CN112098384A

CN112098384A - Simple method for rapidly predicting whether water quality is biologically stable

Info

Publication number: CN112098384A
Application number: CN202011003367.0A
Authority: CN
Inventors: 王秋华; 张卫风; 许元龙; 王璐璐; 肖坤元; 高爱平; 龙肖霞
Original assignee: East China Jiaotong University
Current assignee: East China Jiaotong University
Priority date: 2020-09-22
Filing date: 2020-09-22
Publication date: 2020-12-18
Anticipated expiration: 2040-09-22
Also published as: CN112098384B

Abstract

A simple method for quickly predicting whether water quality is biologically stable is characterized in that specific bacteria are quantitatively added into a water sample to be detected and pure water in equal quantity, then ATP fluorescence detection is carried out, the detection results of the ATP fluorescence detection and the pure water are compared, if the ATP fluorescence detection and the ATP fluorescence detection are obviously different, the biological instability of the water quality is predicted, and if the ATP fluorescence detection and the ATP fluorescence detection are not obviously different, the biological instability of the water quality is. The prediction method is simple, convenient and quick, and the evaluation index of the method is high in application and popularization value and practical.

Description

Simple method for rapidly predicting whether water quality is biologically stable

Technical Field

The evaluation method can be applied to water supply treatment and drinking water pipe network water quality biological stability analysis, and belongs to the field of municipal engineering water supply and drainage monitoring.

Background

Biostability of drinking water refers to the potential of biodegradable organic matter in drinking water to support the growth of heterotrophic bacteria, i.e., the maximum likelihood that organic nutrients in the water will support the growth of bacteria when the nutrients become limiting factors for the growth of heterotrophic bacteria, which can be expressed in terms of the concentration of nutrients that limit the growth of microorganisms equivalent thereto. When the factory water contains a certain amount of organic matters, the color and turbidity of the water are increased due to the growth of organisms in the water, bad smell is generated, bacteria randomly attached to the pipe wall can grow by utilizing a nutrient medium in the water to form a biological membrane, and the corrosion and scaling of the pipe wall are induced; the corrosion and scaling of the pipe wall can reduce the water delivery capacity of the pipe network, so that the power consumption of a secondary pump station is increased, and even pipe explosion is caused; the propagation of pathogenic microorganisms in the biomembrane and the pipe network water can also pose a direct threat to the health of the drinkers.

The indexes for drinking water biostability research are BDOC (biodegradable soluble organic carbon), AOC (absorbable organic carbon), BGP (bacterial growth potential) and MAP (biologically available phosphorus). The AOC index is taken as the main index, but the measuring method is complex, some researches also use BDOC as the evaluation index of the water quality biological stability, but the measuring time is long and still cannot meet the requirement of practical application. And no judgment standard for whether the water quality is biostable or not is available up to now.

BDOC (biodegradable soluble organic carbon) measurement methods can be divided into two types, static culture and dynamic culture. The static culture mainly includes a suspension culture method in which indigenous bacteria are inoculated and a sand culture method in which a biofilm is attached. The method comprises the specific steps of firstly removing insoluble substances from a water sample to be tested through membrane filtration, culturing at 20 ℃ under a dark room condition after inoculation (28 days in a suspension culture method and 10 days in a biological sand culture method), simultaneously measuring the change quantity of DOC (soluble organic carbon) values in the water sample, and calculating the difference of the DOC values before and after culture to obtain BDOC when the DOC values are constant. The method is simple and easy to implement, but has long measuring time, and in order to overcome the defect, a dynamic culture method appears, and at present, a closed cycle measuring method and a plug flow measuring method are mainly used, the methods take a biological membrane as a measuring flora structure, improve the capacity of degrading organic matters, and can shorten the measuring time to 2-3 days, even within hours, but the dynamic culture method can only measure one water sample at a time, and hardly meets the requirements of conventional research batch tests, and the BDOC reflects only the amount of organic carbon consumed by biological growth and cannot directly reflect the amount of biomass synthesized by biological growth.

