CN113493248A

CN113493248A - Method for determining constructed wetland biofilm development state based on extracellular enzyme

Info

Publication number: CN113493248A
Application number: CN202110769274.7A
Authority: CN
Inventors: 陶玲; 李谷; 彭亮; 代黎黎
Original assignee: Yangtze River Fisheries Research Institute CAFS
Current assignee: Yangtze River Fisheries Research Institute CAFS
Priority date: 2021-07-07
Filing date: 2021-07-07
Publication date: 2021-10-12
Anticipated expiration: 2041-07-07
Also published as: CN113493248B

Abstract

The invention discloses an ectoenzyme-based method for measuring the development state of an artificial wetland biofilm, and belongs to the technical field of artificial wetland water treatment. The method comprises the following steps: the method comprises the steps of measuring the activity of extracellular enzymes related to the degradation of nitrogen, phosphorus and organic matters in the surface of the root system of the constructed wetland, the substrate of the root area and the substrate biomembrane of the non-root area, and evaluating the development state and the process of the constructed wetland biomembrane by using evaluation indexes such as enzyme type number, enzyme diversity index, enzyme comprehensive index and the like. By utilizing the method, the activity and the degradation capability of the artificial wetland biofilm in the starting operation process can be analyzed and evaluated simply, quickly and comprehensively, the development degree and the stabilization process of the biofilm in the starting period of the artificial wetland can be analyzed, the maturation stabilization state and the treatment efficiency of the wetland biofilm can be predicted, and theoretical basis and practical reference are provided for quick starting and application optimization of the artificial wetland for treating the culture tail water.

Description

Method for determining constructed wetland biofilm development state based on extracellular enzyme

Technical Field

The invention belongs to the technical field of artificial wetland water treatment and biomembrane activity evaluation, and particularly relates to an artificial wetland biomembrane development state evaluation method based on extracellular enzymes.

Background

The artificial wetland is a water treatment technology which is developed in the 50 s of the 20 th century, has low investment, high efficiency and is easy to maintain. Compared with the traditional water treatment technology, the constructed wetland has the advantages of economy, environmental protection, high efficiency, low consumption, easy maintenance and management and the like, so the constructed wetland is widely applied to the treatment of various waste water such as domestic sewage, industrial waste water, agricultural waste water, mine waste water, landfill leachate, surface runoff and the like. In recent years, artificial wetlands are also gradually applied to the treatment of the culture tail water in order to reduce the influence of the culture tail water rich in nitrogen and phosphorus nutrients on the receiving water body.

