 3min filtrated air in titration process, the CO lost in this time ₂measure the CO by 30min ₂uptake converts;

(2) typical case's repetition is chosen in cultivation after terminating, and is chosen by all bacterium colonies, uses NaOH and Na afterwards ₄p ₂o ₇mixing digestion agent carries out HA second extraction, NaOH and Na ₄p ₂o ₇concentration is 0.1molL ^-1first 40mL digestion agent is used slowly to rinse HA solid medium, flushing limit, limit is with 0.45 μm of filtering with microporous membrane, this process lasts 4h, use 100mL mixing digestion agent to divide afterwards and soak HA solid medium 5 times, during the 4th, nutrient culture media is transparent, and now lixiviate work completes substantially, soak time is 24h, and all extracting solutions are all transferred to 150mL volumetric flask for measuring infrared spectrum.

Further, respiratory capacity carbon refers to the C amount that degradation bacteria is consumed by respiration.Calculate according to the following formula:

RC＝(2V ₁-V ₂-V ₀)W×6

In formula: V ₁, V ₂, V ₀represent blank, 3min CO respectively ₂loss amount, process titration consume HCl volume mL; W is HCl concentration molL ^-1; 6 is conversion coefficient;

Microbes biomass carbon refers to the C amount of degradation bacteria body, adopts stifling cultivation to measure, converts, be calculated as follows in units of ware:

MBC＝(M ₁-M ₀)/k

In formula: M ₁, M ₀the CO of the stifling and not stifling nutrient culture media release of representative respectively ₂amount mg ware ^-1; K is constant, gets 0.411.

Experimental result uses Excel 2013, SAS 8.1 to complete data processing and variance analysis, and infrared spectrum absorpting peak area uses OPUS 6.5 software to calculate.

Compared with prior art, beneficial effect of the present invention is:

1) upgrowth situation of No. 3 bacterium is best, at 129 ~ 226CFU ware ^-1between, illustrate that No. 3 bacterium have very strong adaptive faculty to HA.In each combined treatment, No. 1 bacterium growing way is stablized, and drop is less, and No. 2 bacterium growing way in combined treatment is the poorest, and especially with obvious in T12 and T123, proportion is only 25.00% and 1.94% of total clump count.In combined treatment, between each bacterium colony, nothing covers phenomenon mutually, illustrates and can coexist between different bacterium colony.

2) all there is significant difference (P < 0.05) in each process respiratory capacity carbon, show as combined treatment and be greater than single bacterium colony process, the respiratory capacity carbon of T1 comparatively T2 and T3 exceeds 105.69% and 26.87%, illustrates that the respiration capability of No. 1 bacterium is stronger.The MBC of T3, T1 and T13 is apparently higher than other process (P < 0.05), and the MBC of T2 is minimum, is only 26.61% of T3, and the solid carbon ability of visible 1, No. 3 bacterium is comparatively strong, and No. 2 solid carbon abilities of bacterium are more weak.T23 and T13 total degradation rate apparently higher than other process (P < 0.05), be respectively 59.78% and 56.71%, T123, T12, T3 and T1 total degradation rate between 42.68% ~ 50.91%, T2 is only 21.73%.

3) cultivate the display of end HA infrared spectrum, each process polysaccharide and alcohols material increase, especially obvious with T12, T123 amplification.Each process containing No. 1 bacterium has stronger degradation capability to aromatic carbon, makes HA structure be tending towards simple, and degree of humification reduces.No. 2 bacterium 1151/1637, (1045+1075)/1637 and 135,7/1,637 3 place's absorption peak strength area ratio all higher than level before degraded, but amplification processes lower than other.

Accompanying drawing explanation

Fig. 1 is scanning electron microscope (SEM) photograph, and Fig. 1 a is B.licheniformis, Fig. 1 b be M.resistens, Fig. 1 c is S.maltophilia;

Fig. 2 is degradation bacteria clump count variation diagram in time;

Fig. 3 is respiratory capacity carbon changing trend diagram in time;

Fig. 4 is that degraded terminates rear HA infrared spectrum.

Embodiment

Below in conjunction with the drawings and specific embodiments, technical scheme of the present invention is described in more detail.