The AOC (absorbable organic carbon) measuring method is proposed by Van der Kooij, D, and mainly takes common bacteria Pseudomonas fluorescens (Pseudomonas fluorescens) P17 and Spirillum sp NOx in a water supply network as test strains to inoculate a water sample, the maximum growth bacterial quantity is obtained by culturing for 3-14 days under specific conditions, the maximum growth value of the test bacteria in the water sample is converted into an AOC value by using a standard curve, and the AOC value is expressed in the form of the carbon concentration of standard matrix sodium acetate. From the above, the conventional AOC measurement is a long process and the steps are complicated, so that the method is difficult to popularize in practical application, how to shorten the test time and simplify the steps are main problems in AOC bioassay research.

The BRP (bacterial growth potential) index is proposed by Sathasivan et al based on AOC bioassay, and is mainly different from the traditional AOC bioassay in that natural indigenous water bacteria are used to replace specific pure strains for inoculation, and the BRP value is expressed by the maximum value of bacterial growth, so that the determination also needs at least about 3 days. Compared with specific pure strains, the native bacterial colony is adopted as a test strain, so that the strain is more easily adapted to a local water sample and has stronger absorption capacity on assimilable organic carbon; the maximum value of the bacterial growth is used for replacing the AOC value, so that the establishment of a standard curve and the calculation of growth factors can be avoided, and the time and the labor are saved. The nature of BRP is derived from AOC bioassay in terms of the method and mechanism of determination, which is limited by poor comparability.

MAP (biologically available phosphorus) index was proposed by Lehtola et al for evaluating the level of biologically available phosphorus in water by establishing a standard curve between the concentration of disodium hydrogenphosphate standard substrate and the maximum value of growth of P17 strain by batch culture, the slope of which is a growth factor, measuring the maximum value of growth of P17 strain by inoculating and culturing P17 strain in a water sample supplemented with a non-phosphorus-containing inorganic salt solution and a sufficient amount of organic carbon, and converting the maximum value of growth into a concentration form expression against standard disodium hydrogenphosphate, i.e., a MAP value, using the growth factor. The MAP method is characterized in that standard substrate carbon acetate in the traditional AOC bioassay is replaced by sodium dihydrogen phosphate, and assimilable organic carbon is replaced by bioavailable phosphorus to evaluate the water quality biological stability, and mainly reflects the influence of phosphorus sources on the water quality biological stability, but the maximum value of the measured bacterial quantity growth still needs a long time, and the measuring steps are complicated.

Disclosure of Invention

The invention aims to overcome the limitation and the defect of the prior method for measuring the biological stability of the water quality of the drinking water and solve the problem that the judgment standard for judging whether the water quality is biologically stable does not exist. The traditional method for measuring the maximum bacterial quantity of biological growth needs long-time culture, has the defects of complicated measuring steps, long time, high cost, inapplicability to practical popularization and the like, and the measured result cannot be timely applied to water supply treatment and monitoring and analysis of the water quality biological stability of a drinking water pipe network.

The technical scheme provided by the invention is as follows:

a simple method for quickly predicting whether water quality is biologically stable includes quantitatively adding specific bacteria into water sample to be detected and pure water in equal quantity, carrying out ATP fluorescence detection, comparing detection results of ATP fluorescence detection and pure water, and predicting that water quality is biologically unstable if obvious difference exists, otherwise, determining that water quality is stable.

The specific bacteria refer to bacteria which are cultured at normal temperature for 1 month and are in a growth maintaining stage.

The water sample to be detected is tap water which is a terminal of a water supply system; or pure water, etc.

The significant difference refers to that when the repeated test value ranges are compared for more than 3 times, the test value ranges are not overlapped and have obvious intervals among different water samples; there is no significant difference if the overlap or adjacency is close.

The detection result and the judgment standard of the detection method for whether the water sample is biostable or not are as follows: whether the water sample can increase the reproduction of bacteria added into the water sample (the bacteria regrowth), if the regrowth happens, the organism is unstable, and if the regrowth does not happen, the organism is stable.

