NL2027762A

NL2027762A - Highly cadmium-tolerant strain and isolation and screening method thereof

Info

Publication number: NL2027762A
Application number: NL2027762A
Authority: NL
Inventors: Li Wen; Chen Yinyuan; Wang Tao
Original assignee: Xuzhou Inst Technology
Priority date: 2020-03-17
Filing date: 2021-03-17
Publication date: 2021-07-30
Also published as: CN111925950A; NL2027762B1

Abstract

The present invention relates to the technical field of soil remediation, and specifically provides a highly cadmium-tolerant strain and an isolation and screening method 5 thereof. The strain is deposited in the Guangdong Microbial Culture Collection Center (GDMCC) on May 28, 2018, with an accession number of GDMCC No: 60378, and has a 26S rDNA sequence shown in SEQ NO. 1. The present invention overcomes the deficiencies of the prior art and isolates the highly cadmium-tolerant strain from heavy metal contaminated soil samples. The strain has better cadmium resistance than that 10 reported in existing literature. In addition, the method for isolating and screening the highly cadmium-tolerant strain features low instrumentation requirement, simple operation, safety, and rapidness. The highly cadmium-tolerant strain of the present invention is of important significance for the remediation of soil and water contaminated by heavy metals, and has substantial social and economic value.

Description

-1- HIGHLY CADMIUM-TOLERANT STRAIN AND ISOLATION AND

SCREENING METHOD THEREOF

TECHNICAL FIELD The present invention relates to the technical field of soil remediation, and specifically belongs to a highly cadmium-tolerant strain and an isolation and screening method thereof.

BACKGROUND In some areas of China, the soil is heavily polluted. Soil pollution is usually a combined pollution that is dominated by a certain kind of pollutant and accompanied by other pollutants. Reportedly, nearly 25 million hm? cultivated land in China is polluted by heavy metals, such as Cd, As, Cr, and Pb, which accounts for about 1/5 of the total cultivated land area. Herein, cadmium-lead combined pollution, which is dominated by cadmium pollution, has seriously endangered the grain yield and quality safety of Hunan Province and other major grain production bases in China. Therefore, soil cadmium pollution has become an important environmental problem that poses a serious threat to human health and national food security. At the same time, heavy metal pollution in water is becoming more and more serious. In some watersheds, pollutant cadmium exceeds the standard as high as 64%. Bioremediation is deemed as a method with a wide range of sources, low disposal cost, no pollution and high remediation efficiency. In particular, microbial remediation technology is increasingly favored by scholars. Microorganisms that remediate the heavy metal pollution are dominated by indigenous fungi and bacteria. Tolerance to heavy metal pollution varies with different microorganisms, usually fungi > bacteria > actinomycetes. Indigenous microorganisms in contaminated soil tend to be highly resistant to heavy metals, and the microorganisms isolated therefrom are adapted to the local natural environment, so they can effectively remediate the heavy metal pollution. However, no strain with good

2- heavy metal resistance has been reported so far. Therefore, seeking a strain with high heavy metal resistance is of great significance for soil and water remediation and environmental protection.

SUMMARY An objective of the present invention is to provide a highly cadmium-tolerant strain and an isolation and screening method thereof. The present invention overcomes the deficiencies of the prior art and isolates and purifies a highly cadmium-tolerant strain from contaminated soil.

To solve the above problems, the technical solutions adopted by the present invention are as follows: A highly cadmium-tolerant strain is provided, where the strain is deposited in the Guangdong Microbial Culture Collection Center (GDMCC) on May 28, 2018, with an accession number of GDMCC No: 60378, and has a 26S rDNA sequence shown in SEQNO. 1. Further, the strain may have a maximum cadmium-tolerant concentration of 22,000 mg/L in a solid medium, and a maximum cadmium-tolerant concentration of 18,000 mg/L in a liquid medium. A method for isolating and screening the highly cadmium-tolerant strain is provided, including the following specific steps: step 1, isolation and purification of the strain: selecting a multiple heavy metal contaminated soil sample to prepare a soil suspension, and spreading a supernatant evenly on a solid purification medium supplemented with 100 mg/L cadmium chloride; picking single colonies separately, and using streak plate method to separately plate the colonies into solid media with cadmium concentrations of 100, 150, and 200 mg/L to select a colony with the strongest cadmium tolerance; purifying the single colony thereof by the plate streaking method for four consecutive times; and step 2, screening of the highly cadmium-tolerant strain: plating purified strains onto the same selective medium with serial concentration gradients including cadmium

-3- concentrations of 250-24,000 mg/L, successively, and culturing at room temperature, picking strains resistant to high-concentration cadmium to obtain the highly cadmium- tolerant strain of the present invention, being numbered QN-3. Further, the soil suspension may be prepared by mixing the soil sample with sterile water in a weight ratio of 1:10, shaking at 150 r/min and room temperature for 30 min, and being left to stand for 10 min.

