CN108504695B - Extracellular polymer and preparation method thereof, heavy metal treatment agent and heavy metal wastewater treatment method - Google Patents
Extracellular polymer and preparation method thereof, heavy metal treatment agent and heavy metal wastewater treatment method Download PDFInfo
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Abstract
The invention relates to the technical field of environmental microorganisms and wastewater treatment, in particular to an extracellular polymer and a preparation method thereof, a heavy metal treatment agent and a heavy metal wastewater treatment method. The invention discloses a preparation method of an extracellular polymer, which comprises the following steps: carrying out exogenous sulfur induction treatment on the aerobic heterotrophic bacteria to obtain extracellular polymers; the exogenous sulfur is one or more of sodium sulfide, sodium sulfite, sodium thiosulfate and sodium dithionite. The preparation method is simple to operate and environment-friendly, and experimental data show that the composition proportion of the extracellular polymeric substance is correspondingly changed after the induction of the exogenous sulfur, and the extracellular polymeric substance is Cu2+And Zn2+The adsorption capacity of the polymer almost reaches more than 1g/g, the excellent adsorption effect is shown, and the technical problem that the existing microbial extracellular polymer has poor capability of removing heavy metals is solved.
Description
Technical Field
The invention relates to the technical field of environmental microorganisms and wastewater treatment, in particular to an extracellular polymer and a preparation method thereof, a heavy metal treatment agent and a heavy metal wastewater treatment method.
Background
Heavy metal contamination refers to environmental contamination caused by heavy metals or compounds thereof. Heavy metal pollution in water mainly comes from human social activities, including mining and metallurgy, metal processing, chemical engineering, electroplating, use of pesticides and chemical fertilizers, pollution accidents and the like, the consequences are mainly water pollution, and the harm degree of the water pollution depends on the concentration and chemical form of heavy metals in the environment, food and organisms. Heavy metals are pollutants which are extremely harmful to the ecological environment, cannot be biodegraded after entering the environment, often participate in food chain circulation and are finally accumulated in organisms, damage the normal physiological metabolic activity of the organisms and harm the health of human bodies, for example, the zinc poisoning is caused by excessive zinc intake of the human bodies, and symptoms such as nausea and bellyache appear; gastrointestinal poisoning can occur when drinking large quantities of drinking water contaminated with copper. Metal pollution is a prominent problem in the environmental field of China at present.
Heavy metal wastewater is usually treated by chemical adsorption and other technologies, but when the concentration of heavy metals is at a lower level, the removal effect of the heavy metals by the methods is not obvious, and secondary pollution is generated. In recent years, more and more researchers have focused attention on extracellular polymeric substances.
Extracellular Polymers (EPS) are high molecular polymers produced by microorganisms, and are composed of organic Substances such as polysaccharides, proteins, nucleic acids, uronic acids, humic acids, lipids, and the like. The extracellular polymeric substance contains abundant functional groups, such as carboxyl, amino, hydroxyl, carbonyl and the like, and the functional groups can be complexed and chelated with metal elements in water, so as to capture and adsorb heavy metals in water. Aerobic heterotrophic bacteria are bacteria which can utilize various organic matters as carbon sources, are widely present in the environment, and can generate a large amount of extracellular polymers on the surface.
At present, the research on the extraction of the microbial extracellular polymer is less at home and abroad, and the removal capability of the extracted extracellular polymer to heavy metals is weaker.
Disclosure of Invention
The invention provides an extracellular polymer and a preparation method thereof, a heavy metal treatment agent and a heavy metal wastewater treatment method, and solves the technical problem that the existing microbial extracellular polymer has poor capability of removing heavy metals.
The specific technical scheme is as follows:
the invention provides a preparation method of an extracellular polymer, which comprises the following steps: carrying out exogenous sulfur induction treatment on the aerobic heterotrophic bacteria to obtain extracellular polymers;
the exogenous sulfur is one or more of sodium sulfide, sodium sulfite, sodium thiosulfate and sodium dithionite.
Preferably, the exogenous sulfur is sodium sulfide or sodium thiosulfate.
Preferably, the aerobic heterotrophic bacteria are selected from the group consisting of Bacillus vallismortis, Bacillus subtilis, and Pseudomonas aeruginosa, and more preferably Bacillus vallismortis.
Preferably, the concentration of the exogenous sulfur is 10mg/L to 140mg/L, more preferably 10mg/L to 120mg/L, even more preferably 40mg/L to 60mg/L, and most preferably 40mg/L and 60 mg/L.
