CN103451103A - High-cadmium-adsorption filamentous fungus Paecilomyces lilacinus XLA, and preparation method and application thereof - Google Patents
High-cadmium-adsorption filamentous fungus Paecilomyces lilacinus XLA, and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a high-cadmium-adsorption filamentous fungus Paecilomyces lilacinus XLA, and a preparation method and an application thereof. The preparation method comprises the following steps: 1, weighing a pot experiment soil sample used for heavy metal restoration and having a content of heavy metal cadmium of 50mg/Kg, adding to sterile water with glass beads, oscillating on a constant temperature shaking table, allowing an obtained solution to stand, taking an obtained supernatant, and carrying out serial dilution; 2, coating a Martin flat containing 60mM of cadmium with different dilution rates of dilutions, allowing the obtained flat to stand for a moment, inverting on a constant temperature culturing box, and culturing; 3, carrying out streaking purification of single colonies growing on the flat, coating the Martin flat with the obtained single colonies, and culturing; 4, carrying out separating screening to obtain the Paecilomyces lilacinus XLA stably resisting cadmium, and preserving on a PDA inclined plane for later use. The Paecilomyces lilacinus XLA is preserved and has a preservation number of CCTCC NO:M2012135. Viable and non-viable organism of the XLA can efficiently adsorb and remove the heavy metal cadmium in heavy metal sewage, so the XLA has the advantages of low cost and simple technology, and has an efficient restoration potential of heavy metal polluted soil and water.
Description
Technical field
The invention belongs to water body environment and administer field.The filamentous fungus Paecilomyces lilacinus XLA that more specifically relates to a kind of high absorption cadmium also relates to the preparation method of a kind of filamentous fungus Paecilomyces lilacinus XLA of high absorption cadmium simultaneously, also relates to the purposes of a kind of filamentous fungus Paecilomyces lilacinus XLA of high absorption cadmium.
Background technology
Since 20th century, industrialized development is played huge pushing effect to progress of human society, but has brought serious environmental pollution also to water body and soil simultaneously, especially heavy metal contamination, and then have influence on human health (Hsu et al., 2006; Beyer, 1985), a series of heavy metal poisoning, heavy metals exceeding standard event, (Luigi C., et al, 2006 such as minamata disease that the Japan caused such as cadmium pollution " itai-itai ", mercury pollution cause; Ma little Fan etc., 2004).Heavy metal contamination is one of current topmost environmental problem.
Cadmium (Cd) is the nonessential metallic element of a kind of organism, under low consistency conditions, just can produce toxicity to cell.Due to the mankind's activity, cadmium in entered environment, causes serious cadmium pollution by all means.Cadmium has stable accumulation and is difficult for the characteristics of eliminating in environment and in organism.The maximum characteristics of cadmium Transport And Transformation are be difficult for by the organism decomposition and inversion and excrete, and are difficult for moving with water, but up transmit step by step and make the human body chronic poisoning by inrichment along food chain.The compound of cadmium in 1992 is confirmed as IA level carcinogens by IARC (IARC); Classified as the 6th toxic substance be detrimental to health (Huang Baosheng, 2005) by U.S. toxicant management committee (ATSDR).At present, tactful become a huge challenge (BastaN.T., et al.2001 are administered in the cadmium pollution of finding soil and water; McGregorD B, et al., 2000).
The soil of heavy metal contamination and the treatment process of water body are had to chemical method, ion exchange method, absorption method, electrolytic process, chemistry redox method, electrochemical treatment method, reverse osmosis method and membrane separation process etc., but all because technology is loaded down with trivial details, easily cause the high limiting factor of secondary pollution mistake relative to processing cost can't drop into (Kratochvil D in production application, Volesky B, 1998).Biosorption process restoration of soil polluted by heavy metal and water body refer to by the interaction between organism and heavy metal particles and alleviate the harm (Volesky B., 1999) of heavy metal for environment.At biological renovation method in biomaterial used, fungal material has that biomass is large, the heavy metal adsorption capacity is large, easy many merits (Gao Wei etc., 2005 such as recovery of metal after absorption; Bhainsa K.C., Souza S.F., 1999; Sud D, et al., 2008; Wang J, Chen C, 2006; Goksungur Y, et al., 2005).And the microbial metabolism material is as exocellular polysaccharide, chitin etc. also participate in absorption and immobilization process (May H.et al, 1993 of heavy metal; Gadd GM, White C, 1993; Brim H., et al, 2000; Galun M., et al, 1983).This mainly is based on the functional group on microorganism cells surface, as hydroxyl, and carbonyl, sulfydryl and amine groups etc., these functional groups can be combined effectively with heavy metal cation, thereby have adsorption effect (Volesky, B. to heavy metal, Holan, Z.R, 1995).Fungi is the focus of Pollution abatement research and utilization to the biological adsorption of heavy metal.
