CN113930477A - Method for detecting autotrophic arsenic-oxidizing functional microorganisms contained in plant root endophytes - Google Patents

Method for detecting autotrophic arsenic-oxidizing functional microorganisms contained in plant root endophytes Download PDF

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CN113930477A
CN113930477A CN202111071599.4A CN202111071599A CN113930477A CN 113930477 A CN113930477 A CN 113930477A CN 202111071599 A CN202111071599 A CN 202111071599A CN 113930477 A CN113930477 A CN 113930477A
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endophyte
solution
culture
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liquid
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孙蔚旻
孙晓旭
裘浪
黄端仪
徐锐
刘华清
李永斌
高文龙
颜耕
徐芙清
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Institute of Eco Environmental and Soil Sciences of Guangdong Academy of Sciens
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Abstract

The invention discloses a method for detecting autotrophic arsenic-oxidizing functional microorganisms contained in plant root endophytes. The invention can accurately judge the functional microorganism participating in the heavy (similar) metal redox process in the plant endophyte by a comprehensive means of combining separation, culture and DNA-SIP technology. On the basis of efficiently extracting and separating the endophytes in the plant tissues, the endophyte liquid obtained by the enlarged culture and separation of the liquid culture medium is utilized, so that the microbial abundance and the activity of the endophyte liquid are ensured. Meanwhile, by taking DNA-SIP as a means, the microorganisms driving heavy metal redox can be directly and effectively anchored, q-PCR amplification and 16S rRNA high-throughput sequencing are further utilized, and whether the functional bacteria driving the heavy (like) metal redox process exist in the plant endophyte community can be accurately judged by analyzing the abundance change of the heavy metal redox functional microorganism group and the metabolic information of the key functional endophyte microorganisms in the microcosm system.

Description

Method for detecting autotrophic arsenic-oxidizing functional microorganisms contained in plant root endophytes
Technical Field
The invention belongs to the field of microbial ecology, and particularly relates to a method for detecting autotrophic arsenic-oxidizing functional microorganisms contained in plant root endophytes.
Background
Endophytes of plants live mainly in the plant body and can produce various biological effects on host plants. Researches show that the endophyte of the plant can enhance the resistance of host plants to abiotic stress such as temperature, water and metal pollution and biotic stress such as insects, pathogenic bacteria and nematodes, and can provide required nutrients such as nitrogen sources for the plants, thereby promoting the growth of the plants and regulating and controlling the physiological process of the plants.
In recent years, endophytes of plants are widely concerned at home and abroad due to unique ecological characteristics, and a large number of researches show that the endophytes participate in the physiological action and various metabolisms of plants to bring various changes and functions to the plants, for example, the endophytes can promote the host plants to relieve heavy metal stress through plant growth regulating hormones, ACC deaminase, chitinase and the like generated by the host plants, and simultaneously reduce the heavy metal toxicity of the plants by changing the biological effectiveness or toxicity of the heavy metals.
Since most of endophytes cannot be obtained by pure culture technology, although community constitution of the endophytes is found by molecular biology technology, the heavy metal tolerance mechanism of the endophytes at the roots of the plants is still unclear, and the ecological interaction effect between the endophytes with specific functions and the heavy metal polluted soil-plants needs to be further researched.
Disclosure of Invention
On the basis of efficiently extracting and separating plant tissue endophytes with disinfected surfaces, endophyte liquid with stable activity is obtained through liquid culture medium amplification culture, meanwhile, a culture system for driving heavy metal morphological change by endophyte liquid microorganisms is established by taking a microcosm environment and a DNA-SIP as means, and finally, whether functional bacteria driving heavy (similar) metal redox process exist in a plant endophyte community is judged by selecting different processing heavy layer DNAs to perform q-PCR amplification analysis and high-throughput sequencing of a 16S rRNA gene V4-V5 area, so that the method has important significance for the mechanism that the centi-clearer plant endophyte community promotes plant growth and resists heavy metal.
