Register      Login
Soil Research Soil Research Society
Soil, land care and environmental research
RESEARCH ARTICLE

Abundance and composition of ammonia-oxidising bacteria and archaea of a degraded lake wetland, Inner Mongolian Plateau, China

Linhui Wu A B C , Lihua Hui A , Xiaoyi Wang C , Jingyu Li A , Jingli Yu B and Ji Zhao B D
+ Author Affiliations
- Author Affiliations

A College of Life Sciences of Inner Mongolia University, 235 West University Blvd, Hohhot 010021, Inner Mongolia Autonomous Region, China.

B College of Environment and Resources of Inner Mongolia University, 235 West University Blvd, Hohhot 010021, Inner Mongolia Autonomous Region, China.

C CSIRO Land and Water, Urrbrae, SA 5064, Australia.

D Corresponding author. Email: ndzj@imu.edu.cn

Soil Research 51(6) 554-560 https://doi.org/10.1071/SR13160
Submitted: 23 May 2013  Accepted: 11 August 2013   Published: 6 November 2013

Abstract

Wetlands are one of the most highly threatened ecosystems on Earth, and their loss and degradation are regarded as major environmental problems. The degradation rate of lake wetlands of the Huitengxile grassland, Inner Mongolia, has increased over recent years. In this study, soil samples were collected in a degraded lake wetland of the Huitengxile grassland. The abundance and composition of soil ammonia-oxidising bacteria (AOB) and ammonia-oxidising archaea (AOA) were assessed by quantitative real-time PCR, cloning, and sequencing approaches. Four sampling sites were selected according to the order of water withdrawal. The lakeshore sample appeared to have the highest copy numbers of AOB amoA genes, and the lowest AOB population size was found in the lakebed sample. Similar to AOB, the AOA were most abundant in the lakeshore sample, and the population size in the lake centre sample was the lowest. Ex2cept in one lakebed sample, AOB were more abundant than AOA in all other samples, with AOB to AOA ratios ranging from 11 to 13. Phylogenetic analysis of the amoA gene fragments showed that all AOB sequences from different sites were affiliated to class β-Proteobacteria, order Nitrosomonadales, family Nitrosomonadaceae and can be grouped into two clusters. Little difference was found in AOB community composition among different samples, which indicated that AOB community composition was stable during the drying process of the degraded lake. However, the AOA community compositions were very different between samples. All AOA sequences fell into four clusters. Cluster 1 was dominant in the sample from the centre of the lake, and cluster 2 was dominant in the lakeshore sample, indicating a difference in the community composition of AOA in response to the drying up of the lake. No clear relationship was found between the AOA and AOB community populations and soil physio-chemical properties. This study suggested that the AOA community in wetland systems is more sensitive than the AOB community to the drying process of the wetland ecosystem.

Additional keywords: abundance, ammonia monooxygenase, ammonia-oxidising archaea (AOA), ammonia-oxidising bacteria (AOB), diversity, wetlands.


References

Avrahami S, Conrad R (2003) Patterns of community change among ammonia oxidizers in meadow soil upon long-term incubation at different temperatures. Applied and Environmental Microbiology 69, 6152–6164.
Patterns of community change among ammonia oxidizers in meadow soil upon long-term incubation at different temperatures.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3sXotlKjsL4%3D&md5=3c5f773936ecdbef3c352af8513e3d58CAS | 14532075PubMed |

Avrahami S, Conrad R, Braker G (2002) Effect of soil ammonium concentration on N2O release and on the community structure of ammonia oxidizers and denitrifiers. Applied and Environmental Microbiology 68, 5685–5692.
Effect of soil ammonium concentration on N2O release and on the community structure of ammonia oxidizers and denitrifiers.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD38Xos1ajsbY%3D&md5=b2a9a3fe778a26f63f503599d454cf50CAS | 12406765PubMed |

Avrahami S, Liesack W, Conrad R (2003) Effects of temperature and fertilizer on activity and community structure of soil ammonia oxidizers. Environmental Microbiology 5, 691–705.
Effects of temperature and fertilizer on activity and community structure of soil ammonia oxidizers.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3sXntFyqtr0%3D&md5=c74c7e38ab89b10ccbb93381f9d7c9e7CAS | 12871236PubMed |