At present, along with socioeconomic development, heavy metal contamination is day by day serious, sets up economy, and heavy metal contamination reparation system seems particularly important efficiently.Microbe species is various, and breeding rapidly; Microorganism cells after absorption also can pass through chemical reagent desorption, midwifery thing material back and forth.Simultaneously, the microorganism of repairing for heavy metal can reduce heavy metal concentration to some heavy metal absorption, precipitation, oxidation and reductive action, thereby reduces toxicity.The present invention utilizes microorganism to produce these characteristics of patience being subject to the physical environment heavy metal stress, and the Cadmium resistance microorganism is screened, and obtains and provide the efficient tolerance bacterial strain that can be used for biological treating heavy metal contamination.Simultaneously, the present invention for the treatment of heavy metal-polluted water, finds that this bacterial strain has good processing power and uses potentiality by this bacterial strain.The present invention is intended to provide for the microorganism repairing heavy metal pollution resistant strain that various heavy is had to relative high absorption capacity and stable performance.
Summary of the invention
The objective of the invention is to be to provide a kind of filamentous fungus Paecilomyces lilacinus XLA.The present invention has separated a strain filamentous fungus from the pot experiment soil sample for the heavy metal cadmium reparation.The remarkable adsorbing heavy metal cadmium of this bacterial strain energy under laboratory condition; Simultaneously, the invention still further relates to the bacterium active condition on the impact of absorption and this bacterium in the practical application aspect the heavy metal-polluted water reparation, result show filamentous fungus provided by the present invention the soil and water of heavy metal contamination administer and commercial applications on all have broad prospects.
Another object of the present invention is the preparation method who has been to provide a kind of filamentous fungus Paecilomyces lilacinus XLA, and this preparation method is simple and efficient, and the thalline obtained has the ability of high Adsorption of Heavy Metals, and adsorption process is not subject to the restriction of nutritional condition.
Further object of the present invention is to be to provide the application of a kind of filamentous fungus Paecilomyces lilacinus XLA in heavy metal cadmium is administered.Filamentous fungus XLA viable bacteria and dead bacterium can be adsorbed the heavy metal cadmium removed in heavy metal containing sewage efficiently, reach the purpose of administering heavy metal contamination, effectively reduce production costs, simplify treatment process, thereby provide possibility for preparing on a large scale the heavy metal polluted waste water water purification agent, possessed the potentiality of commercialized development.
In order to realize above-mentioned purpose, the present invention adopts following technical measures:
The preparation method of a kind of filamentous fungus Paecilomyces lilacinus XLA, its step is separation, purifying and multiple sieve.Detailed process is:
A, take the pot experiment soil sample 10g repaired for heavy metal that heavy metal cadmium content is 50mg/Kg in 90ml the sterilized water with 5 0.5mm granulated glass spherees, 200r/min vibration 30min on 28 ℃ of constant-temperature tables, after standing 1min, get supernatant liquor and carry out 10 times of serial dilutions;
B, the diluent of the different extension rates of 100 μ L is coated on the Martin's flat board that contains the 60mM cadmium, in static a moment, is inverted in 28 ℃ of constant incubators and cultivates 5d;
C, by the microorganism grown on the flat board separation and purification of being rule, then again be applied to and above-mentionedly cultivated containing on Martin's flat board of cadmium;
D, after 10 separation screenings, preserve the bacterial strain that cadmium is had to the stable resistance ability, on the PDA inclined-plane, preserved, standby.