Preferably, the induction treatment time is 2h to 48h, more preferably 24 h.
Preferably, before the exogenous sulfur induction treatment of the aerobic heterotrophic bacteria, the method further comprises the following steps: performing activated culture on the aerobic heterotrophic bacteria;
the volume percentage of the inoculation amount of the activated culture strain is 2-5%, and the preferable volume percentage is 5%.
More preferably, the components of the culture medium for activation culture comprise peptone, beef extract and sodium chloride, the pH value of the culture medium is 7.2 +/-0.2, the culture temperature is 20-40 ℃, and the oscillation speed is 130-200 rpm.
Further preferably, the culture temperature is 35 ℃ and the shaking speed is 150 rpm.
Preferably, after the activated culture of the aerobic heterotrophic bacteria and before the exogenous sulfur induction treatment of the aerobic heterotrophic bacteria, the method further comprises:
carrying out oxygen consumption treatment on the aerobic heterotrophic bacteria;
the time of the oxygen consumption treatment is 1-3 h.
More preferably, the oxygen-consuming treatment time is 2 h.
Preferably, after the exogenous sulfur induction treatment is performed on the aerobic heterotrophic bacteria, before the extracellular polymeric substance is obtained, the method further comprises the following steps:
exogenous sulfur was removed and then the cells were removed.
Preferably, the removed cells are: adding a salt solution with the volume percentage of 2% into the aerobic heterotrophic bacteria subjected to the exogenous sulfur induction treatment, and centrifuging at the temperature of 4 ℃.
More preferably, the removing of the bacterial cells is specifically: adding 2% of salt solution into the aerobic heterotrophic bacteria subjected to exogenous sulfur induction treatment, fully mixing, heating in a water bath, centrifuging at 4 ℃, filtering, and collecting supernatant. Wherein the salt solution is EDTA-NaCl solution (pH is 8.0 +/-0.2), the water bath temperature is 50-60 ℃, the centrifugation speed is 10000-12000 rpm, the centrifugation time is 10-15 min, and the filtering material is a cellulose acetate filtering membrane with the diameter of 0.22-0.45 mu m.
More preferably, the method of removing exogenous sulfur is: centrifuging the aerobic heterotrophic bacteria subjected to exogenous sulfur induction treatment for 15min at 4 ℃ and 4000rpm for three times, and cleaning with sterile water after each centrifugation to wash off the residual culture medium and exogenous sulfur on the bacteria.
The invention also provides the extracellular polymeric substance prepared by the preparation method.
The invention also provides a heavy metal treatment agent which comprises the extracellular polymeric substance.
The aerobic heterotrophic bacteria induced by exogenous sulfur can be prepared into pure EPS biological adsorbent, and can also be prepared into living or inactivated biological adsorbent containing microbial cells.
The invention also provides a heavy metal wastewater treatment method, which comprises the following steps: adding the extracellular polymer or the metal treatment agent to wastewater containing heavy metals;
the heavy metal is one or more of copper, zinc, lead, cadmium and chromium, and more preferably copper or zinc.
The preparation method is simple to operate and environment-friendly, and experimental data show that after the induction of the exogenous sulfur, the composition proportion of the extracellular polymeric substance is correspondingly changed, particularly the proportion of protein is increased, wherein the protein content is in the exogenous stateThe highest concentration was obtained when sodium sulfide was added at a concentration of 40mg/L and sodium thiosulfate at a concentration of 60mg/L, and in addition, extracellular polymeric substance was added to Cu2+And Zn2+Almost all the amounts of the components (A) and (B) were 1g/g or more, and an excellent adsorption effect was exhibited.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without inventive exercise.
FIG. 1 is a bar graph of extracellular polymeric substances induced by sodium sulfide in Bacillus vallismortis at various concentrations in example 1 of the present invention;
FIG. 2 is a graph showing the adsorption performance of extracellular polymeric substances generated by Bacillus vallismortis induced by sodium sulfide of different concentrations on heavy metals in example 1 of the present invention;
FIG. 3 is a bar graph showing the induction of extracellular polymeric substances by different concentrations of sodium thiosulfate in Bacillus vallismortis in example 1 of the present invention;
FIG. 4 is a graph showing the measurement of the adsorption performance of extracellular polymeric substances generated by Bacillus vallismortis induced by sodium thiosulfate with different concentrations on heavy metals in example 1 of the present invention;
FIG. 5 shows Cu concentrations in comparative examples of the present invention2+Inducing the bacillus vallismortis to generate an extracellular polymer to heavy metal adsorption performance measurement diagram;
FIG. 6 shows different Zn concentrations in comparative examples of the present invention2+Inducing the bacillus vallismortis to generate an extracellular polymer to heavy metal adsorption performance determination diagram.