Further, the solid purification medium may include the following components: 15 g of glucose, 5 g of yeast extract, 2 g of aerofloat, 4 g of xanthate, 3 mL of magnesium sulfate heptahydrate, 2 mL of ammonium sulfate, 4 mL of potassium chloride, 0.3 g of manganese sulfate, 20 g of agar, and 6 mL of bromophenol blue.

Further, the selective medium may be a Potato Dextrose Agar (PDA) solid medium or a PDA liquid medium, and specifically include the following components: 20 g of sucrose, 20 g of agar, 200 g of potato, and 1,000 mL of pure water, with cadmium chloride concentration gradients of 100 mg/L, 200 mg/L, 250 mg/L, 300 mg/L, 400 mg/L, 500 mg/L, 600 mg/L, 700 mg/L, 800 mg/L, 900 mg/ L, 1,000 mg/L, 1,600 mg/L, 1,800 mg/L, 2,000 mg/L, 2,200 mg/L, 2,400 mg/L, 2,500 mg/L, 2,600 mg/L, 4,000 mg/L, 6,000 mg/L, 8,000 mg/L, 10,000 mg/L, 11,000 mg/L, 12,000 mg/L, 13,000 mg/L, 14,000 mg/L, 15,000 mg/L, 16,000 mg/L, 17,000 mg/L, 18,000 mg/L, 19,000 mg/L, 20,000 mg/L, 22,000 mg/L, and 24,000 mg/L.

Compared with the prior art, the present invention has the following implementation effects: As for the highly cadmium-tolerant strain and the isolation and screening method thereof provided by the present invention, the highly cadmium-tolerant strain is isolated from heavy metal contaminated soil, and the strain has better cadmium resistance than that reported in existing literature.

In addition, the method for isolating and screening the highly cadmium-tolerant strain features low instrumentation requirements, simple operation, is secure and safe, and rapidity.

The highly cadmium- tolerant strain of the present invention is of important significance for the remediation of soil and water contaminated by heavy metals, and has substantial social and economic value.

4-

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic diagram of colonies in the preliminary screening of a soil suspension provided by the present invention; FIG. 2 is a broken line graph of the growth of the strain QN-3 provided by the present invention in liquid media with different cadmium concentrations; FIG. 3 is a broken line graph of the inhibitory rate of cadmium against the growth of strain QN-3 provided by the present invention; FIG. 4 is a schematic diagram of the cell morphology of the QN-3 provided by the present invention; FIG. 5 is a schematic diagram of the colony morphology of the strain QN-3 provided by the present invention; FIG. 6 1s an electropherogram of 26S rDNA of the strain QN-3 provided by the present invention; FIG. 7 is a phylogenetic tree of 26S rRNA gene sequences of the strain QN-3 provided by the present invention and model strains related thereto; and FIG. 8 is scanning electron micrographs of the bacterial cells of the strain QN-3 provided by the present invention.

DETAILED DESCRIPTION OF THE EMBODIMENTS The present invention will be further described below with reference to the examples, but the present invention is not limited thereto. Any improvement made without departing from the purpose of the present invention falls within the protection scope of the present invention. Example 1 A highly cadmium-tolerant strain was provided; the strain was deposited in the Guangdong Microbial Culture Collection Center (GDMCC) on May 28, 2018, with an accession number of GDMCC No: 60378, and had a 26S rDNA sequence shown in SEQ NO. 1. The isolation and screening method thereof was as follows:

5- step 1, isolation and purification of the strain: a multiple heavy metal contaminated soil sample was selected to prepare a soil suspension, and a supernatant was spread evenly on a solid purification medium supplemented with 100 mg/L cadmium chloride; single colonies were picked separately, and streak plate method was used to separately plate the colonies into solid media with cadmium concentrations of 100, 150, and 200 mg/L to select a colony with the strongest cadmium tolerance; the single colony thereof was purified by the plate streaking method for four consecutive times; step 2, screening of the highly cadmium-tolerant strain: purified strains were plated onto PDA solid media with serial concentration gradients containing cadmium concentrations of 250-24,000 mg/L, successively, and cultured at room temperature.