The purpose of the invention is realized by the following technical scheme:
a method for detecting autotrophic arsenic-oxidizing functional microorganisms contained in plant root endophytes comprises the following steps:
(1) removing the foreign bacteria on the root surface of the plant
Shaking off the soil attached to the surface of the plant root, and sequentially placing the plant root in MgSO4Solution, MgSO 20 containing Tween 204Solution, MgSO4Solution, sodium hypochlorite solution containing Tween 20, MgSO4Carrying out vortex oscillation or ultrasonic treatment in the solution to remove the plant root surface bacteria;
MgSO4very easily absorb water, using MgSO4Can dehydrate bacteria, thereby achieving the purpose of sterilization. Further, MgSO4And is also a strong oxidizing agent, which can further disrupt the cellular structure of the bacteria. Vortex oscillation or ultrasonic treatment aims to ensure that the buffer solution is fully contacted with the roots of the plants, so that the sterilization is more thorough.
MgSO as described in Steps (1) and (2)4The solution preferably has a concentration of 10 mM;
MgSO containing Tween 20 as described in step (1)4The solution, Tween 20 preferably has a concentration of 0.01% (v/v);
the sodium hypochlorite solution containing the Tween 20 in the step (1), wherein the concentration of the sodium hypochlorite is preferably 1% (v/v), and the concentration of the Tween 20 is preferably 0.01% (v/v);
(2) extraction and isolation of endophytes
Removing root surface impurity bacteria of plant root in MgSO4Breaking cell wall in the solution, homogenizing, filtering with filter cloth, and collecting filtrate; centrifuging the filtrate at 500 Xg for 10min, centrifuging the supernatant at 9500rpm for 15min, centrifuging to obtain weight difference, settling the heavy bacterial cells to the bottom, and keeping the thallus cell precipitate;
resuspending the thallus cell precipitate in sterile NaCl solution, slowly adding into iohexol solution, centrifuging at 15000 Xg for 60min, layering thallus cell precipitate in iohexol gradient density by centrifugation, displaying endophyte cells as milky white band, and recovering milky band part; removing the recovered iohexol, adding an equal volume of sterile NaCl solution, centrifuging at 7500 Xg for 20min, removing the supernatant, adding sterile NaCl solution, and resuspending; finally, centrifuging at 7500 Xg for 20min, taking the precipitate, adding sterile NaCl solution for resuspension, namely the recovered endophyte;
the pore diameter of the filter cloth in the step (2) is preferably 25 μm;
the concentration of the sterile NaCl solution in the step (2) is preferably 0.8% (W/V);
the concentration of the iohexol solution in the step (2) is preferably 1.3 g/mL;
(3) expanded culture of endophyte
Adding the separated plant root endophyte into an R2A liquid culture medium, and carrying out aerobic culture for 10-16h to obtain endophyte bacterial liquid;
the composition of the R2A liquid medium is as follows: 0.5g/L of yeast extract powder; peptone 0.5 g/L; 0.5g/L of casein hydrolysate; glucose 0.5 g/L; 0.5g/L of soluble starch; dipotassium hydrogen phosphate 0.3 g/L; anhydrous magnesium sulfate 0.024 g/L; 0.3g/L of sodium pyruvate;
(4) establishment and analysis of endophyte liquid oxidized As (III) -DNA-SIP microcosm cultivation system
4.1, adding the endophyte bacterial liquid obtained in the step (3) into an inorganic salt culture medium (MSM), and adding As (III) and13/12c-labelled NaHCO3As an electron acceptorSealing body and electron donor with aerobic membrane, and shake culturing;
the experiment included four groups of treatments:
A、13C-NaHCO3+ As (III) + endophytic bacteria liquid (C)13Experimental group C);
B、12C-NaHCO3+ As (III) + endophytic bacteria liquid (C)12Experimental group C);
C、13C-NaHCO3+ endophytic bacteria solution (As-free control group);
D、13C-NaHCO3+ as (iii) (sterile control group);
during the shaking culture period, carrying out non-destructive sampling at a plurality of time points, and detecting the concentrations of As (III) and As (V) in different treatments; after 15-20 days of culture, destructive sampling is carried out, and then differently processed DNA is extracted for subsequent SIP analysis;
the nondestructive sampling is to sample by using a needle tube without opening a bottle cap, so that the original domestication environmental conditions of a reaction system are kept to the maximum extent, and the reaction is continued;
the destructive sampling is to open a cover for sampling, the original domestication environmental condition is not changed after air enters a sample bottle, and the experiment is finished;
if a carbon source or As (III) is largely consumed during the culture period, corresponding components are supplemented to strengthen the metabolism of the microorganism;
preferably, the processing of the first and second sets of treatments,13C-NaHCO3the concentration of the mixed solution is 8mM,12C-NaHCO3the concentration is 8mM, the initial concentration of As (III) is 1-5mM, and 3.33mL of endophyte liquid is added in each liter of culture system.
