CN110951642B - Method for reducing and adsorbing heavy metal chromium by fixed microorganisms - Google Patents

Method for reducing and adsorbing heavy metal chromium by fixed microorganisms Download PDF

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CN110951642B
CN110951642B CN201911309712.0A CN201911309712A CN110951642B CN 110951642 B CN110951642 B CN 110951642B CN 201911309712 A CN201911309712 A CN 201911309712A CN 110951642 B CN110951642 B CN 110951642B
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microbial inoculum
chromium
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heavy metal
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CN110951642A (en
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迟慧梅
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Southeast University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00—Biological treatment of water, waste water, or sewage
    • C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/04—Enzymes or microbial cells immobilised on or in an organic carrier entrapped within the carrier, e.g. gel or hollow fibres
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2101/00—Nature of the contaminant
    • C02F2101/10—Inorganic compounds
    • C02F2101/20—Heavy metals or heavy metal compounds
    • C02F2101/22—Chromium or chromium compounds, e.g. chromates
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
    • Y02W10/00—Technologies for wastewater treatment
    • Y02W10/10—Biological treatment of water, waste water, or sewage

Abstract

The invention belongs to the technical field of environmental biology, and particularly relates to a method for reducing and adsorbing heavy metal chromium by fixed microorganisms, which utilizes gel microspheres to fix a bacillus cereus and rhodococcus ruber mixed microbial inoculum so as to reduce heavy metal chromium pollutants; the mixed microbial inoculum can utilize ferric oxide as an electron donor to reduce the ferric oxide into ferrous ions so as to promote the reduction of hexavalent chromium; the invention also provides a method for immobilizing the mixed microbial inoculum, which is used for preparing the mixed microbial inoculum by taking the active carbon loaded ferric oxide as a carrier, adsorbing the mixed microbial inoculum and promoting the mixed microbial inoculum to grow by utilizing the ferric oxide; meanwhile, sodium alginate and polyvinyl alcohol are used as embedding agents to embed and adsorb carriers of the mixed microbial inoculum, and microbial inoculum immobilized beads are prepared; the microbial agent immobilized pellet can provide a stable growth environment for the mixed microbial agent, is suitable for a toxic environment, and efficiently reduces and adsorbs heavy metal ions such as chromium.

