Testing Different Membrane Filters for 16S rRNA Gene-Based Metabarcoding in Karstic Springs
Abstract
:1. Introduction
2. Materials and Methods
2.1. Sampling and Sample Preparation
2.2. Physicochemical Measurements and Laboratory Analysis
2.3. 16S rRNA Protocol
2.4. Statistical Analysis
3. Results
3.1. Chemical Analysis
3.2. Microbial Diversity Inferred by 16s RNA Gene-Based Metabarcoding
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Membrane Code | Fabric | Producer | Porosity (µm) | Sterile * | Price ** |
---|---|---|---|---|---|
PES | polyethersulfone | Millipore | 0.22 | sterilized | High |
PVDF | polyvinylidene fluoride | Millipore | 0.22 | sterilized | High |
NC | cellulose nitrate | Macherey-Nagel | 0.20 | sterilized | Low |
NYLON | nylon | Fioroni | 0.22 | sterilized | Low |
MCE_a | mixed cellulose ester | Fioroni | 0.22 | sterile | Low |
MCE_b | mixed cellulose ester | Whatman | 0.20 | sterile | Medium |
S-Pak | mixed cellulose ester | Merck | 0.20 | sterile | Low |
PSR009 | cellulose acetate and nitrate | Nahita | 0.22 | sterilized | Low |
PSR010 | cellulose acetate and nitrate | Nahita | 0.45 | sterilized | Low |
Parameter | Unit | Spring | ||
---|---|---|---|---|
Banpotoc | Baita | Rapoltel | ||
TDS | mg/L | 1210 | 298 | 987 |
DOC | mg/L | 17.7 | 1.4 | 19.3 |
pH | - | 6.3 | 7.6 | 6.5 |
Bicarbonates | mg/L | 1122 | 256 | 988 |
Alkalinity | mmol/L | 18.4 | 4.2 | 16.2 |
Na | mg/L | 28.5 | 6 | 8.1 |
Ca | mg/L | 280 | 72.9 | 219 |
Mg | mg/L | 37 | 4.3 | 33.9 |
K | mg/L | 5.7 | 0.45 | 2.4 |
Ba | µg/L | 671 | 13.6 | 109 |
Sr | µg/L | 351 | 92.7 | 240 |
Mn | µg/L | 74.6 | 1.5 | 25.7 |
Ni | µg/L | 13.6 | 5.7 | 13.4 |
Cr | µg/L | 1.48 | 2.61 | 1.41 |
NH4+ | mg/L | 0.57 | 0.12 | 0.04 |
Cl− | mg/L | 4.53 | 2.55 | 2.2 |
SO42− | mg/L | 2.2 | 19.6 | 6 |
NO3− | mg/L | <0.2 | 2.4 | <0.2 |
NO2− | mg/L | 0.32 | <0.05 | <0.05 |
Spring | Code | Fabric | Membrane Code | OTUs | Diversity Indices | |
---|---|---|---|---|---|---|
Chao1 | Shannon | |||||
Banpotoc | AE20 | polyethersulfone | PES | 186 | 188.6250 | 2.8280 |
AE21 | polyvinylidene fluoride | PVDF | 226 | 264.2778 | 2.5738 | |
AE22 | cellulose nitrate | NC | 228 | 306.9643 | 2.6665 | |
AE23 | nylon | NYLON | 264 | 294.3571 | 2.7375 | |
AE24 | mixed cellulose ester | MCE_a | 251 | 318.0000 | 2.7688 | |
AE25 | mixed cellulose ester | MCE_b | 241 | 257.5000 | 2.8318 | |
AE26 | mixed cellulose ester | S-Pak | 261 | 320.2941 | 2.9322 | |
AE4 | cellulose acetate and nitrate | PSR009 | 263 | 267.5000 | 4.9879 | |
AE27 | cellulose acetate and nitrate | PSR010 | 115 | 126.5500 | 1.2992 | |
Baita | BE11 | polyethersulfone | PES | 1280 | 1699.2250 | 6.4304 |
BE12 | polyvinylidene fluoride | PVDF | 1335 | 1679.3142 | 6.8048 | |
BE18 | cellulose nitrate | NC | 1368 | 1652.6980 | 6.6819 | |
BE16 | nylon | NYLON | 1538 | 1803.8583 | 7.1422 | |
BE15 | mixed cellulose ester | MCE_a | 1271 | 1569.4933 | 7.0039 | |
BE13 | mixed cellulose ester | MCE_b | 1566 | 1734.7447 | 8.5057 | |
BE19 | mixed cellulose ester | S-Pak | 1455 | 1752.9286 | 7.9813 | |
BE17 | cellulose acetate and nitrate | PSR009 | 794 | 1009.5301 | 4.9305 | |
BE14 | cellulose acetate and nitrate | PSR010 | 718 | 915.3356 | 4.3866 | |
Rapoltel | CE29 | polyethersulfone | PES | 200 | 202.0000 | 4.8509 |
CE31 | polyvinylidene fluoride | PVDF | 221 | 224.7500 | 5.1441 | |
CE32 | cellulose nitrate | NC | 235 | 242.2000 | 4.4465 | |
CE30 | nylon | NYLON | 174 | 174.6000 | 3.9005 | |