4.2, performing ultra-high speed centrifugation on the extracted DNA, collecting each layered component, measuring the Buoyancy Density (BD) of each layered component, and removing CsCl precipitate; addition of ddH to the layered Components2Dissolving DNA by using O, finally obtaining purified DNA layers, and performing q-PCR amplification on each layer component by using an aioA gene (arsenic oxidation functional gene) primer;
4.3, selecting and processing the layered components with higher q-PCR amplification values in A and B, and performing high-throughput sequencing on the 16S rRNA gene V4-V5 region; comparing the sequencing data with the existing 16S rRNA database, classifying the data with more than 97% of similarity into a small group (OTU), and analyzing the microbial community diversity and abundance difference between treatments;
and (4) excluding the treatment of the control C, and determining the OTU with higher relative abundance in the treatment A as the autotrophic arsenic-oxidizing functional microorganism compared with the treatment B.
Compared with the prior art, the invention has the following advantages and effects:
the method for detecting the microorganisms with autotrophic arsenic oxidation function in the plant root endophyte provided by the invention can accurately judge the functional microorganisms participating in the heavy (similar) metal redox process in the plant root endophyte by combining the comprehensive means of separation and culture and DNA-SIP technology. The endophyte liquid obtained by the enlarged culture and separation of the liquid culture medium ensures the microbial abundance and activity of the endophyte liquid. Meanwhile, by taking DNA-SIP as a means, the method can directly and effectively anchor microorganisms driving heavy metal oxidation reduction, further utilizes q-PCR amplification and 16S rRNA high-throughput sequencing, and can accurately judge whether the plant endophyte community has heavy (like) metal oxidation functional bacteria or not by analyzing abundance change of the heavy metal oxidation functional microorganism group and metabolic information of key functional endophyte microorganisms in a microcosm system, thereby having important significance for understanding the heavy metal oxidation reduction driving process and the heavy metal resistance mechanism of the plant endophyte.
Drawings
FIG. 1 shows the result of coating isolated plant root endophytes.
FIG. 2 is13Concentration change curves of As (III) and As (V) in the C + As (III) + endophytic bacteria liquid treatment system.
FIG. 3 is12Concentration change curves of As (III) and As (V) in the C + As (III) + endophytic bacteria liquid treatment system.
FIG. 4 is13As (III) and As (V) concentration profiles in the C + As (III) treatment system.
FIG. 5 is the results of the relative abundance of the aioA gene in the DNA of each buoyancy density component of the different treatment groups at day 15.
FIG. 6 is13C/12q-PCR amplification in C + As (III) + endophytic bacteria liquid treatment groupHigher numbers of layered OTU relative abundance results.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
Examples
A method for detecting whether a plant root endophyte contains autotrophic arsenic oxidation functional microorganisms or not comprises the following steps:
separation of endophytes from plant roots
1. Pretreating the roots of plants:
(1) gently shaking off soil attached to the root of a ciliate desert-grass growing in Guangxi river basin and polluted by arsenic in one mining area for a long time, and transferring the root system to a container containing 10mL of MgSO 24(10mM) solution in centrifuge tube, vortex and shake for 5min, remove turbidity repeatedly, then transfer to Tween 20 (0.01%, V/V) in 10mM MgSO 204Vortex and oscillate in the solution for 5min, and ultrasonically treat for 15 min; while retaining the last buffer coating, more microbial growth was observed (FIG. 1 a);
(2) following the above procedure, 10mL MgSO was again added4Vortex shaking (10mM) solution for 5min, transferring to 1% NaClO solution containing Tween 20 (0.01%, V/V), vortex shaking for 20min, and ultrasonic processing for 15 min; finally, the plant roots were transferred to 10mL MgSO4Vortex shaking in (10mM) solution, repeating the operation for 5 times, reserving the last buffer solution coating, and observing that only a few microorganisms grow (figure 1b), thereby basically achieving the purpose of removing root surface bacteria;
the coating observation method was as follows: the solid LB plate medium was smeared with 100. mu.L of the remaining buffer, cultured at 25 ℃ for 7 days, and the production of microorganisms in the plate was observed.