Description

Method for reducing and adsorbing heavy metal chromium by fixed microorganisms
Technical Field
The invention relates to a method for reducing and adsorbing heavy metal chromium by using immobilized microorganisms, belonging to the technical field of environmental biology.
Background
The dissimilatory iron reducing bacteria are a general name of a microorganism which can take Fe (III) as a unique electron acceptor, reduce Fe (III), oxidize an organic carbon source and obtain energy from the organic carbon source for self growth. The dissimilatory iron reducing bacteria can reduce various metal ions in the processes of bacterial respiration and bacterial growth, and remove organic matters and heavy metals. Iron reduction is an important microbial metabolic pathway and also an important link in the process of geochemical cycle. Fe (II) generated during reduction of the dissimilatory iron catalyzes reduction of Cr (VI), which provides possibility for treatment of Cr (VI) pollution.
Chromium is mainly used in the industries of metal processing, electroplating, tanning and the like, and a large amount of chromium pollution is also generated in the agricultural livestock and poultry breeding process. Heavy metal chromium is an important industrial raw material, the annual output of China exceeds 16 ten thousand tons, and the generated hexavalent chromium waste reaches 3500 tons. This causes serious pollution of the soil and water near the plant area and where the waste is dumped. Chromium in the water environment generally exists in the forms of Cr (VI) and Cr (III), Cr (VI) has strong fluidity, causes long-term pollution to water and soil, has great harm to human bodies, and has small harm to human bodies after being reduced into trivalent chromium. Therefore, the removal of hexavalent chromium in water is one of the main directions for solving the problem of heavy metal pollution.
The heavy metal removal methods mainly include physical adsorption and chemical precipitation methods, such as ion exchange, membrane separation, reverse osmosis, and the like. The methods generally have the problems of high energy consumption, high operation cost, complex operation, low efficiency in treating low-concentration heavy metal and the like. Physical and chemical remediation methods also cause secondary pollution of water and soil. Bioremediation becomes a research hotspot for removing heavy metal pollution, and microbial remediation becomes a main heavy metal removal means with the advantages of no secondary pollution, high speed, low cost, high efficiency and the like. Because the heavy metal-containing sewage has high toxicity and poor living environment, the selection of tolerant microorganisms to reduce, adsorb and precipitate soluble heavy metal ions, and the selection of tolerant microorganisms and the fixation of microorganisms become the key to solve the problem of toxic environment.
Disclosure of Invention
The technical problem is as follows:
the invention mainly aims to provide a microbial agent for reducing hexavalent chromium, an iron oxide compound for adsorbing trivalent chromium and a preparation method of immobilized beads of the iron oxide compound, and aims to provide a solution for removing chromium in a sewage environment.
The technical scheme is as follows:
in order to realize the purpose of the invention, the technical scheme adopted by the invention is as follows:
a mixed bacterial agent, which comprises Bacillus cereus (Bacillus cereus) DIF1 and Rhodococcus ruber (Rhodococcus ruber) DIF 2; the Bacillus cereus (Bacillus cereus) DIF1 is preserved in the China center for type culture Collection in 2018, 5 and 15 days, and the preservation unit address is as follows: the preservation number of the strain is CCTCC NO of M2018274 at Wuhan university in Wuhan, China; the Rhodococcus ruber (Rhodococcus ruber) DIF2 is preserved in the China center for type culture Collection in 2018, 5 months and 15 days, and the preservation unit address is as follows: the preservation number of the strain is CCTCC NO of M2018275 at Wuhan university in Wuhan, China.
Further, the preparation of the mixed microbial inoculum comprises the following steps:
(1) respectively activating bacillus cereus DIF1 and Rhodococcus ruber DIF2, performing suspension culture by using an LB liquid culture medium, and performing shake culture at 30 ℃ and 120 revolutions for 12 hours;
the LB liquid culture medium comprises the following components: 10g/L of tryptone, 5g/L of yeast extract, 10g/L of sodium chloride and pH 7.0.
(2) And putting the suspension culture of the bacillus cereus DIF1 and the rhodococcus ruber DIF2 with the same amount into a fresh LB liquid culture medium for mixed culture for 12 hours to prepare a mixed microbial inoculum for later use.
Further, the mixed microbial inoculum reduces Fe in a dissimilatory manner 3+ And/or Cr 6+ Application of the aspect.
A preparation method of immobilized beads of microbial agents comprises the following steps: bacillus cereus DIF1 and Rhodococcus ruber DIF2 are mixed to prepare a microbial inoculum, an active carbon carrier loaded with ferric oxide is used for adsorbing the mixed microbial inoculum, and an embedding medium is used for embedding the microbial inoculum carrier to form a microbial inoculum immobilized pellet, so that the reduction adsorption of chromium is realized.
Further, the preparation method of the iron oxide loaded activated carbon comprises the following steps:
(1) mixing 40gFe (NO3) 3 ·9H 2 Dissolving O in 500mL of deionized water, adding 2-4g of active carbon, and stirring;
(2) dropwise adding 500mL of 1mol/L sodium hydroxide solution, stirring, and stopping dropwise adding sodium hydroxide when the pH of the solution is adjusted to 7-8;
(3) and centrifuging the precipitated product for 20min at 4000g, washing with ultrapure water, centrifuging again, sterilizing, and drying at 100 ℃ for later use.
Further, the preparation method of the microbial agent immobilized bead comprises the following steps:
(1) immersing iron oxide loaded activated carbon into the microbial inoculum suspension culture solution 3 for 8-12 hours according to the mass ratio of 1.8-2.5%;
(2) adding polyvinyl alcohol into the obtained suspension at a concentration of 20-30g/L, simultaneously adding sodium alginate at a concentration of 20-30g/L, and uniformly mixing to obtain a thallus embedding solution;
(3) slowly adding 50-80g/L CaCl into the thallus embedding liquid 2 And (3) crosslinking for 7-10 hours in the crosslinking solution, solidifying into balls, and washing with pure water to obtain immobilized balls of the microbial agent.
Further, the microbial agent immobilized beads are used for reducing Cr in microorganisms 6+ Application of the aspect.
Further, the embedding agent is sodium alginate and/or polyvinyl alcohol.
Advantageous effects
Based on the technical scheme, the invention has the beneficial effects that:
when the chromium reduction microbial agent is used for reducing hexavalent chromium, the advantages of economy, high efficiency, no secondary pollution and the like are achieved, the hexavalent chromium with the concentration of 2mg/L can be completely degraded under the aerobic condition, and the chromium reduction microbial agent can be used for removing the hexavalent chromium in various sewage.
The invention adopts an immobilization means to fix the microbial agent, can play a role of buffering the change of the external environment, can provide a stable growth environment for microorganisms, is more suitable for a toxic environment, treats hexavalent chromium ions with higher concentration, and prolongs the service time and the times of the microbial agent.
Drawings
FIG. 1 is a chart of the efficiency of reducing chromium by using a mixed microbial inoculum of DIF1 and DIF 2;