Detailed Description
The embodiment of the invention provides an extracellular polymer and a preparation method thereof, a heavy metal treatment agent and a heavy metal wastewater treatment method, and solves the technical problem that the existing microbial extracellular polymer is poor in capability of removing heavy metals.
In order to make the objects, features and advantages of the present invention more obvious and understandable, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is obvious that the embodiments described below are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The extracellular polymeric substance and the preparation method thereof, the heavy metal treatment agent and the heavy metal wastewater treatment method provided by the invention are all available in the market.
The extracellular polymeric substance and the preparation method thereof, the heavy metal treatment agent and the heavy metal wastewater treatment method provided by the invention are further described below.
EXAMPLE 1 exogenous Sulfur Induction of extracellular polymers
In the embodiment, Bacillus vallismortis sp (which belongs to aerobic heterotrophic bacteria and is obtained by separation and purification in a laboratory) is selected. The specific preparation steps of the extracellular polymeric substance are as follows:
taking out the Bacillus vallismortis preserved at-18 ℃, thawing to room temperature, performing activation culture, and shaking at constant temperature for 48h to obtain Bacillus vallismortis suspension. During induction culture, the bacterial suspension is respectively inoculated into a liquid culture medium which is autoclaved for 20min at 121 ℃ under aseptic condition according to the inoculation amount of 5% (the culture medium is contained in conical flasks, each conical flask contains 100mL of the culture medium), the liquid culture medium is vibrated for 2h at constant temperature in a shaking table, after the vibration is finished, the sterilized sodium sulfide solution is injected into the liquid culture medium according to the inoculation amount of 2% under aseptic condition, so that the sulfur source concentration in 10 conical flasks is respectively 0, 10, 20, 30, 40, 50, 60, 80, 100 and 120mg/L, and exogenous sulfur induction culture is carried out for 24h under the conditions of 35 ℃ and 150 rpm. And (3) centrifuging the culture of the bacillus vallismortis subjected to induced culture at different exogenous sulfur concentrations for three times at the temperature of 4 ℃ and the rotation speed of 4000rpm for 15min, and cleaning the culture medium remained on the thalli by using sterile water after each centrifugation. And after three times of centrifugation, deionized water is added to prepare a bacterial suspension. And then adding an EDTA-NaCl solution into the bacterial suspension according to the proportion of 2% (V/V), fully mixing, placing in a water bath at 50 ℃ for heating, then centrifuging at 4 ℃ and 12000rpm for 15min, filtering the collected supernatant by using a 0.22 mu m acetate fiber filter membrane, dialyzing for 12h to obtain a specific extracellular polymer generated by the bacillus vallismortis after induction of different exogenous sulfur concentrations, and storing in a refrigerator at-18 ℃.
EXAMPLE 2 determination of major Components of extracellular polymers
In the determination of the main components of the extracellular polymer, the following methods were used: the total sugar content is determined by anthrone colorimetry, the protein content is determined by Coomassie brilliant blue G-250 method, and the nucleic acid content is determined by diphenylamine method. Total sugars, proteins and nucleic acids together characterize the composition of extracellular polymers.
As can be seen from FIGS. 1 and 3, in the examples of the present invention, Bacillus vallismortis was induced at different concentrations of S than before induction2-Under the induction, the content of protein in the specific extracellular polymeric substances generated by the bacillus vallismortis is highest when the concentration of the added sodium sulfide is 40mg/L and the concentration of the sodium thiosulfate is 60mg/L, the change trend of total sugar and nucleic acid is not obvious, and in general, the total amount of the extracellular polymeric substances after the induction by the exogenous sulfur is in an increasing trend, so that the protein is more obvious in expression.
Example 3 extracellular Polymer on heavy Metal Cu2+Measurement of adsorption Property
2mL of the specific extracellular polymer produced by the induction of exogenous sulfur in Bacillus vallismortis in example 1 was measured and added to 20mL of Cu2+Adjusting pH to about 5.0 in wastewater with concentration of 20mg/L, oscillating at 150rpm for 2 hr, loading the mixture into dialysis bag with molecular weight cutoff of 4000, dialyzing with deionized water for 12 hr, and measuring Cu after dialysis with flame atomic absorption spectrophotometer2+Concentrations were run in blank with uninduced extracellular polymer. 3 replicates were set for each sample and the experimental data averaged.