According to aforesaid method, the pot experiment soil sample of applicant from repairing for heavy metal cadmium, utilize the fungi selective medium to be screened, separation has obtained a strain can be the filamentous fungus containing growing on Martin's rose bengal medium of 60mM cadmium, and the applicant is by its called after XLA.Through identifying that this filamentous fungus is Paecilomyces lilacinus (Paecilomyces lilacinus), the preservation of this bacterial strain and product spore substratum are the PDA substratum.The applicant delivers this bacterial strain to Chinese Typical Representative culture collection center (CCTCC) preservation in the Wuhan University of Wuhan City, Hubei Province on April 24th, 2012, address: Wuhan, China Wuhan University, deposit number: CCTCC NO:M2012135, Classification And Nomenclature: Paecilomyces lilacinus XLA Paecilomyces lilacinus XLA.
The characteristic of filamentous fungus Paecilomyces lilacinus XLA is as follows:
1. biological characteristics: filamentous fungus, obligate is aerobic, and 20 ℃ ~ 28 ℃ of optimum growth temperatures, grow rapider, but producing the decline of spore ability more than 35 ℃, in the PDA substratum, sporulation quantity is large, and bacterium colony presents felted, the middle part protuberance, cultivate early stage bacterium colony and present white, and the later stage produces spore and becomes burgundy.
2. genetics characteristic: combining form is learned and is observed and molecular biosciences identifies that this bacterium of confirmation is Paecilomyces lilacinus (Paecilomyces lilacinus).
3. culture condition: the substratum used when preserving this fungi and obtaining its spore is the PDA substratum; What this fungi of separation screening was used is Martin's substratum: glucose 10g, peptone 5g, MgSO
40.5g, distilled water 1000ml, pH nature, 121 ℃ of high pressure steam sterilization 30min.For adsorption experiment and test it to mineral salts medium for heavy metal tolerance (MM): glucose 20g, ammonium sulfate 5g, potassium primary phosphate 15g, anhydrous magnesium sulfate 0.6g, Calcium Chloride Powder Anhydrous 0.6g, iron vitriol 0.005g, manganese sulfate monohydrate 0.0016g, Zinc Sulphate Heptahydrate 0.0014g, cobalt chloride 0.002g, distilled water 1000ml dissolves, and regulates pH to 5.5,115 ℃ of high pressure steam sterilization 15min.Cultivating the form that this bacterium can spore is inoculated: the tween-80 with 2% volume ratio rinses the spore on the PDA inclined-plane, inoculates after then with the tween-80 of 0.2% volume ratio, being diluted to the spore concentration of needs, then carries out 28 ℃, the 150r/min shake-flask culture.
4. functional performance: there is the tolerance to various heavy.Filamentous fungus XLA to the tolerance detected result of different heavy metal ion is: under the liquid culture condition to cadmium (Cd
2+) 130mM, cobalt (Co
2+) 45mM, copper (Cu
2+) 80mM, zinc (Zn
2+) 220mM, chromium (Cr
3+) 60mM, chromium (Cr
6+) 5mM, nickel (Ni
2+) heavy metal such as 24mM has tolerance preferably, next 265mM, 50mM, 80mM, 145mM, 35mM, 4mM, the 16mM of being respectively of solid culture condition.
The application of a kind of filamentous fungus Paecilomyces lilacinus XLA in the biological restoration of heavy metal-polluted water and soil, the steps include:
(1) absorption of static cadmium:
Strain X LA is cultivated on the PDA substratum to 4d and produce a large amount of spores, prepare spore suspension, then be inoculated in the fresh mineral salts medium of 200 milliliters and cultivate 5d according to the ratio of 1:100, after filtering the collection thalline and carrying out the vacuum lyophilization processing, thalline is broken up to loose condition (of surface), then carried out the adsorption experiment of cadmium.
