CN103424370B - The detection method of Phanerochaete chrysosporium viable cell biomass under heavy metal stress - Google Patents
The detection method of Phanerochaete chrysosporium viable cell biomass under heavy metal stress Download PDFInfo
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Abstract
The invention discloses the detection method of Phanerochaete chrysosporium viable cell biomass under a kind of heavy metal stress, comprise the following steps: (1) bacterium ball is cultivated: added by Phanerochaete chrysosporium in liquid culture medium and carry out constant temperature culture, then add heavy metal solution and carry out Stress treatment; (2) chromogenic reaction: the Phanerochaete chrysosporium bacterium ball obtained after getting above-mentioned heavy metal stress joins in aseptic tetrazolium bromide solution, chromogenic reaction 0.5h ~ 4h is carried out at 30 DEG C ~ 50 DEG C, then add rapidly hydrochloric acid solution cessation reaction, obtain reacted mixed liquor; (3) centrifugal treating: by mixed liquor centrifugal treating, removes supernatant, obtains lower sediment thing; (4) extraction detects: in sediment, add extractant extract, and measures the absorbance of gained extract, the viable cell biomass of Phanerochaete chrysosporium under calculating heavy metal stress.Detection method of the present invention is simple, convenient, application is wide and practical.
Description
Technical field
The present invention relates to microbial technology field and field of environmental toxicology, particularly relate to Phanerochaete chrysosporium viable cell biomass and active detection method under a kind of heavy metal stress.
Background technology
At present, Heavy Metals In Environment has become a global problem.Heavy metal in environment, by the enrichment also lasting existence in vivo of food chain effect, threatens ecologic environment and human health day by day.Utilize the heavy metal in microbiological treatment industrial waste water, have that expense is low, little to environmental impact, efficiency advantages of higher, more and more receive the concern of numerous scientific and technical personnel.In recent years, white-rot fungi has extensively and the degradation capability of uniqueness various xenobiotics with it, and is extensively studied, wherein, especially with in the majority to the research of its type culture Phanerochaete chrysosporium the good adsorption effect of heavy metal ions in wastewater.
In processing procedure, pollutant also has certain toxic action to Phanerochaete chrysosporium.These toxic pollutantss have certain inhibiting effect to the growth of Phanerochaete chrysosporium, breeding, metabolism and activity, and can cause the death of Phanerochaete chrysosporium cell, thus greatly reduce treatment effeciency.Organism waste water processing procedure requires that microorganism has enough viable cell biomass or cytoactive, could realize higher treatment effeciency.
Some traditional detection methods, such as dry weight in wet base method, blood cell plate counting method, the method for plate culture count and fluorescent microscope counting method etc. have been widely used in the activity detecting microorganism, but traditional method has shortcomings, such as: blood cell plate counting method, the method for plate culture count are wasted time and energy, and often occur the inaccurate situation of technology; Fluorescent microscope counting method uses various dyestuff to dye to cell, with microscopic examination dead/different colours that presents of living cells and counting, the color that after this just usually occurs dyeing, dead/living cells presents is distinguished not obvious, situation about easily obscuring.In addition, Phanerochaete chrysosporium is a kind of filamentous fungi, in Liquid Culture process, its mycelia is easy to be wound the mycelium pellet that diameter is about 3mm, even if its mycelia is also be intertwined to form large block or large sheet under static culture conditions, mycelia and nutrient solution can not form homogeneous system, and therefore traditional method is not all suitable for its viable cell biomass of mensuration.And simple cell weight (dry weight or weight in wet base) can not reflect physiological status, the aging degree of thalline in system for handling, cell even can not be distinguished anyway.Thus, setting up a kind of Fast Measurement Phanerochaete chrysosporium viable cell biomass, the detection method of effective evaluation mycelia active, seeming very important for better regulating and controlling waste water treatment efficiency.