2. And (3) endophyte separation:
removing the root surface sundry fungi in the step 1, transferring the cut plant root system to a wall breaking machine, and adding 10mL MgSO4(10mM) the solution is broken up to give a homogenate; meanwhile, filtering homogenate by using a filter cloth with the diameter of 25 mu m to achieve the aim of removing plant tissue residues; the filtrate was then centrifuged (500 Xg, 10min) and centrifuged again(9500rpm,15min), then abandoning the supernatant, because the endophyte bacteria have heavier thallus mass, and depositing to the bottom of the centrifuge tube after centrifugation.
The cell pellet was resuspended in 25mL of 0.8% sterile NaCl solution and then slowly transferred over 10mL iohexol solution (ρ ═ 1.3g/mL), followed by high speed centrifugation (15000 × g, 60 min); under the action of centrifugal force, the endophyte is concentrated at a certain specific position and forms a milky strip, at the moment, the milky strip part is recovered, an equal volume of sterile NaCl solution is added, centrifugation is carried out (7500 Xg, 20min), and supernatant is removed; adding 20mL of 0.8% sterile NaCl solution for resuspension, centrifuging again (7500 Xg, 20min), and discarding the supernatant, wherein the steps are to wash off residual iohexol; and finally, resuspending the precipitate obtained after centrifugation in sterile physiological saline to obtain the extracted live bacteria in the roots. The results of the plating culture are shown in FIG. 1 c.
Second, the enlargement culture of endophyte
Culturing the separated plant root living bacteria (obtained in step one (2)), namely adding 10mL of R2A liquid culture medium which is sterilized and cooled under high pressure into a 50mL sterile centrifuge tube under the environment of a sterile super clean bench, and then taking 100uL of OD600Transferring the intraradicular bacterial liquid with the value of 0.15 into a liquid culture medium, performing aerobic culture for 10-16h in a constant-temperature shaking table at 25 ℃ by inclined oscillation (200rpm), and culturing until the logarithmic phase of the bacterial liquid or turbidity is observed visually; a blank control was also set to monitor whether the liquid medium was contaminated.
The composition of the R2A liquid medium is as follows: 0.5g/L of yeast extract powder; peptone 0.5 g/L; 0.5g/L of casein hydrolysate; glucose 0.5 g/L; 0.5g/L of soluble starch; dipotassium hydrogen phosphate 0.3 g/L; anhydrous magnesium sulfate 0.024 g/L; sodium pyruvate 0.3 g/L.
Third, the establishment and analysis of endophyte liquid oxidized As (III) -DNA-SIP microcosm cultivation system
1. Adding 100uL of the endophyte bacterial liquid after enlargement culture into a penicillin bottle containing 30mL of inorganic salt Medium (MSM) solution under a sterile super clean bench, and adding As (III) and13/12c-labelled NaHCO3As electron acceptor and electron donor, respectively. Finally, sealing the culture chamber with an aerobic membrane, and culturing the microcosm culture system in an incubator at 25 ℃ under shaking (200 rpm).
As (III) Oxidation Microcosmic design Process includes four treatments:
(1)8mM 13C-NaHCO3+1mM As (III) +100uL of endophyte;
(2)8mM 12C-NaHCO3+1mM As (III) +100uL of endophyte;
(3)8mM 13C-NaHCO3+100uL of endophyte liquid;
(4)8mM 13C-NaHCO3+1mM As(III)；
as (III) is NaAsO2Solutions, each treatment was repeated 3 times.