0.002g freeze-drying thalline is added in the 50mL centrifuge tube that contains the 20mL cadmium solution and carries out, be placed in 28 ℃ of constant-temperature tables, 150r/min reacts 240min, in reaction process respectively 0,5,15,30,45,60,120,180 and 240min get 100 μ L supernatant liquors, carry out the cadmium content analysis by the sampling Graphite Furnace Atomic Absorption spectrophotometer after dilution.
(2) the attached experiment of absorption-desorption:
Select thalline after frozen dried to carry out the adsorption experiment of cadmium.Concrete adsorption experiment condition: cadmium solution concentration is that 500mg/L, pH 4.0,0.1g/L freeze-drying thalline fully adsorb 4h, adsorb saturated rear supernatant sampling, with the content of cadmium in supernatant liquor before and after the absorption of sampling Graphite Furnace Atomic Absorption spectrophotometric determination, calculate the content of the cadmium that is adsorbed onto the thalline surface after diluted sample.Filter and collect the rear sample of absorption and carry out lyophilize.Choose respectively the EDTA, 0.1mol/L sodium hydroxide of deionized water, 0.1mol/L nitric acid, 0.1mol/L hydrochloric acid, 2% mass volume ratio as the desorption agent, system reaction 4h with 0.01g adsorption sample/20ml desorption agent, select the suitableeest desorption agent, and desorption kinetics under the suitableeest desorption agent effect is studied.
(3) impact of the active condition of bacterial strain on cadmium absorption:
The spore that strain X LA is produced in the activation of PDA substratum in 4 days is afterwards collected and is made suspension, then be inoculated in the fresh mineral salts medium of 200ml and cultivate 5d according to the ratio of 1:100, filter and collect thalline (cultivating the triangular flask of collecting the active condition thalline is the triangular flask of bottom with rib, and cultivating the triangular flask of collecting dead bacterium is common triangular flask).Viable bacteria is removed moisture after collecting as far as possible, gets 80 ℃ of sub-fractions and dries the thalline water content of surveying to constant weight.Dead bacterium carries out the rear sieve of crossing 300 μ m apertures of lyophilize-high pressure steam sterilization-lyophilize-grinding and makes dead bacterium powder after collecting.Respectively the bacterium of two states carried out to the cadmium adsorption experiment, experiment is carried out in the 50mL centrifuge tube that contains the 20mL cadmium solution, and the absorption system condition is viable bacteria: pH 5.0, cadmium concentration 300mg/l, absorption 120min, viable bacteria dry weight 0.5g/l; Dead bacterium: pH 5.0, cadmium concentration 300mg/l, absorption 45min, the heavy 0.5g/l of dead bacterium.After thalline absorption reaches capacity, with the content of cadmium in supernatant liquor before and after the absorption of sampling Graphite Furnace Atomic Absorption spectrophotometric determination, thereby obtain its adsorptive power.
The thalline that absorption is saturated reclaims, and the metal elutriant carries out sour digestion process after removing the cadmium that is combined in the bacterium surface, uses equally the content of cadmium in sampling Graphite Furnace Atomic Absorption spectrophotometric determination thalline.The specific acid digestive process is as follows: dry cell weight 0.1-0.5g weighs and adds the alimentary canal bottom, drips the 1ml deionized water sample is soaked.Add the 5ml vitriol oil, add about 0.5ml perchloric acid to alimentary canal with placing after sample blending to spend the night again, 180 ℃ of digesting low temperature digest 0.5h, and 300 ℃ of high temperature disappears to boil to clear, colorless and digested.
(4) heavy metal out of electroplating wastewater removes experiment:
The contriver has carried out respectively a kind of filamentous fungus Paecilomyces lilacinus XLA viable bacteria and the experiment of dead bacterium to cadmium removal ability in the electroplating wastewater gathered: by embodiment 3.4, describing and collect viable bacteria and prepare dead bacterium, two kinds of thalline are all for removing the cadmium ion of electroplating wastewater.