Summary of the invention
Technical matters to be solved by this invention overcomes the deficiency that prior art exists, and provides a kind of simple, convenient, feasible, effective, detection method that can measure and reflect Phanerochaete chrysosporium viable cell biomass under heavy metal stress more accurately.
For solving the problems of the technologies described above, the technical solution used in the present invention is the detection method of Phanerochaete chrysosporium viable cell biomass under a kind of heavy metal stress, comprises the following steps:
(1) bacterium ball is cultivated: added in liquid culture medium by Phanerochaete chrysosporium (Phanerochaete chrysosporium) and carry out constant temperature culture, then Stress treatment is carried out to containing adding heavy metal solution in the liquid culture medium of Phanerochaete chrysosporium, after filtration after washing, obtain the Phanerochaete chrysosporium bacterium ball after heavy metal stress;
(2) chromogenic reaction: get the Phanerochaete chrysosporium bacterium ball after above-mentioned heavy metal stress and join in aseptic tetrazolium bromide (MTT) solution, at 30 DEG C ~ 50 DEG C, carry out chromogenic reaction 0.5h ~ 4h(tetrazolium bromide after chromogenic reaction, obtain tetrazolium bromide-formazan), then add rapidly hydrochloric acid solution cessation reaction, obtain reacted mixed liquor;
(3) centrifugal treating: above-mentioned mixed liquor is carried out centrifugal treating, removes supernatant, obtains lower sediment thing;
(4) extraction detects: in above-mentioned sediment, add extractant extract, be extracted liquid, measures the absorbance of extract, characterizes and the viable cell biomass of Phanerochaete chrysosporium under calculating heavy metal stress.
In above-mentioned method, Phanerochaete chrysosporium is cultured to growth logarithmic phase in (1) by described step, cultivation temperature preferably 30 DEG C ~ 39 DEG C.
In above-mentioned method, the time preferred 2h ~ 48h of Stress treatment described in step (1), the temperature of described Stress treatment preferably 30 DEG C ~ 39 DEG C.
In above-mentioned method, heavy metal solution described in step (1) is preferably containing cadmium, lead, copper, one or more heavy metal solution in chromium; When heavy metal solution is cadmium-containing solution, the concentration of cadmium ion preferably 1 μm of ol/L ~ 500 μm ol/L in heavy metal solution.
In above-mentioned method, the preferred 1g/L ~ 5g/L of concentration of aseptic tetrazolium bromide solution described in step (2), the preferred 0.2mol/L ~ 1mol/L of concentration of described hydrochloric acid solution.
In above-mentioned method, the preferred 8000rpm ~ 10000rpm of rotating speed centrifugal described in step (3), preferred 2min ~ 10min of centrifugal time.
In above-mentioned method, the preferred dimethyl sulfoxide (DMSO) of extractant, methyl alcohol, acetone or isopropyl alcohol, particularly preferably isopropyl alcohol described in step (4).
In above-mentioned method, the time preferred 2h ~ 4h of extraction described in step (4).
In above-mentioned method, step (1) ~ step (3) is required all needs sterilization treatment with vessel, and agents useful for same solution all configures by sterile liquid.