FIG. 2 is a reaction scheme of the present invention for reducing and adsorbing heavy metal chromium by immobilized microorganisms;
FIG. 3 is a graph of the efficiency of reducing chromium by gel microspheres;
Detailed Description
In order to better understand the present invention, the following examples are further provided to illustrate the present invention, but the present invention is not limited to the following examples.
Example 1: preparing a hexavalent chromium reducing microorganism mixed microbial inoculum and performing chromium reduction experiment (I) preparing the mixed microbial inoculum:
(1) two chromium reducing bacteria, namely bacillus cereus DIF1 and rhodococcus ruber DIF2 are selected and respectively inoculated in an LB liquid culture medium; culturing at 28-30 deg.C under 120 rpm for 8-12 hr with OD600 of 0.8-1.0;
the LB culture medium formula is tryptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L and PH 7.0;
(2) selecting 10ml of each of two bacterial solutions, inoculating the two bacterial solutions into 100ml of sterilized fresh culture medium, continuously culturing for 8-12 hours under the condition of the step (1), wherein the OD600 value reaches 1.0-1.2, and the bacterial solutions are reserved;
(II) hexavalent chromium reduction experiment:
the mixed microbial inoculum is used for Cr (VI) reduction capability verification experiment: preparing LB solution, adding 2mmol/L anhydrous sodium chromate into the solution. Sterilizing at high temperature, adding into sterile 100ml serum bottle, adding Bacillus cereus DIF1, Rhodococcus ruber DIF2, and mixed microbial inoculum of DIF1 and DIF2, sealing, and culturing in 30 deg.C incubator in dark. Every 12h, extracting a sample in a sterile operation table, and respectively detecting Cr of the sample by using an ultraviolet spectrophotometer according to a method for detecting a chromium reduction standard curve 6+ The concentration changes, and the result shows that Cr is between 0 and 24 hours 6+ The concentration is reduced rapidly, the chromium reduction capability of the bacillus cereus DIF1 in 96h is about 1.44mmol/L, the chromium reduction capability of the rhodococcus ruber DIF2 in 96h is about 1.36mmol/L, and the mixed microbial inoculum of DIF1 and DIF2 is 1.58mmol/L, which is higher than the reduction efficiency of pure bacteria. See fig. 1.
Example 2: the preparation of the gel microsphere and the preparation of the activated carbon loaded iron oxide with chromium reduction effect comprise the following steps:
(1) mixing 40gFe (NO3) 3 ·9H 2 Dissolving O in 500mL of deionized water, adding 2-4g of active carbon, and stirring;
(2) dropwise adding 500mL of 1mol/L sodium hydroxide solution, stirring, and stopping dropwise adding sodium hydroxide when the pH of the solution is adjusted to 7-8;
(3) and centrifuging the precipitated product at 4000g for 20min, washing with pure water, centrifuging again, sterilizing, and drying at 100 ℃ for later use. (II) preparing gel microspheres, comprising the following steps:
(1) immersing the prepared active carbon loaded with ferric oxide into a microbial inoculum suspension culture solution for 8-12 hours according to the mass ratio of 1.8-2.5%;
(2) adding alginic acid with the mass concentration of 2-3% and polyvinyl alcohol with the mass concentration of 2-3% into the bacterial liquid obtained in the step (1) to prepare a thallus embedding liquid;
(3) slowly adding 50-80g/L CaCl into the thallus embedding liquid in the step (2) 2 And (3) crosslinking for 7-10 hours in the crosslinking solution, solidifying into balls, and washing with pure water to obtain immobilized balls of the microbial agent.
The obtained microbial agent immobilized pellet has the following characteristics:
(1) the two chromium reducing bacteria, namely the bacillus cereus and the rhodococcus ruber, are gram-positive bacteria, and after being mixed, the chromium reducing efficiency is higher than that of a single strain, the hexavalent chromium tolerance is improved, and the environmental toxicity adaptability is improved;
(2) the ferrite compound is loaded on the activated carbon, so that the specific surface area is increased, and the adsorption capacity to chromium ions is increased;
(3) the iron reduction microbial agent reduces the iron oxide compound into ferrous iron ions, the ferrous iron ions reduce the hexavalent chromium ions into trivalent chromium ions, the hexavalent chromium ions are oxidized into trivalent iron ions, and the microorganisms continue to reduce the trivalent iron ions into the ferrous iron ions, so that the reduction can be carried out circularly. The removal efficiency of hexavalent chromium ions is increased, as illustrated in fig. 2.
(III) application of immobilized beads of microbial agent prepared by the preparation method in reduction of hexavalent chromium
Preparing LB solution, adding 2mmol/L anhydrous sodium chromate into the solution. Sterilizing at high temperature, adding into sterile 100ml serum bottle, and adding microorganism into the serum bottleThe immobilized pellet of the microbial inoculum is sealed and placed in a constant temperature incubator at 30 ℃ for dark culture. Every 12h, extracting a sample in a sterile operation table, and respectively detecting Cr of the sample by using an ultraviolet spectrophotometer according to a method for measuring a chromium reduction standard curve 6+ The concentration changes, and the result shows that Cr is between 0 and 24 hours 6+ The concentration is reduced rapidly, the chromium reduction capacity of the immobilized pellet of the microbial agent exceeds 90 percent in 96 hours, and is higher than the reduction efficiency of a mixed microbial agent of DIF1 and DIF 2. See fig. 3.
The above embodiments are merely preferred embodiments of the present invention, and should not be construed as limiting the present invention, the scope of the present invention should be defined by the claims, and equivalents including technical features described in the claims are intended to be included in the scope of the present invention, that is, equivalent modifications within the scope of the present invention are also within the scope of the present invention.
Sequence listing
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<120> method for reducing and adsorbing heavy metal chromium by fixed microorganism
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gataacattt tgaaccgcat ggttcgaaat tgaaaggcgg cttcggctgt cacttatgga 180
tggacccgcg tcgcattagc tagttggtga ggtaacggct caccaaggca acgatgcgta 240
gccgacctga gagggtgatc ggccacactg ggactgagac acggcccaga ctcctacggg 300
aggcagcagt agggaatctt ccgcaatgga cgaaagtctg acggagcaac gccgcgtgag 360
tgatgaaggc tttcgggtcg taaaactctg ttgttaggga agaacaagtg ctagttgaat 420
aagctggcac cttgacggta cctaaccaga aagccacggc taactacgtg ccagcagccg 480
cggtaatacg taggtggcaa gcgttatccg gaattattgg gcgtaaagcg cgcgcaggtg 540
gtttcttaag tctgatgtga aagcccacgg ctcaaccgtg gagggtcatt ggaaactggg 600
agacttgagt gcagaagagg aaagtggaat tccatgtgta gcggtgaaat gcgtagagat 660
atggaggaac accagtggcg aaggcgactt tctggtctgt aactgacact gaggcgcgaa 720
agcgtgggga gcaaacagga ttagataccc tggtagtcca cgccgtaaac gatgagtgct 780
aagtgttaga gggtttccgc cctttagtgc tgaagttaac gcattaagca ctccgcctgg 840
ggagtacggc cgcaaggctg aaactcaaag gaattgacgg gggcccgcac aagcggtgga 900
gcatgtggtt taattcgaag caacgcgaag aaccttacca ggtcttgaca tcctctgaaa 960
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attgacgggg gcccgcacaa gcggcggagc atgtggatta attcgatgca acgcgaagaa 900
ccttacctgg gtttgacata caccggaccg ccccagagat ggggtttccc ttgtggtcgg 960
tgtacaggtg gtgcatggct gtcgtcagct cgtgtcgtga gatgttgggt taagtcccgc 1020
aacgagcgca acccttgtcc tgtgttgcca gcacgtaatg gtggggactc gcaggagact 1080
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Claims (6)