1 materials and methods

1.1 soil sample collections

For examination soil sample from forest farm, Ma Lian beach, Yongshou County, Shaanxi, according to the principle that typicalness is close with Restoration stage, in the 0-10cm soil of 5 kinds of vegetation patterns (farmland, Chinese pine, locust tree, sea-buckthorn, mixed forest) top layer, respectively get 3 soil samples.Soil sample is divided 2 part process, a part, in room-dry, crosses 2mm sieve series for HA.Another part, in 4 DEG C of refrigerations, treats used time incubated at room temperature 24h, crosses 2mm sieve series for soil bacteria suspension.

The Isolation and ldentification of 1.2 Soil HA degradation bacteria

Extract Soil HA according to Wen Qixiao method therefor, utilize each vegetation pattern HA that soil extracts to be that unique C, N source makes nutrient culture media, concrete composition comprises: HA about 0.2300 g, NaCl 0.25g, Agar 1.00g, d H ₂o 50.0ml, regulates pH to be 7.3.After 0.1MPa steam sterilizing 30min, sterile working is transferred to 90mm double dish, every ware 15.0mL.Research institute's HA nutrient culture media carbon content is 28.34mg.Claim 10.00g soil sample in 250mL triangular flask, add 100.0mL sterile distilled water, after shaking table concussion 30min, draw 100uL supernatant and be evenly applied to above-mentioned HA solid medium, cultivate 14d, period determines the degradation rate (table 1) of degradation bacteria to HA.Bacterial classification is separated after bacterium colony is obvious, research before author, obtains Bacillus licheniformis (99.65%), Rhizobium nepotum (99.78%), Microbacteriumresistens (98.71%), Stenotrophomonas maltophilia (99.23%), Streptomycesazureus (99.78%) 5 strain degradation bacteria altogether.

1.3 experiments are selected with bacterium

Account for the bacterial classification clump count sum selected in experiment that overall proportion is comparatively large, growing way preferably standard (table 1), choose B.licheniformis, M.resistens and S.maltophilia 3 strain degradation bacteria, No. 1, numbering, No. 2, No. 3 (Fig. 1) respectively, if 7 process T1, T2, T3, T12, T13, T23, T123, this 3 strain degradation bacteria of further research and the degraded situation of combination to C in the HA of arable land thereof, for ease of expressing, T1, T2, T3 are collectively referred to as single bacterium colony process, T12, T13, T23, T123 are collectively referred to as combined treatment.Arable land HA solid medium component is with 1.2.0.1MPa steam sterilizing 30min, slightly cool sterile working and be transferred to 90mm double dish, every ware 15.0mL, 4 repetitions are established in each process.Containing C in the HA of arable land is 41.08%, is 4.32% containing N.

To take a morsel degradation bacteria with oese bacterium colony point after No. 1, bacterial classification, No. 2, No. 3 purifying, (HA fluid nutrient medium is except without except agar to be transferred to sterilizing small test tube containing 10.0mL arable land HA fluid nutrient medium, component is with aforementioned), cultivate in 37 DEG C of shaking tables, every 24h gets 15 μ L constant volumes in 150mL volumetric flask, after fully shaking up, get 100 μ L bacterium liquid and be evenly applied to HA solid medium.Observe each degradation bacteria clump count in the medium (colony forming unit, CFU), quantity counts in units of ware.After each degradation bacteria clump count is stable (the 5th day, Fig. 2), T1, T2, T3 respectively get the HA fluid nutrient medium constant volume of the corresponding degradation bacteria of 15.0 μ L in 150mL volumetric flask, fully shake up, and draw 100 μ L and are applied to HA solid medium.T12 gets each 7.5 μ L constant volumes of HA fluid nutrient medium containing No. 1, No. 2 degradation bacteria in 150mL volumetric flask, and the same T1 of all the other processes (T23, T13 are in like manner), T123 then respectively gets 5.0 μ L, and operating process is with above-mentioned.Every ware HA is 28.34mg containing C, and bacterium liquid extension rate is 1 × 10 ⁴doubly.

Table 1 is for examination bacterial classification morphological feature

1.4 experimental programs are formulated and process

Respiratory capacity carbon (respiration carbon, RC) assay method is as follows.Combined by 90mm double dish, junction uses Arabic gum sealing, and outer tire layer sealed membrane is to ensure impermeability.HA solid medium sticks to top, with 1.0molL ^-1naOH 10.0mL collect degradation bacteria breathe the CO discharged ₂, every 24h 0.5molL ^-1hCl titration once (the concrete concentration Na of HCl of left and right ₂b ₄o ₇10H ₂o demarcates), cultivate 14d in 28 DEG C.Be culture media supplemented 3min filtrated air in titration process, the CO lost in this time ₂measure the CO by 30min ₂uptake converts.