The screening results thereof are shown in Table 1. Table 1 The maximum cadmium tolerance of all strains in solid culture Strain Cadmium concentration (mg/L) No. 230 300 400 600 700 800 900 1000 10000 11000 22000 24000 OND AHF AE HE + - - - ON-8 +++ + - - - - - - - - - - QN-7 a + - - - - - - - QN-6 deeb ee - ~ ~ - - - - - - QN-5 Fd Eh + - ~ - - - - - - QN-4 bb Ab bb + - - - - - - - - QN-3 EE RRR eR RE bE ++ + - OQN-2 +H - - - - - - - - - - QN-1 tt AE + - - - - - - - - JZP-7 ++ - - - - - - - - - . JZP-6 HA +++ HH + - - - - - - - - JZP-5 ++ - . . . . . . . . . JZP-4 + ++ Fb tt +++ tt Ht ++ + - - JZP-3 ++ - . - . . . - - - - - JZP-2 a A + - - - - - - - JZP-1 ee + ~ ~ - - - - - - NOTE: +++ denotes vigorous growth; ++ denotes fair growth; + denotes the strain slightly grown; - denotes the strain hardly grown It can be seen from Table 1 that: as the cadmium concentration increases, the growth of all strains is inhibited, and the plate count is gradually decreasing.

Strains QN-8, QN-6, QN-2, JZP-7, JZP-5, and JZP-3 are poorly tolerant to cadmium, strains QN-9, QN-7,

6- QN-5, QN-4, QN-1, JZP-4, JZP-6, JZP-2, and JZP-1 are well tolerant to cadmium, strain QN-3 is the most tolerant to cadmium. The QN-3 can grow on a plate with a cadmium concentration of 22,000 mg/L. So far, the strain has better cadmium tolerance compared with that reported in literature. Therefore, the QN-3 is finally selected as the strain with maximum cadmium tolerance. Example 2 Based on Example 1, strains with better cadmium tolerance were plated onto PDA liquid media with serial concentration gradients containing cadmium concentrations of 250-24,000 mg/L, successively, and cultured at room temperature. The screening results thereof are shown in Table 2. Table 2 The tolerance of all strains to liquid cadmium concentration Strain Cadmium concentration (mg/L) No. 500 700 1200 1800 2400 3400 4000 10000 18000 25000 QN-9 1458 1.095 - - - - - - - - QN-7 1253 0.236 0.081 0052 - - - - - - ON-5 02 - - - - - - - - - QN-4 1250 0.030 0002 0019 - - - - - - QN-3 2.571 2.290 2.214 2.133 2.105 2061 2016 1259 0.106 0.009 ON-1 1058 - - - - - - - - - JZP-6 0703 - - - - . - - - . JZP-4 0.544 0.383 0.886 0398 - - - - - - JZP-2 1224 1196 0932 0031 - - - - - - JZP-1 0042 - - - - - - - - - NOTE: - denotes no growth, and the results listed in the table are absorbance values It can be seen from Table 2 that: as the cadmium concentration increases, the growth of all strains is inhibited. QN-9, QN-5, QN-1, JZP-6, and JZP-1 grow poorly, QN-7, QN-

4. JZP-4, and JZP-2 grow well, and QN-3 grows the best. Therefore, the QN-3 is finally selected as the strain with maximum liquid cadmium tolerance, which is the same as solid. Example 3 Cadmium tolerance test of strains:

7- The highly cadmium-tolerant strain QN-3 selected was cultured on liquid media with different cadmium concentrations. The growth of the QN-3 in liquid media with different cadmium concentrations is shown in FIG. 2. The inhibitory rate of cadmium against the growth of QN-3 is shown in FIG. 3.

From FIG. 2, the ODso value gradually decreases with the increase of cadmium concentration; the maximum cadmium tolerance of the QN-3 is 18,000 mg/L. From FIG. 3, the inhibitory rate of cadmium against bacterial cells increases with the increase of cadmium concentration and is completely inhibited when the cadmium concentration reaches 25,000 mg/L. Therefore, the maximum cadmium tolerance of the QN-3 in liquid medium is 18,000 mg/L.

Example 4 Identification and testing of strains:

1. The highly cadmium-tolerant strain QN-3 selected was observed under a microscope. The cell morphology of the QN-3 is shown in FIG. 4. The QN-3 cells are oval and quasi-circular, showing typical yeast morphology.