A kind of filamentous fungus Paecilomyces lilacinus XLA to the actual experimental system that removes of cadmium in electroplating wastewater is: 20mL electroplating wastewater (Cd
2+: 0.13mg/L), dry weight 0.01g/l thalline, 28 ℃ and 150r/min process after 2h to filter and collect supernatant liquor, measure the content of cadmium before and after absorption according to preceding method.The contriver utilizes the dead bacterium of a kind of filamentous fungus Paecilomyces lilacinus XLA and viable bacteria flocculant for treating to process sewage later again in addition, to understand the actual application ability of a kind of filamentous fungus Paecilomyces lilacinus XLA in removing sewage of low concentration.
The applicant shows rapidly and efficiently adsorbing heavy metal cadmium of this bacterial strain after utilizing filamentous fungus XLA to carry out static cadmium adsorption experiment.This bacterium has notable difference to the heavy metal cadmium absorption property under the different activities state, and wherein the adsorptive power of active thalline is higher.The remove the results show of the XLA carried out subsequently to heavy metal out of electroplating wastewater, this filamentous fungus XLA is efficient adsorption remove the cadmium in polluted-water in actual applications.
The present invention compared with prior art, has the following advantages and effect:
(1) the thalline raw material easily obtains, with low cost, and treatment process is simple.
(2) utilize the freeze-drying thalline of filamentous fungus Paecilomyces lilacinus XLA not only can remove the heavy metal ion in sewage by efficient adsorption as biological adsorption agent, also possess good adsorbent reactivation ability simultaneously, possess the potential of commercial applications.
(3) filamentous fungus Paecilomyces lilacinus XLA has height endurability and high characterization of adsorption to various heavy, and the heavy metal in environment is had to higher crystallized ability, reduces its biological toxicity.This bacterial strain can be survived under high density heavy metal level, therefore the Paecilomyces lilacinus XLA of usining can realize the long-term immobilized while of heavy metal in soil as the heavy metal-polluted soil sorbent material, avoid repeatedly applying the sorbent material of heavy metal in environment, possess the potentiality of efficient restoration of soil polluted by heavy metal and water body.
The accompanying drawing explanation
The static cadmium adsorptive capacity measurement result schematic diagram that Fig. 1 is a kind of filamentous fungus XLA freeze-drying thalline.
Fig. 2 is the selection schematic diagram of a kind of filamentous fungus XLA to the desorption agent of cadmium.
Fig. 3 is the desorption kinetic results schematic diagram of a kind of filamentous fungus XLA to cadmium.
Wherein, experiment adopts the EDTA that the optimum solution sorbent material is 2% mass volume ratio.
Fig. 4 be the dead bacterium of a kind of filamentous fungus XLA and viable bacteria to electroplating wastewater and throwing out after in waste water cadmium remove result schematic diagram.
Specific embodiments
Below narration is embodiment according to embodiments of the present invention, should be noted that embodiments of the invention only have illustration for the present invention, and there is no restriction.The standard operating instructions of relative dna and the medicine used are with reference to " molecular cloning experiment guide " described content (referring to J. Pehanorm Brooker etc., 2002, molecular cloning experiment guide, the third edition, Jin Dongyan etc. (translating), Science Press, Beijing).Related other various experimental implementation in the present invention, be the routine operation technology of this area, the part that there is no special instruction in literary composition, those of ordinary skill in the art can be implemented with reference to the various common tool books before the present patent application day, scientific and technical literature or relevant specification sheets, handbook etc.
Embodiment 1: the preparation method of a kind of filamentous fungus Paecilomyces lilacinus XLA the steps include: separation, purifying and multiple sieve.
1) cadmium separation resistant strain XLA
A, take the pot experiment soil sample 10g repaired for heavy metal that heavy metal cadmium content is 50mg/Kg in 90ml the sterilized water with 5 0.5mm granulated glass spherees, 200r/min vibration 30min on 28 ℃ of constant-temperature tables, after standing 1min, get supernatant liquor and carry out 10 times of serial dilutions;
B, the diluent of the different extension rates of 100 μ L is coated on the Martin's flat board that contains the 60mM cadmium, in static a moment, is inverted in 28 ℃ of constant incubators and cultivates 5d;
C, by the microorganism grown on the flat board separation and purification of being rule, then again be applied to and above-mentionedly cultivated containing on Martin's flat board of cadmium;
D, after 10 separation screenings, preserve the bacterial strain that cadmium is had to the stable resistance ability, on the PDA inclined-plane, preserved, standby.