According to the water flow mode, the artificial wetland can be divided into three types, namely Surface Flow (SF), horizontal subsurface flow (HF) and Vertical Flow (VF). The adsorption, degradation and transformation of pollutants when the artificial wetland is used for treating sewage are mainly completed by a biological membrane. The process of removing pollutants in the artificial wetland is a series of biological oxidation-reduction reaction processes catalyzed by a plurality of enzymes generated by microorganisms in a biological membrane. Microorganisms in the artificial wetland are mainly attached to the surfaces of a substrate and a root system biomembrane, and the biomembrane can be used as a key index for measuring the development degree of the artificial wetland due to the special characteristics and performance of the biomembrane. In addition, the development degree of the biological membrane directly influences the treatment efficiency of the wetland system, the growth condition and activity of the biological membrane directly influence the purification function and operation effect of the wetland, and particularly, how to promote the development of the biological membrane in the early stage of wetland construction and improve the degradation performance of the biological membrane are the key points of improving the treatment effect of the artificial wetland system. The actual action of the constructed wetland biofilm is not the total amount of the observed biofilm but only the biomass part with stronger biological activity. Therefore, in order to fully exert the function of the wetland biofilm, it is necessary to study an accurate and simple method for measuring the microbial activity of the wetland biofilm and evaluating the development state of the wetland biofilm. The existing method for evaluating the growth and activity of the biological membrane mainly comprises a method for measuring the content of extracellular polysaccharide and extracellular protein, a DGGE molecular biological evaluation method, a hydraulic conductivity index, a thickness index of the biological membrane, a dehydrogenase activity index and the like. The thickness of the biofilm does not reflect the function of the biofilm, because when the biofilm is small, all the biofilms are active, the activity is increased due to the increase of the thickness of the biofilm, when the thickness of the biofilm is increased to be larger than the optimal thickness, although the total amount of the biofilm is still increased, the activity is reduced, and researches show that after the thickness of the biofilm growing on the surface of the carrier is increased to a certain degree, the microorganisms of the part of the biofilm close to the surface of the carrier, namely the inert biolayer, are difficult to obtain nutrients, the activity is poor, the microorganisms do not participate in biochemical reaction basically, the active biolayer wrapping the inert biolayer has stronger activity, and the removal of pollutants mainly depends on the microorganisms in the layer. Excessive biofilm thickness can adversely result in inefficient processing. The biofilm is required to be in an appropriate thickness, too thick is easy to block, too low is not active, so that no report of an appropriate biofilm thickness exists at present, and the optimal biofilm thickness of different systems is inconsistent, so that a uniform standard is difficult to determine. The evaluation indexes such as DGGE molecular biology method, hydraulic conductivity, extracellular polysaccharide content, extracellular protein content and the like are complex to operate, the detection time is long, and the activity of the wetland biofilm cannot be quickly reflected. The extracellular enzyme is a limiting step in the material degradation process and can reflect the activity of a biological membrane and the degradation capability and the operation condition of the materials in the wetland. The evaluation method based on the dehydrogenase activity can reflect the active microbial biomass in the biological membrane and the degradation activity of the biological membrane on organic matters, but the selected enzyme type is single, the degradation activity of the biological membrane on specific types of organic matters cannot be reflected, the metabolic type activity of the biological membrane of the wetland cannot be reflected, and the real condition of the development of the artificial wetland is difficult to reflect. Therefore, an evaluation method and a comprehensive evaluation index system for the activity and the development state of the subsurface flow constructed wetland based on a plurality of extracellular enzymes are needed to be established, the enzymes and the metabolism change are monitored simultaneously to reflect the activity of the biofilm according to the metabolism characteristics of the constructed wetland substrate and the root system microorganisms, and the method is helpful for further deepening the understanding of the purification mechanism of the constructed wetland so as to guide the operation management and the evaluation of the constructed wetland.

Disclosure of Invention

In order to solve at least one of the above technical problems, the technical solution adopted by the present invention is as follows:

the invention provides an artificial wetland biofilm development state determination method based on extracellular enzymes, which comprises the following steps:

simultaneously measuring the activity of extracellular enzyme related to the degradation of nitrogen, phosphorus and organic matters in the surface of the artificial wetland root system, the substrate of the root area and the substrate biomembrane of the non-root area,

analyzing the metabolic activity and metabolic pathway of the constructed wetland biomembrane based on the activity of the extracellular enzyme, thereby obtaining the development state of the constructed wetland biomembrane.

In some embodiments of the invention, the extracellular enzymes associated with the degradation of nitrogen, phosphorus and organic matter include phosphatases, esterases, peptidases and glycolytic enzymes.

In some embodiments of the invention, the phosphatase comprises alkaline phosphatase, acid phosphatase, naphthol-AS-BI-phosphohydrolase.

In some embodiments of the invention, the esterase comprises a lipid esterase, a lipase and an esterase.

In some embodiments of the invention, the peptidases include leucine arylamine enzymes, valine arylamine enzymes, and cystine arylamine enzymes.

In some embodiments of the invention, the sugar fermentation enzyme comprises a β -galactosidase, N-acetyl-glucamine, α -glucosidase, β -glucuronidase, α -mannosidase and fucosidase.