Choose a typical case after cultivation terminates to repeat, all bacterium colonies are chosen, uses NaOH and Na afterwards ₄p ₂o ₇mixing digestion agent carries out HA second extraction, NaOH and Na ₄p ₂o ₇concentration is 0.1molL ^-1.First use 40mL digestion agent slowly to rinse HA solid medium, flushing limit, limit is with 0.45 μm of filtering with microporous membrane, and this process lasts 4h, this measure is to intercept residual degradation bacteria, to eliminate its impact on infrared spectrum as far as possible.(during the 4th, nutrient culture media is transparent to use 100mL mixing digestion agent to divide 5 immersion HA solid mediums afterwards, now lixiviate work completes substantially), soak time is 24h, and all extracting solutions are all transferred to 150mL volumetric flask for measuring infrared spectrum.Concrete secondary lixiviate step is shown in that Tatzber made a search.Remain 3 and be recycled and reused for mensuration microbes biomass carbon (microbial biomass carbon, MBC).

The assay method of 1.5 indexs of correlation

Bran & Luebbe AutAnalyel type elemental analyser is used to measure containing C, N percentage in HA; Clump count adopts the method for plate culture count; HA infrared spectrum adopts BRUKER company's T ensor27 type Fourier transform infrared spectrometer (Fouriertransform infrared spectrometer, FTIR) to measure; Scanning electron microscope uses Hitachi, Ltd S-4800 type field emission scanning electron microscope.

Respiratory capacity carbon refers to the C amount that degradation bacteria is consumed by respiration.Calculate according to the following formula:

RC＝(2V ₁-V ₂-V ₀)W×6

In formula: V ₁, V ₂, V ₀represent blank, 3min CO respectively ₂loss amount, process titration consume HCl volume (mL); W is HCl concentration (molL ^-1); 6 is conversion coefficient.

Microbes biomass carbon refers to the C amount of degradation bacteria body, adopts stifling cultivation to measure, converts in units of ware.Be calculated as follows:

MBC＝(M ₁-M ₀)/k

In formula: M ₁, M ₀the CO of the stifling and not stifling nutrient culture media release of representative respectively ₂amount (mg ware ^-1); K is constant, generally gets 0.411.

2 results of the present invention

2.1 different disposal degradation bacteria quantative attributies

Single bacterium colony process degradation bacteria quantity has larger difference, and T1, T2 are 53.95% and 26.98% of T3.The degradation bacteria total amount of combined treatment shows as T13 > T123 > T23 > T12, T123, T23, T12 are respectively 79.23%, 59.62% and 21.54% of T13, No. 3 bacterium degradation bacteria quantity are the highest, account for 86.92% of T13, T23, T123 total amount, 83.23%, 82.52% respectively, when inoculating quantity and reducing 1/2 and 1/3, in T13, T123, the degradation bacteria quantity of No. 3 bacterium is still suitable with T3, visible, when using HA as unique C, N source, its adaptive faculty is the strongest.No. 1 bacterium degradation bacteria quantity in corresponding combined treatment remains relatively stable, and No. 2 bacterium upgrowth situation in T12 and T123 is poor, and proportion is only 25.00% and 1.94%, and this illustrates the producing level of No. 2 bacterium to HA lower (table 2).Different strain is to the degradation capability of HA difference to some extent, and this and the adaptive faculty of degradation bacteria to HA have substantial connection, and the degradation bacteria that adaptive faculty is stronger often can become sociales.Containing in each process of No. 3 bacterium in this research, its clump count is all the highest, illustrates that it has obligate degradation capability to HA.The upgrowth situation of T1 is also better, this is because No. 1 bacterium and organic decomposition have direct relation, it can accelerate the conversion of organic nutrient in soil, although proportion is less in T12, T13, T123, but colonial morphology is obvious, illustrate that it can produce coordinative role with No. 2, No. 3 bacterium and ecological niche is separated, which avoid keen competition between population.No. 2 bacterium clump count in corresponding process is minimum, and this is relevant with HA its desirable C source non-.