The prediction method is simple, convenient and quick, and the evaluation index of the method is high in application and popularization value and practical.

Drawings

FIG. 1 schematic diagram of the change of bacterial viability in water (ATP test result of equal bacterial viability)

FIG. 2 is a schematic diagram of the change of bacterial activity of water with different concentrations of nutrients

FIG. 3 water sample result chart of ATP fluorescence test in example 1

FIG. 4 water sample result chart of ATP fluorescence test in example 2

Detailed Description

The technical scheme of the invention is further explained by combining the drawings and the embodiment.

It is well known in the art that bacteria multiply and grow in nutrient rich waters, increasing the overall population; the metabolism speed is reduced in the water with less nutrition, the bacteria can only maintain growth, and the total number of cells is kept unchanged for a longer time period; the growth of the nutrient-poor water can not be maintained, and the metabolic abnormality and even the endogenous energy metabolism phenomenon can be caused.

When the number of bacteria in the water body does not increase, the water quality is considered to be biostable.

The technical scheme of the invention is based on the following findings and research results:

through experimental research, the invention takes the ultrapure water as a sample with stable water quality, obtains the characteristic of bacterial activity change in the ultrapure water under experimental test conditions, and finds that the water quality conforming to the characteristic also has biological stability, and the water quality with obvious difference from the characteristic is unstable biologically. (principle of establishment of method is shown in FIG. 1 and FIG. 2)

And (3) under the ultrapure water test and under specific conditions, the vitality change characteristic of bacteria (bacteria in a growth maintenance stage) is used as a defined standard for whether the water quality is biostable, so that the water quality biostable of the tested water body is predicted. Theoretical explanation:

adding a certain amount of the specific bacteria culture solution in the growth maintaining stage into the ultrapure water to form the bacteria ultrapure water solution. The growth activities of the original bacteria culture solution and the bacteria ultrapure water solution with the same quantity are respectively tested by utilizing an ATP fluorescence detection technology (the two tested samples are ensured to contain the same bacteria quantity), and the test results show that the test results of the original bacteria culture solution and the bacteria ultrapure water solution are obviously different. After bacteria enter the ultrapure water, the growth activity of the bacteria is obviously changed along with the water quality in the new environment and the water quality in the primary storage environment.

The test results show that the test results of the original bacteria culture solution and the bacteria ultrapure water solution have obvious difference, as shown in figure 1: the ATP test result of the activity of the equivalent bacterial amount; JC: adding ultrapure water ATP test result of the bacterial culture solution; j: ATP test results of primary bacteria culture.

Exploration and validation of further application aspects:

the same ATP test was also performed with bacteria added to drinking water samples of different dilution ratios, with ultrapure water as the diluent. By comparing the results of the test immediately after the addition of the bacteria, the test after 3 hours, the test after 6 days and the test after 14 days (fig. 2), it was found that in a short period of time (3 hours), the bacterial viability of all the tap water-containing water samples increased due to the nutrients of the tap water, and the 3 hour test value was taken as the initial bacterial load in the experiment. Results of the 6d curve and the 14d curve show that the bacterial load in the water samples of ultrapure water (C), 0.05 times diluted tap water (0.05S), 0.1 times diluted tap water (0.1S) and 0.2 times diluted tap water (0.2S) at the later stage of culture is reduced to be below the initial bacterial load of 3h, and no bacterial load increase occurs, so that the samples can be judged to be biostable; the test results of 0.4 times diluted tap water (0.4S), 0.6 times diluted tap water (0.6S), 0.8 times diluted tap water (0.8S) and original tap water (S) showed that the bacterial count was greater than or equal to 3 hours, and the increase in bacterial count was evident, and thus, it was judged that the sample was biologically unstable. The immediate test result (curve 0) shows that the bacterial viability test results of the biostable water sample and the biostable water sample have significant differences (when comparing the repeated test value ranges of 3 times, the test value ranges of different water samples have no overlap and have significant intervals, namely significant differences, and if the overlap or the adjacent are very close, the bacterial viability test results of different water samples have no significant differences). In the immediate test result (0 curve), it is found that compared with the ultrapure water C, the change of the bacterial activity of the biostable water samples (0.05S, 0.1S, and 0.2S) is not significantly different, the change range of the test result of the water samples with poor biostability (0.4S, 0.6S, 0.8S, and S) is significantly different from that of the above water samples, and the test values of the water samples with 0.4S, 0.6S, 0.8S, and S show a decreasing rule as the faucet water ratio increases.