1. A mixed bacterial agent is characterized in that: the mixed microbial inoculum comprises bacillus cereus (B.) (Bacillus cereus) DIF1 and Rhodococcus ruber (C.), (B.)Rhodococcusruber) DIF 2; two strains are preserved in China center for type culture Collection, Bacillus cereus (Bacillus cereus) ((B))Bacillus cereus) The preservation number of DIF1 is CCTCC NO: m2018274; rhodococcus ruber (C)Rhodococcusruber) The preservation number of DIF2 is CCTCC NO: m2018275.
2. The method for reducing Fe by dissimilatory reaction of the mixed microbial inoculum according to claim 1 3+ And/or Cr 6+ Application of the aspect.
3. A microbial agent-immobilized pellet containing the mixed bacterial agent of claim 1.
4. The immobilized bead of microbial agent according to claim 3, which is prepared by the following steps: the carrier is active carbon loaded ferric oxide, the mixed microbial inoculum of claim 1 is adsorbed, and embedding and adsorption are carried out through an embedding agent, so that the carrier is obtained.
5. The microbial inoculant-immobilized bead of claim 4, wherein: the embedding agent is sodium alginate and/or polyvinyl alcohol.
6. The microbial inoculant-immobilized beads of claim 3 or 4 for microbial reduction of Cr 6+ Application of the aspect.
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