Example 4 extracellular Polymer on heavy Metal Zn2+Measurement of adsorption Property
2mL of the specific extracellular polymer produced by the induction of exogenous sulfur in Bacillus vallismortis in example 1 was measured and added to 20mL of Zn2+Adjusting the pH value to 5.0 in the waste water with the concentration of 20mg/LOscillating at 150rpm for 2h, filling the mixture into dialysis bag with molecular weight cutoff 4000, dialyzing with deionized water for 12h, and measuring Zn after dialysis with flame atomic absorption spectrophotometer2+Concentrations were run in parallel with a blank test with uninduced extracellular polymer. 3 replicates were set for each sample and the experimental data averaged.
FIGS. 2 and 4 show that the Bacillus vallismortis produces specific extracellular polymeric substance pair Cu under the induction of sodium sulfide and sodium thiosulfate in the embodiment of the invention2+And Zn2+The adsorption capacity of the bacillus vallismortis almost all reaches more than 1g/g, and experimental data show that the Cu is adsorbed by extracellular polymeric substances generated by the bacillus vallismortis under the induction of exogenous sulfur2+And Zn2+The excellent removal effect is shown.
Comparative example 1 heavy Metal Cu2+Inducing extracellular polymeric substances
Taking out the Bacillus vallismortis preserved at-18 ℃, thawing to room temperature, performing activation culture, and shaking at constant temperature for 48h to obtain Bacillus vallismortis suspension. During induction culture, respectively inoculating the bacterial suspension into liquid culture medium (culture medium contained in conical flasks each containing 100mL of culture medium) sterilized at 121 deg.C under high pressure for 20min at sterile condition according to 5% inoculum size, shaking in shaking table at constant temperature for 2 hr, and injecting sterilized Cu into the liquid culture medium under sterile condition according to 2% inoculum size2+Mother liquor, concentration of 10 conical flasks was 0, 10, 20, 30, 40, 50, 60, 80, 100, 120mg/L, Cu was adjusted at 35 ℃ and 150rpm2+And performing induction culture for 24 hours. Mixing the above Cu2+The culture of the dead millet bacillus after the concentration induction culture is centrifuged for three times at the temperature of 4 ℃ and the rotating speed of 4000rpm, the centrifugation time is 15min, and the culture medium remained on the thalli is washed by sterile water after each centrifugation. And after three times of centrifugation, deionized water is added to prepare a bacterial suspension. Adding trace amount of NaCl, stirring gently to make NaCl concentration in each bacterial suspension 0.85%, heating in 80 deg.C hot water for 120min, centrifuging at 4 deg.C and 12000rpm for 20min, filtering the collected supernatant with 0.22 μm filter membrane, concentrating the filtrate to 1/10 of original volume with rotary evaporator, and vacuum concentrating the concentrated solutionFreeze-drying in air to obtain Bacillus vallismortis in different Cu2+Specific extracellular polymers produced after concentration induction.
Comparative example 2 heavy Metal Zn2+Inducing extracellular polymeric substances
Taking out the Bacillus vallismortis preserved at-18 ℃, thawing to room temperature, performing activation culture, and shaking at constant temperature for 48h to obtain Bacillus vallismortis suspension. During induction culture, respectively inoculating the bacterial suspension into liquid culture medium (culture medium contained in conical flasks each containing 100mL of culture medium) sterilized at 121 deg.C under high pressure for 20min at sterile condition according to 5% inoculum size, shaking in shaking table at constant temperature for 2 hr, and injecting sterilized Zn into the liquid culture medium under sterile condition according to 2% inoculum size2+Mother liquor, Zn concentration of 10 conical flasks was 0, 10, 20, 30, 40, 50, 60, 80, 100, 120mg/L at 35 ℃ and 150rpm2+And performing induction culture for 24 hours. Different Zn is added2+The culture of the dead millet bacillus after the concentration induction culture is centrifuged for three times at the temperature of 4 ℃ and the rotating speed of 4000rpm, the centrifugation time is 15min, and the culture medium remained on the thalli is washed by sterile water after each centrifugation. And after three times of centrifugation, deionized water is added to prepare a bacterial suspension. Then adding trace amount of NaCl, stirring gently to make NaCl concentration in each bacterial suspension be 0.85%, heating in 80 deg.C hot water for 120min, centrifuging at 4 deg.C and 12000rpm for 20min, collecting supernatant, filtering with 0.22 μm filter membrane, concentrating filtrate to 1/10 of original volume with rotary evaporator, vacuum freeze drying the concentrated solution to obtain Bacillus vallismortis with different Zn contents2+Specific extracellular polymers produced after concentration induction.