MSM solution composition: 10.55g/L Na2HPO4·12H2O，1.5g/L KH2PO4，0.3g/L NH4Cl，0.1g/L MgCl20.01mg/L vitamin H, 0.02mg/L nicotinic acid, 0.1mg/L vitamin B1, 0.01mg/L p-aminobenzoic acid, 0.005mg/L vitamin B5, 0.05mg/L pyridoxamine hydrochloride, 0.01mg/L cyanocobalamin, 10. mu.L/L HCl (25%, w/w), 1.5mg/L FeCl2·4H2O，0.19mg/L CoCl2·6H2O，0.1mg/L MnCl2·2H2O，0.07mg/L ZnCl2，0.024mg/L NiCl2·6H2O，0.036mg/L NaMoO4·2H2O，0.006mg/L H3BO3，0.002mg/L CuCl2·2H2O；
2. During the microcosm culture, samples were taken at 1-3 day intervals for different treatments, and after filtration, the As (III) and As (V) concentrations were determined by HPLC-hydride generation-atomic fluorescence (HPLC-HG-AFS).
The experimental period was 15 days, and treatments (1), (2) and (4) were sampled at 9 time points on days 0, 3, 4, 5, 8, 10, 11, 13 and 15, respectively.
FIG. 2 shows the results of the variation of As (III) and As (V) concentrations in treatment (1);
FIG. 3 shows the results of the As (III) and As (V) concentration variations in treatment (2);
FIG. 4 shows the results of the As (III) and As (V) concentration variations in treatment (4);
as can be seen from FIGS. 2 and 3, the endophytic bacteria in roots almost exhausted 1mM As (III) added at one time in the system at days 5, 8, 11, and 13.
As can be seen from FIG. 4, there was no significant change in 1mM As (III) in the Microcosmic culture system with the addition of the rootless endophyte.
The results show that the root endophyte liquid microorganism can effectively and rapidly oxidize As (III) to As (V) in the environment of a micro cosmic culture system.
If a large amount of the carbon source or As (III) is consumed during the cultivation, the corresponding components are supplemented in time.
3. On day 15 of the Microcosmic culture System, destructive sampling was performed for treatments (1), (2) and (3), and total DNA of the Microcosmic culture system was extracted using a soil DNA extraction kit. Then carrying out ultra-high speed centrifugal delamination (60000rpm,48h,20 ℃); after centrifugation, the mixed liquid of the centrifuge tube is collected and recovered in layers by using a pump with fixed flow velocity, the BD value of the recovered component in each layer is measured at the same time, then nucleic acid precipitation aid and ethanol are used for precipitation to remove CsCl, and 30uLddH is added2O is the purified recovered component.
In addition, q-PCR amplification of the layered fractions was performed using the known arsenic oxide gene primers aioAF (5 '-ccacttctgcatcggggntgyggta-3') and aioA R (5 '-ggagttgtaggcggccktrttgdat-3').
The BD values of the centrifuged fractions of each layer were plotted on the abscissa and the q-PCR amplification value of the functional gene aioA on the ordinate. As can be seen from FIG. 5, the peaks corresponding to the stratification with higher q-PCR amplification values in the 3 treatment groups each clearly appeared on different BD values.
4. And (3) respectively selecting the layers with higher q-PCR amplification values in the treatment (1) and the treatment (2) of the microcosm culture system on the 15 th day, and performing high-throughput sequencing on the V4-V5 region of the 16S rRNA gene. Sequencing data were subsequently compared to the existing 16S rRNA database, assigning groups with more than 97% similarity to one sub-group (OTU), and analyzing microbial community diversity and abundance differences between the two treatments.