In some embodiments of the invention, the biological membrane comprises a constructed wetland plant root system surface biological membrane, a root zone substrate biological membrane and a non-root zone substrate biological membrane.

In some embodiments of the invention, the activity of the extracellular enzyme is calculated using the number of enzyme types, the enzyme diversity index, and the enzyme combination index.

In some embodiments of the invention, the enzyme type number refers to the number of all enzyme types that show positivity.

In some embodiments of the invention, the enzyme diversity index comprises a shannon diversity index of the enzyme. Further, the shannon diversity index of the enzyme was calculated using the following formula:

H’＝-∑pi*lnpi

wherein, H' represents the aroma diversity index of the enzyme; pi is the ratio of the activity of a particular enzyme to the sum of all enzyme activities.

In some embodiments of the invention, the enzyme integrated enzyme index refers to the sum of the color intensities after all enzyme color reactions.

In the invention, the artificial wetland is used for treating the tail water of pond culture.

The invention has the advantages of

Compared with the prior art, the invention has the following beneficial effects:

(1) according to the invention, by measuring the activity and diversity characteristics of extracellular enzymes related to the degradation of nitrogen, phosphorus and organic matters, the microbial activity change of wetland substrates and plants in the process of adapting to the wetland environment at the start-up biofilm formation stage is inspected, the growth and maturation process of the biomembranes of the wetland from the construction stage to the stabilization stage is analyzed, and the growth state of the biomembranes of the artificial wetland can be simply, rapidly and comprehensively evaluated. Provides a basic basis for optimizing process conditions, strengthening the activity of the biological membrane and assisting the constructed wetland system to quickly form a microbial community in the process of starting the biofilm formation.

(2) The activity state of the biological membrane operated by the artificial wetland is analyzed and represented through enzyme activity evaluation, excessive biological membrane growth and excessive biological membrane thickness are prevented, and a substrate is prevented from being blocked, so that reasonable measures are taken to keep the proper thickness of the biological membrane and the optimal operation state and treatment effect. Meanwhile, according to the degradation characteristics of the constructed wetland substrate and root system microorganisms on pollutants, the content and the types of the cultured pollutants and the like, corresponding microbial agents can be screened and inoculated, the degradation capability of the wetland on the pollutants is improved, the optimization of the constructed wetland is facilitated, and scientific basis is provided for the construction and the operation management of the constructed wetland.

Drawings

Fig. 1 shows a schematic diagram of an artificial wetland for carrying out the test.

FIG. 2 shows the change of the surface of the root system of the constructed wetland, the substrate of the root zone, the extracellular enzyme species number of the substrate of the non-root zone, the enzyme comprehensive index and the enzyme diversity index along with the starting days.

Fig. 3 shows the change of the number of extracellular enzyme species, the enzyme comprehensive index and the enzyme diversity index of the substrate in the non-root area of the constructed wetland along the wetland flow path.

FIG. 4 shows the result of comprehensive exponential linear regression analysis of wetland biofilm biomass and enzymes during the development of constructed wetland biofilms.

FIG. 5 shows the results of linear regression analysis of wetland biofilm biomass and enzyme diversity index during the development of constructed wetland biofilms.

Detailed Description

In order to make the technical problems, technical solutions and advantageous effects solved by the present invention more apparent, the present invention is further described in detail below with reference to the following embodiments.

Examples

The following examples are used herein to demonstrate preferred embodiments of the invention. It will be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function in the invention, and thus can be considered to constitute preferred modes for its practice. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit or scope of the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs and the disclosures and references cited herein and the materials to which they refer are incorporated by reference.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

The experimental procedures in the following examples are conventional unless otherwise specified. The instruments used in the following examples are, unless otherwise specified, laboratory-standard instruments; the test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.