The Cleaning Principle of detection method is the angle from fungal physiology metabolism, and metabolism is closely related with energetic supersession.The key of energetic supersession is that in cell, oxidation operation decomposes generation hydrogen, releases energy through respiratory chain oxidation.Dehydrogenasa is the trunk enzyme system of respiratory chain, and they can be oxidized matrix the hydrogen taken off, and give respiratory chain transmission and give off energy, for biosynthesizing and the vital movement maintaining cell.Therefore measure Dehydrogenase activtity in funginite, the physiologically active of mycelia can be characterized, the viable cell biomass in reflection sample.In the present invention, Phanerochaete chrysosporium after heavy metal stress is joined in tetrazolium bromide solution, it accepts hydrogen in cellular respiration process, the empurpled formazan of shape (Fomazan) particle (MTTF) in cell, and within the specific limits; the granuloplastic amount of formazan is relevant with somatic cells dehydrogenase activity, is also directly proportional to thalline living cells quantity.Therefore, after adopting extractant to extract, by measuring the absorbance of extract, and then the viable cell biomass of reflection thalline.In addition, by measuring dehydrogenasa specific activity in thalline, can characterize microbial activity, its computing formula is as follows:
In formula, DHA---Dehydrogenase activtity (pKat)
γ---MTT reduction reaction initial velocity (A/min)
V---extract volume (ml)
E---MTT molar extinction coefficient [L/ (mol × cm)]
In formula, Specific DHA---dehydrogenasa specific activity (pKat/g)
W---thalline weight in wet base (g)
The activity of dehydrogenasa in thalline can be drawn by above-mentioned formula, above computing method realize based on this index of extract absorbance, namely in computing formula except " γ---MTT reduction reaction initial velocity (A/min) " is except this, the parameters of remainder formula is all the definite value set in mensuration process, only have γ relevant to sample, the unit A/min of γ represents the variable quantity of absorbance per minute, and wherein the time is also setting value, change only have A(absorbance).
Compared with prior art, the invention has the advantages that:
The present invention is the detection method of Phanerochaete chrysosporium viable cell biomass under heavy metal stress, and this detection method is accurate and effective, high specificity; The instrument and equipment used in testing process is few, easy and simple to handle, feasible; Detection method can measure and reflect Phanerochaete chrysosporium viable cell biomass and activity under heavy metal stress more accurately.The present invention is a kind of Fast Measurement Phanerochaete chrysosporium viable cell biomass, and the detection method of effective evaluation mycelia active, provides important leverage for better regulating and controlling waste water treatment efficiency.The present invention is widely used in micro-organisms living cell biomass and active detection under heavy metal stress, and be particularly useful for the viable cell biomass to filamentous fungi and Activity determination, application is wide, practical.
Accompanying drawing explanation
Fig. 1 is the ultraviolet-visible absorption spectroscopy figure of tetrazolium bromide (MTT) in dimethyl sulfoxide (DMSO), methyl alcohol, acetone and isopropyl alcohol extraction agent in the embodiment of the present invention.
Fig. 2 is the ultraviolet-visible absorption spectroscopy figure of tetrazolium bromide-formazan (MTTF) in dimethyl sulfoxide (DMSO), methyl alcohol, acetone and isopropyl alcohol extraction agent in the embodiment of the present invention.
Fig. 3 is the linear relationship chart of extract absorbance and different chromogenic reaction time in the embodiment of the present invention.
Fig. 4 is the linear relationship chart of extract absorbance and viable bacteria weight in wet base in the embodiment of the present invention.
Embodiment
Below in conjunction with Figure of description and concrete preferred embodiment, the invention will be further described, but protection domain not thereby limiting the invention.
Embodiment:
The bacterial classification adopted in the present embodiment for Phanerochaete chrysosporium (Phanerochaete chrysosporium) BKM-F1767(Unite States Standard (USS) type culture collection deposit number be ATCC24725, preferably adopt this bacterial strain, but be not limited thereto).
In the present embodiment, vessel used are all through sterilization treatment, and agents useful for same solution all configures by sterile liquid.
A detection method for Phanerochaete chrysosporium viable cell biomass under heavy metal stress of the present invention, comprises the following steps:
(1) Phanerochaete chrysosporium bacterium ball is cultivated and Cd stress
Phanerochaete chrysosporium spore powder is scraped from slant medium, in sterilized water, makes spore suspension.Be inoculated into by spore suspension in the fluid nutrient medium (Kirk nutrient culture media) in conical flask, fluid nutrient medium 200mL is housed in 500mL conical flask, cultivates in constant temperature oscillation case, cultivation temperature is 37 DEG C, and shaking speed is 150rpm.Cultivate the cadmium-ion solution (1,10,50,100,500 μm of ol/L) of adding a series of concentration gradient after 48h in cultivating system, coerce 24h, then filtration washing obtains the Phanerochaete chrysosporium bacterium ball after Cd stress, and wet bacterium is heavily in table 1.