CE34 | mixed cellulose ester | MCE_a | 214 | 217.3333 | 4.1399 | |
CE33 | mixed cellulose ester | MCE_b | 358 | 360.5000 | 5.0208 | |
CE2 | mixed cellulose ester | S-Pak | 310 | 312.5000 | 5.7927 | |
CE28 | cellulose acetate and nitrate | PSR009 | 260 | 265.1429 | 5.1996 | |
CE35 | cellulose acetate and nitrate | PSR009 | 255 | 255.4286 | 5.0427 | |
CE3 | cellulose acetate and nitrate | PSR010 | 168 | 169.5000 | 3.8749 |
Spring | Sample Code | Membrane Code | Biosafety Risk Species for Humans & Animals | ||
---|---|---|---|---|---|
100–500 reads/L | 501–1000 reads/L | >1001 reads/L | |||
Banpotoc | AE20 | PES | |||
AE21 | PVDF | ||||
AE22 | NC | ||||
AE23 | NYLON | ||||
AE24 | MCE_a | Acinetobacter junii | |||
AE25 | MCE_b | ||||
AE26 | S-Pak | ||||
AE4 | PSR009 | Comamonas testosteroni Enterobacter kobei | |||
AE27 | PSR010 | ||||
Baita | BE11 | PES | |||
BE12 | PVDF | ||||
BE18 | NC | ||||
BE16 | NYLON | ||||
BE15 | MCE_a | ||||
BE13 | MCE_b | ||||
BE19 | S-Pak | ||||
BE17 | PSR009 | ||||
BE14 | PSR010 | ||||
Rapolțel | CE29 | PES | Staphylococcus epidermidis | Acinetobacter junii | |
CE31 | PVDF | Acinetobacter junii Escherichiafergusonii | Staphylococcus epidermidis | ||
CE32 | NC | Acinetobacter junii | |||
CE30 | NYLON | Acinetobacter junii | |||
CE34 | MCE_a | Acinetobacter junii | |||
CE33 | MCE_b | Acinetobacter junii | |||
CE2 | S-Pak | ||||
CE28 | PSR009 | Atopobium vaginae Corynebacterium tuberculostearicum Gardnerella vaginalis Moraxella osloensis | Acinetobacter junii Staphylococcus epidermidis | ||
CE35 | PSR009 | Acinetobacter junii Veillonella dispar | |||
CE3 | PSR010 | Acinetobacter junii |
Spring/ Membrane | Pathogens Identification | Diversity Assessment | Proposed Membrane for Pathogens | Proposed Membrane for Diversity |
---|---|---|---|---|
Banpotoc | cellulose actetate and nitrate (PSR009) | nylon (Nylon) | cellulose actetate and nitrate (PSR009) | S-Pak, cellulose actetate and nitrate (PSR009) |
mixed cellulose ester (MCE_a) | cellulose actetate and nitrate (PSR009) | |||
mixed cellulose ester (S-Pak) | ||||
Baita | mixed cellulose ester (MCE-B) | |||
nylon (Nylon) | ||||
mixed cellulose ester (S-Pak) | ||||
Rapoltel | cellulose actetate and nitrate (PSR009) | mixed cellulose ester (MCE_b) | ||
polyvinylidene fluoride (PVDF) | mixed cellulose ester (S-Pak) | |||
polyethersulfone (PES) | cellulose actetate and nitrate (PSR009) |
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Moldovan, O.T.; Baricz, A.; Szekeres, E.; Kenesz, M.; Hoaghia, M.A.; Levei, E.A.; Mirea, I.C.; Năstase-Bucur, R.; Brad, T.; Chiciudean, I.; et al. Testing Different Membrane Filters for 16S rRNA Gene-Based Metabarcoding in Karstic Springs. Water 2020, 12, 3400. https://doi.org/10.3390/w12123400
Moldovan OT, Baricz A, Szekeres E, Kenesz M, Hoaghia MA, Levei EA, Mirea IC, Năstase-Bucur R, Brad T, Chiciudean I, et al. Testing Different Membrane Filters for 16S rRNA Gene-Based Metabarcoding in Karstic Springs. Water. 2020; 12(12):3400. https://doi.org/10.3390/w12123400
Chicago/Turabian StyleMoldovan, Oana Teodora, Andreea Baricz, Edina Szekeres, Marius Kenesz, Marial Alexandra Hoaghia, Erika Andrea Levei, Ionuț Cornel Mirea, Ruxandra Năstase-Bucur, Traian Brad, Iulia Chiciudean, and et al. 2020. "Testing Different Membrane Filters for 16S rRNA Gene-Based Metabarcoding in Karstic Springs" Water 12, no. 12: 3400. https://doi.org/10.3390/w12123400