2. The highly cadmium-tolerant strain QN-3 was subjected to solid culture. The result of colony morphology is shown in FIG. 5. The QN-3 colony is pinkish-orange, with a smooth surface, a sticky texture, a round prominence, regular edges, and a diameter of 1 to 2 mm. The strain smells pungent.

3. Physiological and biochemical test results of the QN-3: The highly cadmium- tolerant strain QN-3 was spread on media with different carbon sources, and growth thereof was observed. The results are shown in Table 3.

Table 3 Growth status of carbon source assimilation experiment Carbon source Growth status CK ++ Soluble starch + Maltose ++ Citric acid +

8- Glucose ++ Sorbitol + Sucrose ++ Lactose ++ NOTE: ++ denotes vigorous growth, and + denotes poor growth From Table 3, the QN-3 uses carbon sources including maltose, glucose, sorbitol, sucrose and lactose better; the QN-3 uses carbon sources including soluble starch and citric acid poorly.

4. As nitrogen sources, both ammonium sulfate and potassium nitrate can be used by the QN-3.

5. The QN-3 can grow on a vitamin-free medium, that is, the strain can grow in the absence of vitamins.

6. The urea decomposition experiment does not appear dark red, so the QN-3 cannot use urea.

7. The QN-3 does not produce starch analogs.

8. The QN-3 is tolerant to up to 40% sugar.

9. The optimal growth pH of the QN-3 ranges from 4 to 11, and the optimal growth pH is 6.0.

10. When the pH is lower than 2.0, it is difficult for the QN-3 strain to grow and reproduce.

11. The QN-3 strain has some salt tolerance, with the fastest growth in the range of 0- 10% NaCl concentration, and preferably 2%; the strain grows vigorously and steadily before salt concentration of 15% and basically does not grow after salt concentration of 15%.

12. The optimum growth temperature range of the QN-3 strain is 20-30°C, and the optimum growth temperature is about 25°C; the strain does not grow at temperatures below 4°C and above 45°C. Example 5

9- Molecular biological identification of the cadmium-tolerant strain (to select the strain QN-3 with optimal cadmium tolerance for identification)

1. Electropherogram of 26S rDNA of the strain QN-3 Using the genomic DNA of the strain QN-3 as a template, the 26S rDNA thereof was amplified by PCR, followed by 1% agarose gel electrophoresis; the electrophoresis was observed at 150 V and 100 mA for 20 min, and the product was aligned with MK. The result 1s shown in FIG. 6. Aligned with the MK band, the strain QN-3 shows a clear band at 500 bp, indicating that the PCR sequence size of the strain QN-3 is about 500 bp.

2. Sequencing of 16S rDNA of strain ZY-6 The electrophoretic band of the PCR product of strain ZY-6 was cut, recovered, and sequenced. The result is shown in the sequencing list. The sequence length is 1,522 bp.

3. Construction of a phylogenetic tree of the 26S rDNA of the strain QN-3 A phylogenetic tree was constructed based on the 26S rDNA gene sequences of the strain QN-3 and 8 model strains related thereto, as shown in FIG. 7. The strain QN-3 and Cvstobasidium oligophagum (AB702967.1) were in the same branch with 99% homology. Therefore, combined with morphological identification and physiological and biochemical tests, this strain was identified as Cystobasiditm oligophagum. The 26S rDNA sequence of this strain was submitted to the GenBank database of NCBI to obtain the accession number of MK417801.

4. Scanning electron micrograph of highly cadmium-tolerant yeast QN-3 The scanning electron micrographs of the highly cadmium-tolerant yeast QN-3 in 0 mg/L, 100 mg/L and 1,500 mg/L cadmium solutions are shown in FIG. 8. The above content is merely examples and descriptions of the concept of the present invention. Various modifications or supplementations of the specific examples described or substitutions in a similar manner made by those skilled in the art without departing from the concept of the present invention or going beyond the scope as defined in the appended claims shall fall within the protection scope of the present invention.