The different cadmium concentration of table 1 is coerced and to be wet the heavy impact of bacterium on Phanerochaete chrysosporium
| Cadmium concentration (μm ol/L) | 0 | 1 | 10 | 50 | 100 | 500 |
| Thalline weight in wet base (g) | 6.19 | 4.844 | 4.416 | 3.212 | 2.886 | 2.682 |
As shown in Table 1, along with the increase of cadmium concentration, thalline weight in wet base obviously reduces, and illustrates that cadmium inhibits the growth and breeding of Phanerochaete chrysosporium to a certain extent.
The determination of optimum extractant selection and maximum absorption wavelength
Get four groups of centrifuge tubes, often pipe adds 0.2g Phanerochaete chrysosporium bacterium ball, then the aseptic tetrazolium bromide working fluid (hereinafter referred to as tetrazolium bromide working fluid) of 1mL80mg/L is added, 2h is reacted at 50 DEG C, add rapidly the HCl solution cessation reaction of 0.5mL1mol/L, centrifugal 5min under 10000rpm, remove supernatant, the dimethyl sulfoxide (DMSO) (DMSO) of 8mL is added respectively in four groups of centrifuge tubes, the methyl alcohol of 8mL, the acetone of 8mL and the isopropyl alcohol (namely often pipe adds a kind of extractant) of 8mL, extraction 2h, be extracted liquid, with the light absorption value of ultraviolet-visible pectrophotometer scanning extract under each wavelength, the results are shown in Figure 2, its maximum absorption wavelength and maximum light absorption value are in table 2.Separately get four groups of centrifuge tubes, often pipe adds the tetrazolium bromide working fluid of 1mL80mg/L, and in each group of centrifuge tube, add the isopropyl alcohol of the dimethyl sulfoxide (DMSO) of 8mL, the methyl alcohol of 8mL, the acetone of 8mL and 8mL respectively, the light absorption value of each pipe is measured under similarity condition, the results are shown in Figure 1, its maximum absorption wavelength and maximum light absorption value are in table 2.
The maximum absorption wavelength of the different extractant of table 2
From Fig. 1, Fig. 2 and table 2, the effect of extracting of tetrazolium bromide-formazan generated after the tetrazolium bromide of same concentrations and Phanerochaete chrysosporium react in four kinds of extractants is best with acetone, the maximum light absorption value of its excess-three kind (dimethyl sulfoxide (DMSO), methyl alcohol and isopropyl alcohol) is more or less the same, and there is skew slightly the maximum absorption wavelength position of MTTF in different extractant; Comparison diagram 1 and Fig. 2 known, before reaction, the maximum absorption wavelength of tetrazolium bromide falls far short with the maximum absorption wavelength of the rear MTTF of reaction, and corresponding to the maximum absorption wave strong point of MTTF, in Fig. 1, the light absorption value of tetrazolium bromide is close to 0, to show in medium unreacted completely tetrazolium bromide do not disturb the mensuration of MTTF.Consider the toxicity of acetone, in follow-up experiment, we select isopropyl alcohol as extractant.
(2) under Cd stress, the viable cell biomass of Phanerochaete chrysosporium detects (comprising chromogenic reaction, centrifugal treating and extraction to detect)
Phanerochaete chrysosporium bacterium ball after the variable concentrations Cd stress of gained in step (1) is respectively got 0.2g to add in 6 groups of centrifuge tubes respectively, to often organizing in centrifuge tube the tetrazolium bromide working fluid adding 1mL5g/L, 2h is reacted at 50 DEG C, add rapidly the HCl solution cessation reaction of 0.5mL1mol/L again, centrifugal 5min under 10000rpm, remove supernatant, 6mL isopropyl alcohol is added respectively in each group of centrifuge tube, extraction 2h, measures the absorbance of extract, the results are shown in Table 3 in 534nm place.