A strain bacterium called after filamentous fungus XLA in the bacterial strain that the applicant obtains above-mentioned separation.
2) biological characteristics of strain X LA
1. biological characteristics: filamentous fungus, obligate is aerobic, and 20 ℃ ~ 28 ℃ of optimum growth temperatures, grow rapider, but producing the decline of spore ability more than 35 ℃, in the PDA substratum, sporulation quantity is large, and bacterium colony presents felted, the middle part protuberance, cultivate early stage bacterium colony and present white, and the later stage produces spore and becomes burgundy.
2. culture condition: the substratum used when preserving this fungi and obtaining its spore is the PDA substratum; What this fungi of separation screening was used is Martin's substratum: glucose 10g, peptone 5g, MgSO
40.5g, distilled water 1000ml, pH nature, 121 ℃ of high pressure steam sterilization 30min.For adsorption experiment and test it to mineral salts medium for heavy metal tolerance (MM): glucose 20g, ammonium sulfate 5g, potassium primary phosphate 15g, anhydrous magnesium sulfate 0.6g, Calcium Chloride Powder Anhydrous 0.6g, iron vitriol 0.005g, manganese sulfate monohydrate 0.0016g, Zinc Sulphate Heptahydrate 0.0014g, cobalt chloride 0.002g, distilled water 1000ml dissolves, and regulates pH to 5.5,115 ℃ of high pressure steam sterilization 15min.Cultivating the form that this bacterium can spore is inoculated: the tween-80 with 2% volume ratio rinses the spore on the PDA inclined-plane, inoculates after then with the tween-80 of 0.2% volume ratio, being diluted to the spore concentration of needs, then carries out 28 ℃, the 150r/min shake-flask culture.
3) molecular biology identification
Total DNA of the bacterial strain that adopts CTAB method extraction separation screening to obtain, and as pcr template, carry out ITS conserved sequence (the part 18S rRNA gene of the above-mentioned isolated strains of pcr amplification with the ITS1-ITS4 primer, ITS1,5.8S rRNA gene, ITS4,28S rRNA gene), with the Molecular Identification for bacterial strain.Combining form learns observation and molecular biosciences identifies that this bacterium of confirmation is Paecilomyces lilacinus (Paecilomyces lilacinus).The applicant delivers this bacterial strain to Chinese Typical Representative culture collection center (CCTCC) preservation in the Wuhan University of Wuhan City, Hubei Province on April 24th, 2012, and its preserving number is: CCTCC NO:M2012135.
Embodiment 2:
The application of a kind of filamentous fungus Paecilomyces lilacinus XLA in heavy metal cadmium is administered, its application process is:
1) filamentous fungus XLA detects the tolerance of various heavy
The liquid culture condition: the heavy metal mother liquor that adds a certain amount of filtration sterilization in inoculation forward direction mineral salt (MM) liquid nutrient medium, to needs concentration, then, by 1% inoculum size inoculation Paecilomyces lilacinus XLA spore suspension, is cultivated 7d, observes its upgrowth situation.
The solid culture condition: inoculating spores is to mineral salt (MM) liquid nutrient medium, and shaking table is cultured to mycelium pellet and just forms.Mineral salt (MM) solid plate that will contain the finite concentration heavy metal is divided the trisection zone, inoculates the identical mycelium pellet of diameter in each zone with the dibbling method respectively, cultivates 7 days for 28 ℃.Mycelium pellet diameter of every 24 hour record.With Sigmaplot 10.0, analyze on the different concns flat board along with the time changes bacterium colony size variation trend.Can make the substantially unconverted heavy metal concentration of colony diameter be the minimum inhibitory concentration of this bacterium.