Example 1

1 materials and methods

1.1 test site

A group of newly-built artificial wetlands for treating the culture tail water is selected to carry out a test, and a wetland system (shown in figure 1) consists of five stages of undercurrent artificial wetland units with different flow states. The second-level wetland and the fourth-level wetland are upstream and respectively plant the floral leaf arundo donax and canna, and the first-level wetland, the third-level wetland and the fifth-level wetland are surface flow and all plant the reshiving flowers. The substrates in the wetland are all ceramic particles, the artificial wetland is operated under the condition that the hydraulic retention time is 5 hours during the test period, the wetland is fed with water for 12 hours from 6 am to 18 pm every day, the water feeding is stopped in other periods, and the artificial wetland is not operated in rainy days.

1.2 extracellular enzyme assay and analysis

Using enzyme kit method (API ZYM)^TMstrips, BioMerieux, Marcy l "Etoile, France) were assayed for 19 enzyme activities in root surface and matrix surface biofilms. Uniformly taking the matrixes of the upper layer, the middle layer and the lower layer of the non-root area and the root area of the second-stage upflow subsurface flow wetland and the four-stage upflow subsurface flow wetland for 5 times in total of 0d, 20d, 40d, 60d and 80d when the wetland formally starts to operate, and collecting the floral leaf arundo donax and the canna root system planted in the wetland for 4 times in total of 20d, 40d, 60d and 80 d.

Placing 50g of matrix sample into a 250mL conical flask, adding 50mL of sterile water, oscillating for 30min, standing for 10min to prepare matrix eluent, dividing the matrix eluent into two parts, wherein one part is used for measuring the activity of extracellular enzyme, and the other part is used for measuring the biological quantity of a matrix biological membrane by retaining 20mL of matrix eluent. Randomly clipping floral leaf giant reed and canna root systems, placing 5g of root systems in a conical flask, adding 50mL of sterile water, and preparing root surface eluent by the following treatment in accordance with the matrix sample treatment method. Taking 65 mu L of matrix and supernatant of root surface eluent respectively, placing in an API ZYM reagent strip small chamber, culturing at 37 ℃ for 4h after marking, adding a drop of ZYM A reagent and ZYM B reagent respectively after culturing, irradiating under a 1000W bulb for 10s after reacting for 5min, and recording and scoring results according to the color development degree in the small chamber. The number of extracellular enzyme species (N), Shannon (Shannon) index (H') and the integrated enzyme index (SEI) were used to analyze the activity and diversity of the enzymes. The number of extracellular enzyme species (N) refers to the number of all enzymes showing positive, the integrated enzyme index (SEI) refers to the sum of color intensities after color development reaction of all enzymes, and the Shannon diversity index (H ') is calculated by the formula H' ═ Pi × ln (Pi), where Pi is the ratio of the activity of a specific enzyme to the sum of all enzyme activities.

1.3 biofilm Biomass determination

Volatile solids (VSS) are used as an indicator to reflect biofilm biomass. The GF/F glass fiber filter (pore size 0.7 μm) was calcined in a muffle furnace for 5 hours (temperature 450 ℃ C.), and then weighed (W)₀) And marked. For analysis, 20mL of the matrix eluate was suction-filtered under 0.3Pa, and the glass fiber filter membrane with the sample was dried in a constant-temperature drying oven for 48 hours (temperature 60 ℃ C.), and then weighed (W)₆₀) (ii) a Then burning the weighed glass fiber filter membrane in a muffle furnace for 5h (the temperature is 450 ℃), and then weighing (W)₄₅₀). Weighed (to the nearest 0.01mg) on an electronic balance. Biofilm biomass per gram of substrate was calculated. VSS ═ W₆₀-W₄₅₀) S, S is substrate biomass.