Table 3 variable concentrations Cd stress is on the impact of Phanerochaete chrysosporium viable cell biomass
Note: in table, W
0for thalline weight in wet base (g) taken when measuring; A is sample absorbance; A
0for control group (not adding Cd stress) absorbance.
As shown in Table 3, the absorbance of extract reduces along with the increase of the cadmium concentration added, and namely in sample, viable cell biomass is minimizing trend; The heavy metal cadmium added can cause the death of cell, and this effect is all the more obvious along with the increase of cadmium concentration.When equal thalline weight in wet base, the viable cell biomass in each sample differs greatly, and significantly reduces along with the increase of cadmium concentration.This experimental result shows, the inventive method can measure and reflect the viable cell biomass of Phanerochaete chrysosporium under heavy metal stress more accurately.
Meanwhile, by the absorbance of contrast Cadmium treated sample (interpolation cadmium) with control sample (not adding cadmium), the suppression degree of Cd stress to Phanerochaete chrysosporium activity can be drawn.
Wherein, η is suppression degree, A
0for the absorbance of control sample, A is the absorbance for the treatment of samples.
The impact of chromogenic reaction time
Get 5 groups with the Phanerochaete chrysosporium bacterium ball after 10 μm of ol/L Cd stress 24h, often organize 0.2g, add the tetrazolium bromide working fluid of 1mL5g/L, at 50 DEG C, react a period of time, add rapidly the HCl solution cessation reaction of 0.5mL1mol/L, centrifugal 5min under 10000rpm, remove supernatant, in each group of centrifuge tube, add 6mL isopropyl alcohol respectively, extraction 2h, measure the absorbance of extract in 534nm place, the results are shown in Table 4 and Fig. 3.
The table 4 chromogenic reaction time is on the impact of color developing effect
| Reaction time (h) | 0.5 | 1 | 2 | 3 | 4 |
| Absorbance (A) | 0.421 | 0.444 | 0.489 | 0.543 | 0.570 |
As shown in Table 4, along with the increase of chromogenic reaction time, the absorbance of extract presents certain increase.Doing extract absorbance---reaction time curve (Fig. 3) is known, is that in 0.5 ~ 4h, absorbance linearly changed the reaction time in the chromogenic reaction time, coefficient R
2=0.9904.For the consideration of complete degree of reaction and conventional efficient two aspect, our preferred 2h is as optimum reacting time.
The checking of assay method
Take respectively do not add Cd stress Phanerochaete chrysosporium wet thallus 0.04,0.08,0.12,0.16,0.20g, add the thalline of high-temperature inactivation successively, make each sample gross weight be 0.2g, by the absorbance of detection method working sample of the present invention.Investigate the linear relationship between extract absorbance and viable bacteria weight in wet base, each gradient does three Duplicate Samples, and calculates the relative deviation between Duplicate Samples, and investigate the accuracy of the method, measurement result as shown in Figure 4.
As shown in Figure 4, when viable bacteria weight in wet base is in the scope of 0.04 ~ 0.20g, absorbance and viable bacteria weight in wet base present good linear relationship (R
2=0.9984), illustrate that absorbance can reflect viable cell biomass in sample well.Using the straight line in Fig. 4 as typical curve, with viable cell biomass in absorbance characterizing sample, verify the result (i.e. table 3) in above-mentioned the present embodiment step (2), result is as shown in table 5.