Table 1(sees the following form) be the tolerance of Paecilomyces lilacinus XLA to different heavy metal ion.Under the liquid culture condition, different heavy metals are respectively cadmium (Cd to the minimum inhibitory concentration of Paecilomyces lilacinus XLA
2+) 130mM, cobalt (Co
2+) 45mM, copper (Cu
2+) 80mM, zinc (Zn
2+) 220mM, chromium (Cr
3+) 60mM, chromium (Cr
6+) 5mM, nickel (Ni
2+) 24mM; Next 265mM, 50mM, 80mM, 145mM, 35mM, 4mM, the 16mM of being respectively of solid culture condition.This shows that Paecilomyces lilacinus XLA provided by the present invention has stronger tolerance to the murder by poisoning of various heavy.
Table 1 (in the present embodiment filamentous fungus XLA in mineral salts medium to different heavy metal ion tolerance detected results)
2) adsorptive power of filamentous fungus XLA to cadmium
1. the absorption of static cadmium
Strain X LA is cultivated on the PDA substratum to 4d and produce a large amount of spores, prepare spore suspension, then be inoculated in the fresh mineral salts medium of 200 milliliters and cultivate 5d according to the ratio of 1:100, after filtering the collection thalline and carrying out the vacuum lyophilization processing, thalline is broken up to loose condition (of surface), then carried out the adsorption experiment of cadmium.
0.002g freeze-drying thalline is added in the 50mL centrifuge tube that contains the 20mL cadmium solution and carries out, be placed in 28 ℃ of constant-temperature tables, 150r/min reacts 240min, in reaction process respectively 0,5,15,30,45,60,120,180 and 240min get 100 μ L supernatant liquors, carry out the cadmium content analysis by the sampling Graphite Furnace Atomic Absorption spectrophotometer after dilution.Adsorption experiment arranges 3 repetitions.Fig. 1 shows, along with the increase of adsorption time, thalline increases gradually to the unit adsorptive capacity of cadmium, in initial 45min internal adsorption amount, reaches to more than 80% of maximal absorptive capacity, progresses into subsequently the adsorption equilibrium state.XLA adsorptive capacity to cadmium in the 500mg/L cadmium solution reaches 56.34 ± 5.16mg/g dry weight, and the filamentous fungus in proved invention has fast and the ability of efficient adsorption heavy metal cadmium possesses the ability of cadmium pollution soil repair or water body.
2. the attached ability of the absorption-desorption of thalline
Select thalline after frozen dried to carry out the adsorption experiment of cadmium.Concrete adsorption experiment condition: cadmium solution concentration is that 500mg/L, pH 4.0,0.1g/L freeze-drying thalline fully adsorb 4h, adsorb saturated rear supernatant sampling, with the content of cadmium in supernatant liquor before and after the absorption of sampling Graphite Furnace Atomic Absorption spectrophotometric determination, calculate the content of the cadmium that is adsorbed onto the thalline surface after diluted sample.Filter and collect the rear sample of absorption and carry out lyophilize.Choose respectively the EDTA, 0.1mol/L sodium hydroxide of deionized water, 0.1mol/L nitric acid, 0.1mol/L hydrochloric acid, 2% mass volume ratio as the desorption agent, the system reaction 4h with 0.01g adsorption sample/20ml desorption agent, select the suitableeest desorption agent.The choice experiment result that Fig. 2 is the desorption agent, what the desorption effect was best is the EDTA solution of 2% mass volume ratio, adsorption rate reaches 84.7 ± 0.003%.That Fig. 3 shows is the EDTA desorption Cd with 2% mass volume ratio
2+the dynamic experiment result, desorption is a process rapidly, can in 15min, complete.In the invention of this experiment proved, filamentous fungus is processed the effect that can reach recycling by desorption, possesses the potentiality that become the commercialization sorbent material.