FIG. 6 shows the endophyte microbial population in the microcosm culture systemThe first 10 OTUs with the highest abundance in the colony. After exclusion of control treatment (3), comparative analysis13C and12after the abundance of the microbial communities treated by the C experimental group is different, a microcosm system is discovered13The Hydrogenophaga and Shinella enriched in C + As (III) + endophyte liquid treatment may have As (III) oxidizing ability.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims (10)

1. A method for detecting autotrophic arsenic-oxidizing functional microorganisms contained in plant root endophytes is characterized by comprising the following steps:
(1) removing the plant root surface sundry fungus;
(2) extracting and separating endophytes;
(3) performing expanded culture on endophytes;
(4) establishing and analyzing an endophyte liquid oxidized As (III) -DNA-SIP microcosm cultivation system:
4.1, adding the endophyte bacterial liquid obtained in the step (3) into an inorganic salt culture medium, and adding As, (III) and13/12c-labelled NaHCO3As electron acceptor and electron donor, shake culture;
the experiment included four groups of treatments:
A、13C-NaHCO3+ As (III) + endophytic bacteria liquid (C)13Experimental group C);
B、12C-NaHCO3+ As (III) + endophytic bacteria liquid (C)12Experimental group C);
C、13C-NaHCO3+ endophytic bacteria solution (As-free control group);
D、13C-NaHCO3+ as (iii) (sterile control group);
during the shaking culture period, carrying out non-destructive sampling at a plurality of time points, and detecting the concentrations of As (III) and As (V) in different treatments; after 15-20 days of culture, destructive sampling is carried out, and then differently processed DNA is extracted for subsequent SIP analysis;
4.2, performing ultra-high speed centrifugation on the extracted DNA, collecting each layered component, measuring the buoyancy density of the layered component, and removing CsCl precipitate; addition of ddH to the layered Components2Dissolving DNA by using O, finally obtaining purified DNA layers, and performing q-PCR amplification on each layer component by using an aioA gene primer;
4.3, selecting and processing the layered components with higher q-PCR amplification values in A and B, and performing high-throughput sequencing on the 16S rRNA gene V4-V5 region; comparing the sequencing data with the existing 16S rRNA database, classifying the data with more than 97% of similarity into a small group (OTU), and analyzing the microbial community diversity and abundance difference between treatments;
and (4) excluding the treatment of the control C, and determining the OTU with higher relative abundance in the treatment A as the autotrophic arsenic-oxidizing functional microorganism compared with the treatment B.
2. The method of claim 1, wherein: in each of the sets of treatments described in step 4.1,13C-NaHCO3the concentration of the mixed solution is 8mM,12C-NaHCO3the concentration is 8mM, the initial concentration of As (III) is 1-5mM, and 3.33mL of endophyte liquid is added in each liter of culture system.
3. The method of claim 1, wherein: the step (1) is as follows: shaking off the soil attached to the surface of the plant root, and sequentially placing the plant root in MgSO4Solution, MgSO 20 containing Tween 204Solution, MgSO4Solution, sodium hypochlorite solution containing Tween 20, MgSO4And (3) carrying out vortex oscillation or ultrasonic treatment in the solution to remove the plant root surface bacteria.
4. The method of claim 3, wherein: MgSO containing Tween 20 as described in step (1)4The solution, Tween 20 concentration is 0.01% (v/v).
5. The method of claim 3, wherein: the sodium hypochlorite solution containing the Tween 20 in the step (1) has the sodium hypochlorite concentration of 1% (v/v) and the Tween 20 concentration of 0.01% (v/v).
6. The method of claim 1, wherein: the step (2) is as follows:
removing root surface impurity bacteria of plant root in MgSO4Breaking cell wall in the solution, homogenizing, filtering with filter cloth, and collecting filtrate; centrifuging the filtrate at 500 Xg for 10min, centrifuging the supernatant at 9500rpm for 15min, and retaining thallus cell precipitate;
resuspending the thallus cell precipitate in sterile NaCl solution, slowly adding into iohexol solution, centrifuging at 15000 Xg for 60min, layering thallus cell precipitate in iohexol gradient density by centrifugation, displaying endophyte cells as milky white band, and recovering milky band part; removing the recovered iohexol, adding an equal volume of sterile NaCl solution, centrifuging at 7500 Xg for 20min, removing the supernatant, adding sterile NaCl solution, and resuspending; and finally, centrifuging at 7500 Xg for 20min, taking the precipitate, adding sterile NaCl solution for resuspension, and obtaining the recovered endophyte.
7. The method of claim 6, wherein: the concentration of the sterile NaCl solution in the step (2) is 0.8% (W/V).
8. The method of claim 6, wherein: the concentration of the iohexol solution in the step (2) is 1.3 g/mL.
9. The method of claim 1, wherein: the step (3) is as follows: and adding the separated plant root endophyte into an R2A liquid culture medium, and carrying out aerobic culture for 10-16h to obtain an endophyte bacterial liquid.
10. The method of claim 9, wherein: the composition of the R2A liquid medium is as follows: 0.5g/L of yeast extract powder; peptone 0.5 g/L; 0.5g/L of casein hydrolysate; glucose 0.5 g/L; 0.5g/L of soluble starch; dipotassium hydrogen phosphate 0.3 g/L; anhydrous magnesium sulfate 0.024 g/L; sodium pyruvate 0.3 g/L.
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