Table 2 different disposal degradation bacteria quantity

The degraded situation of C in 2.2HA

The respiratory capacity carbon of table 3 HA, MBC and total degradation amount

All there is significant difference (P < 0.05) in each process respiratory capacity carbon, aggregate performance is that combined treatment is greater than single bacterium colony process.Respiratory capacity carbon containing T23, T13 of No. 3 bacterial classification process is the highest, comparatively T1, T2, T3 exceed by a relatively large margin, can be found by the growing way of observing bacterium colony in nutrient culture media, in T23, T13, colony growth is even, there is not the phenomenon that bacterium colony covers mutually, this illustrates that No. 3 bacterium can coexist with No. 1, No. 2 bacterium, and No. 3 bacterium can coexist with other bacterium and can strengthen their activity to have research also to show.This research confirms in single bacterium colony process, and No. 1 bacterium respiration capability is the strongest, and the respiratory capacity carbon of No. 1 bacterium exceeds 105.69% and 26.87% respectively compared with No. 2 bacterium and No. 3 bacterium, and respiratory capacity carbon in time variation tendency is shown in Fig. 3.MBC is directly related with colony counts, all there is significant difference (P < 0.015) each other in the MBC of T3, T1, T13, T23, T123, T2, the MBC of TI, T3 and T13 is significantly higher than other process, visible No. 1 stronger with the solid carbon ability of No. 3 bacterium.Total degradation amount aspect, T23, T13 are the highest, reach 14.75mg and 13.97mg, and be significantly higher than other process (P < 0.05), T2 total degradation amount is minimum, is only 36.34% (table 3) of T23.The degradation rate of each process between 21.73% ~ 59.87%, this with glucose and HA in the situation of C source, the degradation rate of degradation bacteria to farmland, spruce forest, grassland soil HA is close in the result of 17.30% ~ 56.00%.

Research shows, restriction HA palliating degradation degree has 2 large factors, i.e. himself architectural feature and degradation process enzymatic activity.The C part that contains in HA structure comprises fragrant C, fatty C, Small molecular carbohydrates etc.Fat C proportion is comparatively large, and to its producing level depending on degradation bacteria degradation capability, the degradation amount of T2 is minimum, and this may be lower relevant to fatty C producing level with No. 2 bacterium; Fragrance C is difficult to degraded, nonideal C source; Small molecular carbohydrates proportion is lower, and the bacterium that is easily degraded utilized.Palliating degradation degree is higher, degradation bacteria gained energy is more, sufficient energy source is conducive to degradation bacteria and synthesizes relevant digestive enzyme (carbohydrase, proteinase, laccase, lignin peroxidase etc.), the degradation process that these enzymes act on next round provides energy for degradation bacteria, iterative cycles like this, can say that enzyme level determines the adaptive faculty of degradation bacteria to HA to a certain extent, the concrete mechanism of action needs research further.

2.3 degradeds terminate HA diffuse reflectance infrared spectroscopy

According to the position of absorption peak, peak, the infrared spectrum energy reflection molecular structure of complex compound and the information of functional group aspect, can judge by force whether HA exists some functional group and relative content situation thereof.The ownership of this research HA infrared spectrum is shown in corresponding see prior art.Infrared spectrum display (Fig. 4), main absorption peak has 1045cm ^-1(in polysaccharide or polysaccharose substance, C-O stretches), 1075cm ^-1(in alcohol, phenol, C-OH stretches), 1151cm ^-1the asymmetric stretching vibration of C-O-C of ehter bond (in fatty compound and the cycloaliphatic ring), 1375cm ^-1(CH ₃symmetric deformation vibration, C-N is flexible bends with N-H), 1637cm ^-1(aromatic series C=C stretching vibration, in carboxyl and acid amides, C=O vibrates), 3340cm ^-1(O-H stretches).Compared with before degraded, there is change to a certain degree in different disposal spectrum shape, and spectrum shape between respectively processing is similar, illustrates that they have basically identical structure and functional group is formed.Fatty compound, carbohydrates, aromatic, acid amides and oxygen-containing functional group (alcohol, phenol and aliphatic ether) is mainly contained by the structural unit of the known HA of these absorption peaks after degraded terminates.Compared with before degraded, add the organic carbons such as polysaccharide, alcohols, lipid, wherein, compare between each process, T123 polysaccharide, the increase of alcohols organic carbon are comparatively obvious, the increase of T1 and T12 lipid organic carbon is comparatively obvious, and the degraded aromatic carbon degradation amount containing T1, T12, T13 process of No. 1 bacterium process is higher, illustrates that No. 1 bacterium has stronger degradation capability to aromatic carbon structure.