3. the impact of the active condition of bacterial strain on cadmium absorption
The spore that strain X LA is produced in the activation of PDA substratum in 4 days is afterwards collected and is made suspension, then be inoculated in the fresh mineral salts medium of 200ml and cultivate 5d according to the ratio of 1:100, filter and collect thalline (cultivating the triangular flask of collecting the active condition thalline is the triangular flask of bottom with rib, and cultivating the triangular flask of collecting dead bacterium is common triangular flask).Viable bacteria is removed moisture after collecting as far as possible, gets 80 ℃ of sub-fractions and dries the thalline water content of surveying to constant weight.Dead bacterium carries out the rear sieve of crossing 300 μ m apertures of lyophilize-high pressure steam sterilization-lyophilize-grinding and makes dead bacterium powder after collecting.Respectively the bacterium of two states carried out to the cadmium adsorption experiment, experiment is carried out in the 50mL centrifuge tube that contains the 20mL cadmium solution, and the absorption system condition is viable bacteria: pH 5.0, cadmium concentration 300mg/l, absorption 120min, viable bacteria dry weight 0.5g/l; Dead bacterium: pH 5.0, cadmium concentration 300mg/l, absorption 45min, the heavy 0.5g/l of dead bacterium.After thalline absorption reaches capacity, with the content of cadmium in supernatant liquor before and after the absorption of sampling Graphite Furnace Atomic Absorption spectrophotometric determination, thereby obtain its adsorptive power.
The thalline that absorption is saturated reclaims, and the metal elutriant carries out sour digestion process after removing the cadmium that is combined in the bacterium surface, uses equally the content of cadmium in sampling Graphite Furnace Atomic Absorption spectrophotometric determination thalline.The specific acid digestive process is as follows: dry cell weight 0.1-0.5g weighs and adds the alimentary canal bottom, drips the 1ml deionized water sample is soaked.Add the 5ml vitriol oil, add about 0.5ml perchloric acid to alimentary canal with placing after sample blending to spend the night again, 180 ℃ of digesting low temperature digest 0.5h, and 300 ℃ of high temperature disappears to boil to clear, colorless and digested.Table 2(sees the following form) presentation of results, causing viable bacteria and the maximum reason of dead bacterium adsorptive power difference is the thalline state.This experiment confirms that viable bacteria also has the process of accumulating in born of the same parents to cadmium, in actual applications, for the purpose of the repairing heavy metal pollution that reaches efficient economy, take the microbial activity state into consideration.
Table 2 (filamentous fungus XLA viable bacteria and dead bacterium absorption cadmium ability and intracellular accumulation cadmium content in the present embodiment)
Embodiment 3: a kind of filamentous fungus Paecilomyces lilacinus XLA is processing containing the application in heavy metal-containing electroplating effluent, its application process:
By in embodiment 2, describing and collect respectively the XLA viable bacteria and prepare dead bacterium, two kinds of thalline are all for removing the cadmium ion of electroplating wastewater.
XLA to the actual experimental system that removes of cadmium in electroplating wastewater is: 20mL electroplating wastewater (Cd
2+: 0.13mg/L), dry weight 0.01g/l thalline, 28 ℃ and 150r/min process after 2h to filter and collect supernatant liquor, measure the content of cadmium before and after absorption according to preceding method.As can be seen from Figure 4, the XLA viable bacteria removes ability higher than dead bacterium to cadmium, reaches 92.52%.The contriver utilizes the dead bacterium of XLA and viable bacteria flocculant for treating to process sewage later again in addition, to understand the actual application ability of XLA in removing sewage of low concentration.The result demonstration, XLA has equally the ability that removes preferably heavy metal when processing sewage of low concentration, and dead bacterium and viable bacteria are respectively 72.01% and 76.34% to the removal rate of cadmium.This shows in actual heavy metal containing sewage, and filamentous fungus eXLA viable bacteria and dead bacterium can be adsorbed efficiently and remove heavy metal cadmium.Simultaneously, this also illustrates, the life state of XLA bacterial strain is not that heavy metal adsorption is necessary, dry bacterium powder just can apply to the processing of heavy metal polluted waste water, be conducive to reduce production costs, simplify treatment process, thereby provide possibility for preparing on a large scale the heavy metal polluted waste water water purification agent.
Claims (2)
1. a filamentous fungus Paecilomyces lilacinus, is characterized in that, Paecilomyces lilacinus (Paecilomyces lilacinus) XLA, CCTCC NO:M2012135.
2. the application of a kind of filamentous fungus Paecilomyces lilacinus XLA claimed in claim 1 in heavy metal cadmium is administered.
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