Comparative example 3 Cu2+Determination of heavy metal adsorption performance of induced extracellular polymer
Bacillus vallismortis Cu in comparative example 1 was measured2+2mL of specific extracellular polymer generated under induction is added into 20mL of Cu2 +Adjusting pH to about 5.0 in wastewater with concentration of 20mg/L, oscillating at 150rpm for 2 hr, filling the mixture into dialysis bag with molecular weight cutoff of 4000, dialyzing with deionized water for 12 hr, and absorbing with flame atomSpectrophotometric determination of Cu after dialysis2+Concentrations were run in parallel with a blank test with uninduced extracellular polymer. 3 replicates were set for each sample and the experimental data averaged.
Comparative example 4 Zn2+Determination of heavy metal adsorption performance of induced extracellular polymer
Measuring Bacillus vallismortis Zn in comparative example 22+2mL of specific extracellular polymer generated under induction is added to 20mL of Zn2 +Adjusting pH to about 5.0 in wastewater with concentration of 20mg/L, oscillating at 150rpm for 2 hr, filling the mixture into dialysis bag with molecular weight cutoff of 4000, dialyzing with deionized water for 12 hr, and measuring Zn after dialysis with flame atomic absorption spectrophotometer2+Concentrations were run in parallel with a blank test with uninduced extracellular polymer. 3 replicates were set for each sample and the experimental data averaged.
FIGS. 5 and 6 are graphs showing the adsorption amounts of heavy metals to the extracellular polymeric substances after the induction of the heavy metals, and it can be seen from the graphs that the adsorption amounts of copper and zinc to the extracellular polymeric substances after the induction of the heavy metals of copper and zinc reach 1050mg/g and 980mg/g at maximum.
As can be seen from fig. 2, 4, 5 and 6, the exogenous sulfur-induced extracellular polymeric substance has a strong adsorption capacity to heavy metals, and is significantly improved compared to the heavy metal-induced extracellular polymeric substance.
The above-mentioned embodiments are only used for illustrating the technical solutions of the present invention, and not for limiting the same; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
Claims (10)
1. The preparation method of the extracellular polymeric substance is characterized by comprising the following steps: carrying out exogenous sulfur induction treatment on the bacillus vallismortis to obtain extracellular polymeric substances;
the exogenous sulfur is sodium sulfide or sodium sulfite.
2. The method according to claim 1, wherein the exogenous sulfur is present at a concentration of 10mg/L to 140 mg/L.
3. The preparation method according to claim 1, wherein the induction treatment time is 2 to 48 hours.
4. The method according to claim 1, wherein the step of treating Bacillus vallismortis with exogenous sulfur induction further comprises: performing activation culture on the bacillus vallismortis;
the volume percentage of the inoculation amount of the activated culture strain is 2-5%.
5. The method according to claim 4, further comprising, after the activated culture of Bacillus vallismortis and before the exogenous sulfur-inducing treatment of Bacillus vallismortis:
carrying out oxygen consumption treatment on the bacillus vallismortis;
the time of the oxygen consumption treatment is 1-3 h.
6. The method according to claim 1, wherein the step of obtaining extracellular polymeric substance after the exogenous sulfur-inducing treatment of Bacillus vallismortis further comprises:
exogenous sulfur was removed and then the cells were removed.
7. The method according to claim 6, wherein the removed cells are: and adding a saline solution with the volume percentage of 2-5% into the dead bacillus vallismortis subjected to the exogenous sulfur induction treatment, and centrifuging at the temperature of 4-8 ℃.
8. An extracellular polymer produced by the production method according to any one of claims 1 to 7.
9. A heavy metal treatment agent comprising the extracellular polymer according to claim 8.
10. The heavy metal wastewater treatment method is characterized by comprising the following steps: adding the extracellular polymer according to claim 8 or the metal treatment agent according to claim 9 to a wastewater containing a heavy metal;
the heavy metal is one or more of copper, zinc, lead, cadmium and chromium.
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