Table 4 HA infrared spectrum absorpting peak intensity

The integral area of peak region the results are shown in Table 4, except T2, respectively processes at 1045cm ^-1and 1075cm ^-1the light absorption value at two places, before degraded, is respectively 2.4 ~ 2.9 times before degraded and 11.6 ~ 13.2 times, and illustrating in HA degradation process that carbohydrates and fatty compound content have increases largely.Amir research shows, after microorganism compost, in HA, carbohydrates amount increases to some extent, and this research also draws same conclusion.1375cm ^-1absorption peak has increase by a small margin equally before comparatively degrading, this region can reflect that degraded terminates wild Oryza species N element level.All process are at 1637cm ^-1and 3340cm ^-1place's absorption peak area is less with the front difference of degraded, and the former demonstrates the fragrant C of the more difficult decomposition of degradation bacteria, and the latter causes by hydroxyl in digestion agent is more.Relevant research shows, in HA, fragrant C-structure has very strong anti-capacity of decomposition, and be difficult to be utilized by Institute of Micro-biology, this is consistent with this research.The ratio of absorption peak strength may be used for the degraded situation representing HA, also can be used for aromizing and the degree of humification of explaining HA, the each process 1151/1637 of this research, (1045+1075)/1637 and 135,7/1,637 3 place's absorption peak strength area ratio are all higher than level before degraded, these three ratios respectively illustrate under the effect of degradation bacteria, in HA nutrient culture media, polysaccharose substance increases, aromatization degree weakens, degree of humification reduces, and structure is tending towards simplifying.No. 1 bacterium, No. 3 bacterium and be combined in and effectively can decompose complicated organic carbon in degraded HA process and form the organic carbons such as polysaccharide, alcohols, lipid, No. 2 these abilities of bacterium of cultivating separately are comparatively speaking more weak.

The above; be only the present invention's preferably embodiment; protection scope of the present invention is not limited thereto; anyly be familiar with those skilled in the art in the technical scope that the present invention discloses, the simple change of the technical scheme that can obtain apparently or equivalence are replaced and are all fallen within the scope of protection of the present invention.

Claims (2)

1. soil degrading bacteria and combination thereof are to the experimental technique of arable land humic acid degraded, it is characterized in that, comprise the steps:

(1) respiratory capacity carbon measures: combined by 90mm double dish, and junction uses Arabic gum sealing, and outer tire layer sealed membrane is to ensure impermeability, and HA solid medium sticks to top, with 1.0molL ^-1naOH 10.0mL collect degradation bacteria breathe the CO discharged ₂, every 24h 0.5molL ^-1hCl titration once, the concrete concentration Na of HCl ₂b ₄o ₇10H ₂o demarcates, and cultivating 14d in 28 DEG C, is culture media supplemented 3min filtrated air in titration process, the CO lost in this time ₂measure the CO by 30min ₂uptake converts;

(2) typical case's repetition is chosen in cultivation after terminating, and is chosen by all bacterium colonies, uses NaOH and Na afterwards ₄p ₂o ₇mixing digestion agent carries out HA second extraction, NaOH and Na ₄p ₂o ₇concentration is 0.1molL ^-1first 40mL digestion agent is used slowly to rinse HA solid medium, flushing limit, limit is with 0.45 μm of filtering with microporous membrane, this process lasts 4h, use 100mL mixing digestion agent to divide afterwards and soak HA solid medium 5 times, during the 4th, nutrient culture media is transparent, and now lixiviate work completes substantially, soak time is 24h, and all extracting solutions are all transferred to 150mL volumetric flask for measuring infrared spectrum.

2. soil degrading bacteria according to claim 1 and combination thereof are to the experimental technique of arable land humic acid degraded, it is characterized in that, respiratory capacity carbon refers to the C amount that degradation bacteria is consumed by respiration; Calculate according to the following formula:

RC＝(2V ₁-V ₂-V ₀)W×6

In formula: V ₁, V ₂, V ₀represent blank, 3min CO respectively ₂loss amount, process titration consume HCl volume mL; W is HCl concentration molL ^-1; 6 is conversion coefficient;

Microbes biomass carbon refers to the C amount of degradation bacteria body, adopts stifling cultivation to measure, converts, be calculated as follows in units of ware:

MBC＝(M ₁-M ₀)/k

In formula: M ₁, M ₀the CO of the stifling and not stifling nutrient culture media release of representative respectively ₂amount mg ware ^-1; K is constant, gets 0.411;

Experimental result uses Excel 2013, SAS 8.1 to complete data processing and variance analysis, and infrared spectrum absorpting peak area uses OPUS 6.5 software to calculate.