2 results and analysis

2.1 dynamic characteristics of wetland extracellular enzyme activity in startup period

During the starting process, the number (N) of extracellular enzyme types, Shannon indexes (H ') and comprehensive enzyme indexes (SEI) detected by the artificial wetland plant root system surface, root zone matrix and non-root zone matrix biomembrane are gradually increased along with the increase of the running time (figure 2), wherein the number of the enzyme types on the plant root system surface is increased from 6 to 15 from 20d to 80d, the Shannon index (H') is increased from 1.59 to 2.55, and the comprehensive enzyme index (SEI) is increased from 9 to 37; from 0d to 80d, the number of matriptase species in the root zone increases from 5 to 15, the Shannon index (H') increases from 1.61 to 2.55, and the Integrated enzyme index (SEI) increases from 5 to 38; the number of matriptase species in the non-root zone rose from 5 to 9, the Shannon index (H') rose from 1.12 to 2.13, and the Combined enzyme index (SEI) rose from 2.5 to 22. As can be seen from the trend of change, after starting for 60 days, the increase amplitudes of the extracellular enzyme species number (N), the comprehensive enzyme index (SEI) and the Shannon index (H ') of the plant root system and the root zone matrix are gradually reduced and gradually tend to be stable, which indicates that the plant root system and the root zone matrix are basically stabilized at 60 days, the extracellular enzyme species number (N) of the non-root zone matrix is basically stabilized at 60 days, but the comprehensive enzyme index (SEI) and the Shannon index (H') are continuously increased to 80 days and still do not reach the stable state.

As can be seen from the trend of the single enzymes (Table 1 and Table 2), only six enzymes such AS alkaline phosphatase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, esterase, lipoid esterase, leucine arylamine enzyme and the like can be detected on the root area substrate, the non-root area substrate and the root surface of the wetland at the first 20d of wetland initiation, and the degradation capability on organic phosphorus and organic nitrogen is shown. After the wetland is started and operated for 40 days, more sugar fermentation enzymes and peptidase extracellular enzymes are detected in sequence, which shows that extracellular polymers such as protein, polysaccharide and the like are formed and accumulated on the root system and the substrate surface along with the biofilm formation process, and the development of the biomembrane in the starting process is accurately judged and represented to form a stable state, and the dynamic change process of the degradation capability of microorganisms with different functions on the biomembrane on the pollutant is realized.

TABLE 1 non-root zone matrix extracellular enzyme Activity

Note: 1. negative reaction: 0; positive reaction: 1 to 5.

TABLE 2 extracellular enzyme Activity of plant root surface and root zone substrates

Note: 1. negative reaction: 0; positive reaction: 1 to 5.

As shown in fig. 3, in the two-stage upstream wetland, the number of extracellular enzymes, the H' diversity index and the SEI index of the substrate in the non-root zone are gradually increased from the lower layer to the upper layer, and the substrate on the upper layer is the highest, which reflects that the substrate on the upper layer is mature and stable firstly in the development process of the wetland biofilm in the start-up period.

The number of extracellular enzyme species (N), Shannon index (H') and the overall enzyme index (SEI) detected on the surface of the root system, the matrix of the root zone and the biomembrane are all significantly larger than those of the non-root zone (P <0.05), but the difference between the surface of the root system and the matrix of the root zone is not significant (P > 0.05). And by the end of the 80d experiment, the enzyme types detected on the surface of the root system and the substrate in the root area more than the substrate biomembrane in the non-root area are all glycolytic enzymes, including: n-acetyl-beta-glucosaminidase, beta-glucuronidase, alpha-mannosidase and fucosidase. The indexes such as the number of enzymes (N), the enzyme comprehensive index (SEI), the diversity index (H') and the like calculated by the method reflect that after the wetland is started for 20 days, along with the rapid growth and the enhancement of the metabolic capacity of the plant root system, the variety and the activity of the enzyme on the surface of the root system are rapidly improved, meanwhile, the plant root system is firmly combined with the matrix and forms a complex spatial structure together with the matrix in the root system growth process, finally, the activity and the variety of the extracellular enzyme generated by the matrix in the root area and the biofilm on the surface of the root system are basically consistent, a stable state is shown, and the dynamic process that the plant root system promotes the growth of microorganisms in the wetland and the wetland plant root system-matrix interaction forms the biofilm in the starting process is embodied. The results are shown in Table 3.