SEQLTXT

SEQUENCE LISTING <110> XUZHOU INSTITUTE OF TECHNOLOGY <120> HIGHLY CADMIUM-TOLERANT STRAIN AND ISOLATION AND SCREENING METHOD

THEREOF <130> GBSL@44-NL <150> CN 202010185252.1 <151> 2020-03-17 <160> 1 <170> PatentIn version 3.5 <210> 1 <211> 623 <212> DNA <213> Artificial Sequence <220> <223> The sequence is synthesized. <400> 1 aaaagcggag gaaaagaaac taacaaggat tcccctagta acggcgagtg aagtgggaaa 60 agctcaactt tgaaatctgg caccttcggt gtccgagttg tagtctcaag aagtgttttc 120 tgtgctagtc catgtatgag tctgttggaa cacagcgtca tagagggtga caaccccgtt 180 catgacatgg atactagtgc tctgtgatac actctcgaag agtcgagttg tttgggaatg 240 cagctcaaat tgggtggtaa attccatcta aagctaaata ttggcgagag accgatagca 300 aacaagtacc gtgagggaaa gatgaaaagc actttggaaa gagagttaac agtacgtgaa 360 attgttgaaa gggaaacgat tgaagtcaga cgtgcgtgat gcggttcagc tctggttcgc 420 cagagtgtac tccgtatctt tgcaggccaa catcggtttt gtgagccgga taaaggtcag 480 agaaatgtgg caccttcggg tgtgttatag tctctgactg aatacggttt atgggaccga 540 ggaacgcagc gcgccgcaag gcaaaggttc cgaccttttc gcgcttagga tgttggtgaa 600 atggctttaa acgacccgtc ttg 623 Pagina 1

Claims

-10- Conclusies-10- Conclusions

1. Stam die zeer tolerant is voor cadmium, waarbij de stam gedeponeerd is in het Guangdong Microbial Culture Collection Center (GDMCC) op 28 mei 2018, met een toegangsnummer van GDMCC Nr: 60378, en die een 26S-rDNA-sequentie heeft zoals die in SEQ NR. 1 getoond is.1. Strain highly tolerant to cadmium, the strain being deposited at the Guangdong Microbial Culture Collection Center (GDMCC) on May 28, 2018, having an accession number of GDMCC No: 60378, and having a 26S rDNA sequence like that in SEQ NO. 1 is shown.

2. Stam die zeer tolerant is voor cadmium volgens conclusie 1, waarbij de stam een maximale cadmiumtolerantieconcentratie van 22.000 mg/L heeft in een vast medium, en een maximale cadmiumtolerantieconcentratie van 18.000 mg/L heeft in een vloeibaar medium.The strain highly tolerant to cadmium according to claim 1, wherein the strain has a maximum cadmium tolerance concentration of 22,000 mg/L in a solid medium, and a maximum cadmium tolerance concentration of 18,000 mg/L in a liquid medium.

3. Werkwijze voor het isoleren en het screenen van de stam die zeer tolerant is voor cadmium volgens conclusie 1 of 2, waarbij de werkwijze de volgende specifieke stappen omvat: stap 1, het isoleren en het zuiveren van de stam: het selecteren van een aardemonster dat met meerdere zware metalen verontreinigd is om een aardesuspensie te bereiden, en het gelijkmatig spreiden van een supernatans op een vast zuiveringsmedium dat gesupplementeerd is met 100 mg/L cadmiumchloride; het apart van elkaar pikken van enkele kolonies, en het gebruiken van uitstrijkplaatwerkwijze om de kolonies apart van elkaar in het vaste medium met cadmiumconcentraties van 100, 150 en 200 mg/L te platen om een kolonie te selecteren met de sterkste cadmiumtolerantie; het zuiveren van de enkele kolonie daarvan middels de plaatuitstrijkwerkwijze gedurende vier opeenvolgende keren; en stap 2, het screenen van de stam die zeer tolerant is voor cadmium: het uitplaten van gezuiverde stammen op hetzelfde selectieve medium met seriële concentratiegradiënten die achtereenvolgens cadmiumconcentraties van 250 — 24.000 mg/L omvatten, en het kweken bij kamertemperatuur, het pikken van stammen die resistent zijn tegen een hoge cadmiumconcentratie om de stam die zeer tolerant is voor cadmium volgens de onderhavige uitvinding te verkrijgen, die QN-3 genummerd is.The method of isolating and screening the strain highly tolerant to cadmium according to claim 1 or 2, wherein the method comprises the following specific steps: step 1, isolating and purifying the strain: selecting a soil sample contaminated with multiple heavy metals to prepare a soil slurry, and evenly spreading a supernatant on a solid purification medium supplemented with 100 mg/L cadmium chloride; pecking single colonies separately, and using the smear plate method to plate the colonies separately from each other in the solid medium with cadmium concentrations of 100, 150 and 200 mg/L to select a colony with the strongest cadmium tolerance; purifying the single colony thereof by the plating method for four consecutive times; and step 2, screening the strain highly tolerant to cadmium: plating purified strains on the same selective medium with serial concentration gradients comprising cadmium concentrations sequentially from 250-24,000 mg/L, and culturing at room temperature, pecking strains resistant to a high cadmium concentration to obtain the highly cadmium tolerant strain of the present invention, numbered QN-3.