FIG. 2 is a schematic diagram of the change of bacterial activity of water with different concentrations of nutrients. Four curves obtained from bacterial viability tests performed at different time points as shown in figure 2 after addition of bacteria:

immediate testing (0 curve);

test after 3 hours (3hr curve);

test 6 days later (6d curve);

after 14 days the test (14d curve).

Each curve, the abscissa of which represents different test water samples, includes 1 ultrapure water sample and 7 tap water samples with different dilution times, which are respectively:

ultrapure water (C);

tap water (0.05S) with a dilution factor of 0.05;

tap water (0.1S) with a dilution factor of 0.1;

tap water (0.2S) with a dilution factor of 0.2;

tap water (0.4S) with a dilution factor of 0.4;

tap water (0.6S) with a dilution factor of 0.6;

tap water (0.8S) with a dilution factor of 0.8;

the original tap water sample is diluted by 1 (1S);

the ordinate axis is the relative luminescence intensity value (unit: RLU) of the bacterial viability (ATP value).

According to the test and theoretical basis, a defined standard for predicting whether the water quality is biologically stable is established, and a simple method for rapidly predicting whether the water quality is biologically stable is invented, namely, specific bacteria (the specific bacteria refer to bacteria which are cultured at normal temperature for 1 month and are in a growth maintaining stage) are respectively added into a water sample to be detected (such as a tap water terminal of a water supply system) and ultrapure water in equal quantity, then ATP fluorescence detection is carried out, the detection results of the ATP fluorescence detection and the ATP fluorescence detection are compared, if the obvious difference exists (when the detection results are compared with the repeated detection value ranges of 3 times, the detection value ranges of different water samples are not overlapped and have obvious intervals, namely, the obvious difference exists, if the obvious difference exists, or if the obvious difference exists, namely, the water quality is predicted to be biologically unstable, and.

Further, examples are given.

Example 1

Description of the method

Assay apparatus, vessel and material

A constant temperature incubator; an aseptic worktop; an alcohol lamp; an ATP determinator; a 40ml carbonless sample bottle; pipettes (2-200. mu.l, 1ml, 10 ml); milli-Q ultrapure water instrument and pure water instrument.

Test bacterium

The bacteria used in the examples were P17(Pseudomonas fluoroscens) and NOx (Spirillum sp.)

Test preparation

1. Preparation of 1000 Gamma standard carbon solution (carbon acetate concentration 1000mg/L)

NaAc:5.67g/L

2. Preparation of 1000 gamma buffer solution

3. Preparation of 2 Gamma standard carbon solution (carbon acetate concentration 2000 mug/L)

2ml/L of 1000 gamma standard carbon solution

1ml/L of 1000 gamma buffer solution

4. Incubation of inoculated bacterial mother liquors in the test

The P17 and NOX colonies are inoculated into a 2 gamma standard carbon solution and cultured at a constant temperature of 30 ℃ for about one month, and then refrigerated and stored at 4 ℃.

The testing steps are as follows:

1. test bacteria (bacteria in drinking water, mainly P17 and NOX) prepared in advance were taken out.

2. Preparing a water sample: ultrapure water to be detected is prepared, and tap water to be detected is collected (if residual chlorine exists, the residual chlorine is neutralized by sodium thiosulfate). Each tap water sample is divided into two parts, one part is added with bacteria, and the other part is not added with bacteria. Case collection place: the water pipe network end tap water is used for certain campus residence.

3. Preparing a bacterium inoculation water sample: to 1ml of the water sample to be tested, 50. mu.l of the bacterial test solution was added.