TABLE 3 non-root zone substrate, plant root surface and root zone substrate extracellular N, H' and (SEI) differences

Note: p <0.05, nsP > 0.05.

2.2 relationship between wetland extracellular enzymes and substrate biofilm during Start-Up phase

In the wetland starting process, the wetland substrate biofilm biomass is obviously and positively correlated with the comprehensive index and diversity index of the substrate enzyme (R)²0.337, 0.000 and R²0.213 and 0.004) (fig. 4 and fig. 5), which reflects that the process of the biofilm accumulation and the metabolic activity increase gradually in the growth and development of the artificial wetland biofilm is consistent. The invention can detect various enzyme activities of the substrate biomembrane in the artificial wetland and calculate the comprehensive index and diversity of the enzyme to treat the organic matters in the corresponding wastewaterThe efficiency and the removal and degradation capacity of which type of pollutant is predicted. The results indicate that when the artificial wetland is used for treating the culture tail water, different promoters can be added according to the characteristics of the culture tail water to improve the removal of pollutants of corresponding types.

The above results show that:

the invention provides an enzyme comprehensive evaluation method formed by indexes such as artificial wetland enzyme number, enzyme comprehensive index, diversity index and the like, the method is simple and rapid in experimental operation, and the data analysis method is simple and representative, can be used as an effective index for predicting the activity of the artificial wetland matrix biomembrane, analyzes the degradation capability and the activity state of the wetland matrix biomembrane, and provides a new practical method for monitoring the development operation state of the artificial wetland for treating and breeding tail water.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims

1. The method for determining the development state of the constructed wetland biofilm based on the extracellular enzyme is characterized by comprising the following steps of:

2. The method for determining the development status of a biofilm of an artificial wetland based on extracellular enzymes according to claim 1, wherein the extracellular enzymes related to degradation of nitrogen, phosphorus and organic substances include phosphatases, esterases, peptidases and glycolytic enzymes.

3. The method for determining the development status of the biofilm of the artificial wetland based on the extracellular enzymes, according to claim 2, wherein the phosphatase comprises alkaline phosphatase, acid phosphatase, naphthol-AS-BI-phosphohydrolase; the esterase comprises lipoid esterase, lipoid esterase and esterase; the peptidase comprises leucine arylamine enzyme, valine arylamine enzyme and cystine arylamine enzyme; the sugar fermentation enzymes include beta-galactosidase, N-acetyl-glucosaminidase, alpha-glucosidase, beta-glucuronidase, alpha-mannosidase and fucosidase.

4. The method for determining the development state of the constructed wetland biofilm based on the extracellular enzymes as claimed in claim 1, wherein the biofilm comprises a constructed wetland plant root system surface biofilm, a root zone substrate biofilm and a non-root zone substrate biofilm.

5. The method for measuring the biofilm development state in an artificial wetland according to any one of claims 1 to 4, wherein the activity of the extracellular enzyme is calculated by using the number of types of enzymes, an enzyme diversity index and an enzyme combination index.

6. The method for measuring the development status of an artificial wetland biofilm based on extracellular enzymes according to claim 5, wherein the number of enzyme types is the number of all enzymes showing positive activity.

7. The method for determining the development status of an artificial wetland biofilm based on extracellular enzymes according to claim 5, wherein the enzyme diversity index comprises a Shannon diversity index of the enzymes.

8. The method for determining the development state of the constructed wetland biofilm based on the extracellular enzymes according to claim 7, wherein the shannon diversity index of the enzymes is calculated by using the following formula:

H’= −∑pi*lnpi

9. The method for measuring the development status of an artificial wetland biofilm based on extracellular enzymes according to claim 5, wherein the enzyme integrated enzyme index is the sum of color intensities after all enzymes have developed a color reaction.