4. Werkwijze voor het isoleren en het screenen van de stam die zeer tolerant is voor cadmium volgens conclusie 3, waarbij de aardesuspensie bereid wordt middels hetThe method of isolating and screening the highly cadmium tolerant strain according to claim 3, wherein the soil suspension is prepared by

“11 - mengen van het aardemonster met steriel water in een gewichtsverhouding van 1:10, het schudden met 150 r/min en bij kamertemperatuur gedurende 30 min, en het laten staan gedurende 10 min.11 - mixing the soil sample with sterile water in a weight ratio of 1:10, shaking at 150 r/min and at room temperature for 30 min, and standing for 10 min.

5. Werkwijze voor het isoleren en het screenen van de stam die zeer tolerant is voor cadmium volgens conclusie 3, waarbij het vaste zuiveringsmedium de volgende componenten omvat: 15 g glucose, 5 g gistextract, 2 g aerofloat, 4 g xanthaat, 3 mL magnesiumsulfaatheptahydraat, 2 mL ammoniumsulfaat, 4 mL kaliumchloride, 0,3 g mangaansulfaat, 20 g agar en 6 mLbroomfenolblauw.The method of isolating and screening the strain highly tolerant to cadmium according to claim 3, wherein the solid purification medium comprises the following components: 15 g glucose, 5 g yeast extract, 2 g aerofloat, 4 g xanthate, 3 mL magnesium sulfate heptahydrate , 2 mL ammonium sulfate, 4 mL potassium chloride, 0.3 g manganese sulfate, 20 g agar, and 6 mL bromophenol blue.

6. Werkwijze van het isoleren en het screenen van de stam die zeer tolerant is voor cadmium volgens conclusie 3, waarbij het selectieve medium een vast medium van aardappeldextroseagar (PDA) of een vloeibaar medium van PDA is, en specifiek de volgende componenten omvat: 20 g sucrose, 20 g agar, 200 g aardappel en 1.000 mL zuiver water, met cadmiumchlorideconcentratiegradiënten van 100 mg/L, 200 mg/L, 250 mg/L, 300 mg/L, 400 mg/L, 500 mg/L, 600 mg/L, 700 mg/L, 800 mg/L, 900 mg/ L, 1.000 mg/L, 1.600 mg/L, 1.800 mg/L, 2.000 mg/L, 2.200 mg/L, 2.400 mg/L, 2.500 mg/L, 2.600 mg/L, 4.000 mg/L, 6.000 mg/L, 8.000 mg/L, 10.000 mg/L, 11.000 mg/L, 12.000 mg/L,The method of isolating and screening the strain highly tolerant to cadmium according to claim 3, wherein the selective medium is a solid medium of potato dextrose agar (PDA) or a liquid medium of PDA, and specifically comprises the following components: g sucrose, 20 g agar, 200 g potato and 1,000 mL pure water, with cadmium chloride concentration gradients of 100 mg/L, 200 mg/L, 250 mg/L, 300 mg/L, 400 mg/L, 500 mg/L, 600 mg/L, 700 mg/L, 800 mg/L, 900 mg/L, 1,000 mg/L, 1,600 mg/L, 1,800 mg/L, 2,000 mg/L, 2,200 mg/L, 2,400 mg/L, 2,500 mg/L, 2,600 mg/L, 4,000 mg/L, 6,000 mg/L, 8,000 mg/L, 10,000 mg/L, 11,000 mg/L, 12,000 mg/L,

13.000 mg/L, 15 14.000 mg/L, 15.000 mg/L, 16.000 mg/L, 17.000 mg/L, 18.000 mg/L,13,000 mg/L, 15 14,000 mg/L, 15,000 mg/L, 16,000 mg/L, 17,000 mg/L, 18,000 mg/L,

19.000 mg/L, 20.000 mg/L, 22.000 mg/L en 24.000 mg/L.19,000 mg/L, 20,000 mg/L, 22,000 mg/L and 24,000 mg/L.