The checking (with result in table 3 for contrast) of table 5 assay method
As shown in Table 5, calculate with absorbance and calculate two kinds of methods with typical curve and show that the viable cell biomass in sample is substantially identical, illustrating that the inventive method accurately and reliably; Therefore, not add the control group of Cd stress for benchmark, by formula
or typical curve can calculate Phanerochaete chrysosporium viable cell biomass under Cd stress, also can directly reflect by the size of absorbance and characterize Phanerochaete chrysosporium viable cell biomass, in this, as the index evaluating Phanerochaete chrysosporium mycelia, bacterium ball vigor.In above formula, W is viable cell biomass (g); W
0for thalline weight in wet base (g) taken when measuring; A is sample absorbance; A
0for control group (not adding Cd stress) absorbance.
From above result, method of the present invention is utilized to detect the activity of Phanerochaete chrysosporium cell under toxic pollutants is coerced in wastewater treatment, contribute to the physiological response mechanism of the Antagonistic Environment pollutant furtheing investigate Phanerochaete chrysosporium, accelerate and improve the application of Phanerochaete chrysosporium on Environmental Biotechnology.
The above is only the preferred embodiment of the present invention, protection scope of the present invention be not only confined to above-described embodiment, and all technical schemes belonged under thinking of the present invention all belong to protection scope of the present invention.It is noted that for those skilled in the art, improvements and modifications under the premise without departing from the principles of the invention, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (8)
1. the detection method of Phanerochaete chrysosporium viable cell biomass under heavy metal stress, comprises the following steps:
(1) bacterium ball is cultivated: added by Phanerochaete chrysosporium in liquid culture medium and carry out constant temperature culture, then Stress treatment is carried out to containing adding heavy metal solution in the liquid culture medium of Phanerochaete chrysosporium, after filtration after washing, obtain the Phanerochaete chrysosporium bacterium ball after heavy metal stress;
(2) chromogenic reaction: get the Phanerochaete chrysosporium bacterium ball after above-mentioned heavy metal stress and join in aseptic tetrazolium bromide solution, chromogenic reaction 0.5h ~ 4h is carried out at 30 DEG C ~ 50 DEG C, then add rapidly hydrochloric acid solution cessation reaction, obtain reacted mixed liquor;
(3) centrifugal treating: above-mentioned mixed liquor is carried out centrifugal treating, removes supernatant, obtains lower sediment thing;
(4) extraction detects: in above-mentioned sediment, add extractant extract, be extracted liquid, measures the absorbance of extract, the viable cell biomass of Phanerochaete chrysosporium under calculating heavy metal stress;
Described in step (2), the concentration of aseptic tetrazolium bromide solution is 1g/L ~ 5g/L, and the concentration of described hydrochloric acid solution is 0.2mol/L ~ 1mol/L.
2. detection method according to claim 1, is characterized in that, Phanerochaete chrysosporium is cultured to growth logarithmic phase in (1) by described step, and cultivation temperature is 30 DEG C ~ 39 DEG C.
3. detection method according to claim 1, is characterized in that, the time of Stress treatment described in step (1) is 2h ~ 48h, and the temperature of described Stress treatment is 30 DEG C ~ 39 DEG C.
4. detection method according to claim 1, is characterized in that, heavy metal solution described in step (1) is containing cadmium, lead, copper, one or more heavy metal solution in chromium; When heavy metal solution is cadmium-containing solution, in heavy metal solution, the concentration of cadmium ion is 1 μm of ol/L ~ 500 μm ol/L.
5. detection method according to claim 1, is characterized in that, rotating speed centrifugal described in step (3) is 8000rpm ~ 10000rpm, and the centrifugal time is 2min ~ 10min.
6. detection method according to claim 1, is characterized in that, described in step (4), extractant comprises dimethyl sulfoxide (DMSO), methyl alcohol, acetone or isopropyl alcohol.
7. detection method according to claim 1, is characterized in that, described in step (4), the time of extraction is 2h ~ 4h.
8. the detection method according to any one of claim 1 ~ 7, is characterized in that, in step (1) ~ step (3), required vessel all need sterilization treatment, and agents useful for same solution all configures by sterile liquid.
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