4. ATP fluorescence detection: and testing an equivalent inoculation water sample and recording the result at room temperature. The test was repeated 3 times.

5. And (4) analyzing results: the reduced value JL-L of the inoculation tap water (obtained by subtracting the test value L from the test value JL) is compared with the test result JL of the inoculation ultrapure water, and the test results are obviously different (the change amplitudes of the respective 3 times of repeated test data of the two are not overlapped and have obvious distance, as shown in figure 3).

The result chart of the ATP fluorescence test water sample shown in FIG. 3 shows that: each sample was repeated 3 times. L is unanswered bacteria tap water; JL is inoculation tap water; JC is inoculating ultrapure water; JL-L is the test value of the tap water of the transformed strain.

Predictive conclusion of example 1: significant difference results indicate that the test sample is biologically unstable.

In figure 3, the results of 3 times of repeated tests of JL-L and JC show obvious difference, so that the quality of the tap water is predicted to be unstable.

Compared with the prior detection of the biological stability of the drinking water, the method has the advantages of rapidness, convenience, no limitation to specific heterotrophic bacteria and inoculation culture conditions, and capability of providing information to tightly buckle the biological stability of the water quality, and can be effectively applied to water quality regulation and control of drinking water plants and pipe network systems in time.

Example 2

Method descriptions, assay instruments, vessels, materials, etc. are referenced to example 1.

The determination method specifically comprises the following steps:

(1) preparation of test bacteria liquid: the specific bacteria culture solution is used as a test bacteria solution. The specific bacteria refer to bacteria which are cultured at normal temperature for 1 month and are in a growth maintaining stage; the species of bacteria is NOX (Spirillum sp.).

(2) Preparing a water sample: pure water (CS) was prepared with ultrapure water (JC) as a control water sample (pure water does not contain bacteria and therefore does not require conversion).

(3) Preparing a bacterium inoculation water sample: adding the same amount of bacterial test solution (50 mul of bacterial solution/ml of water sample) into the water sample to be tested.

(4) ATP fluorescence detection: pure inoculum water (CS) and ultrapure inoculum water (JC) were tested at room temperature and the results were recorded (see fig. 4). The test was repeated 3 times.

(5) And (4) analyzing results: the characteristics of the results of 3 times of repeated tests of ultrapure water (JC) and pure water (CS) are compared and analyzed (figure 4), and the water quality stability prediction conclusion is obtained by analyzing the results according to the water quality biological stability prediction standard found and established in the invention and having insignificant difference (the ranges of two water samples are overlapped and have no obvious distance).

In the sample ATP determination, the ratio of an ATP reagent to a sample is 50 mu l: 50 μ l.

Other experimental procedures and judgment methods refer to example 1.

This example illustrates that the method of the present invention is also applicable to the prediction of biostability of pure water quality.

Claims

1. A simple method for quickly predicting whether water quality is biologically stable is characterized in that specific bacteria are quantitatively added into a water sample to be detected and pure water in equal quantity, then ATP fluorescence detection is carried out, the detection results of the ATP fluorescence detection and the pure water are compared, if the ATP fluorescence detection and the ATP fluorescence detection are obviously different, the biological instability of the water quality is predicted, and if the ATP fluorescence detection and the ATP fluorescence detection are not obviously different, the biological instability of the water quality is.

2. The method according to claim 1, wherein the specific bacterium is a bacterium in a maintenance growth stage.

3. The method as claimed in claim 1, wherein the sample of water to be tested is tap water, a terminal of a water supply system; or water for water pipe network and pure water.

4. The method of claim 1, wherein the significant difference is that when comparing the range of 3 repeated test values, the range of test values between different water samples does not overlap and has significant spacing; if overlapping or adjacent are very close, there is no significant difference.

5. The method as claimed in claim 1, wherein the determination criteria of the biological stability of the water sample is as follows: whether the water sample can increase the reproduction of bacteria added into the water sample (the bacteria regrowth), if the regrowth happens, the organism is unstable, and if the regrowth does not happen, the organism is stable.