Bernhard AE, Donn T, Giblin AE, Stahl DA (2005) Loss of diversity of ammonia-oxidizing bacteria correlates with increasing salinity in an estuary system. Environmental Microbiology 7, 1289–1297.
Loss of diversity of ammonia-oxidizing bacteria correlates with increasing salinity in an estuary system.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2MXhtVGnur3K&md5=476a63683a64176623668f2f933d9fe6CAS | 16104852PubMed |

Bremner JM, Blackmer AM (1978) Nitrous oxide: emission from soil during nitrification of fertilizer nitrogen. Science 199, 295–296.
Nitrous oxide: emission from soil during nitrification of fertilizer nitrogen.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaE1cXntlGlsw%3D%3D&md5=e4d56491baaa7d9198359769fe44033bCAS | 17759663PubMed |

Cabello P, Dolores Roldan M, Moreno-Vivian C (2004) Nitrate reduction and the nitrogen cycle in archaea. Microbiology 150, 3527–3546.
Nitrate reduction and the nitrogen cycle in archaea.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2cXhtVejs7jI&md5=77031d112bb9761eb7d842f445fa404dCAS | 15528644PubMed |

Chen XP, Zhu YG, Xia Y, Shen JP, He JZ (2008) Ammonia-oxidizing archaea: important players in paddy rhizosphere soil? Environmental Microbiology 10, 1978–1987.
Ammonia-oxidizing archaea: important players in paddy rhizosphere soil?Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1cXpsFOqtrs%3D&md5=336e83523455b4659e32a9ae3148b6a0CAS | 18430011PubMed |

Chouari R, Le Paslier D, Dauga C, Daegelen P, Weissenbach J, Sghir A (2005) Novel major bacterial candidate division within a municipal anaerobic sludge digester. Applied and Environmental Microbiology 71, 2145–2153.
Novel major bacterial candidate division within a municipal anaerobic sludge digester.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2MXjsVyjsro%3D&md5=b7a3d0ccca82a35c1cf21fbf48138e15CAS | 15812049PubMed |

Costanza R, d’Arge R, de Groot R, Farber S, Grasso M, Hannon B, Limburg K, Naeem S, O’Neill RV, Paruelo J, Raskin RG, Sutton P, van den Belt M (1997) The value of the world’s ecosystem services and natural capital. Nature 387, 253–260.
The value of the world’s ecosystem services and natural capital.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK2sXjtlShtbs%3D&md5=99a0ca6308b659fe6d2cf6aac698295eCAS |

De Boer W, Klein Gunnewiek PJA, Veenhuis M, Bock E, Laanbroek HJ (1991) Nitrification at low pH by aggregated chemolithotrophic bacteria. Applied and Environmental Microbiology 57, 3600–3604.

Di HJ, Cameron KC, Shen JP, Winefield CS, O’Callaghan M, Bowatte S, He JZ (2009) Nitrification driven by bacteria and not archaea in nitrogen-rich grassland soils. Nature Geoscience 2, 621–624.
Nitrification driven by bacteria and not archaea in nitrogen-rich grassland soils.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXhtVKktLbE&md5=9f5ffc61e73e995d495fed3b7f2d3532CAS |

Elkins JW, Wosfy SC, McElroy MB, Kolb CE, Kaplan WA (1978) Aquatic sources and sinks for nitrous oxide. Nature 275, 602–606.
Aquatic sources and sinks for nitrous oxide.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaE1MXht1Oiurs%3D&md5=570404056bf5ce3aa66f41e86cdc944bCAS |

Erguder TH, Boon N, Wittebolle L, Marzorati M, Verstraete W (2009) Environmental factors shaping the ecological niches of ammonia-oxidizing archaea. FEMS Microbiology Reviews 33, 855–869.
Environmental factors shaping the ecological niches of ammonia-oxidizing archaea.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXhtVWksbnE&md5=7b85e23c960f7f7a83acea1c652f1c53CAS | 19453522PubMed |

Francis CA, O’Mullan GD, Ward BB (2003) Diversity of ammonia monooxygenase (amoA) genes across environmental gradients in Chesapeake Bay sediments. Geobiology 1, 129–140.
Diversity of ammonia monooxygenase (amoA) genes across environmental gradients in Chesapeake Bay sediments.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3sXpvFSgsrg%3D&md5=c6ba7f10c1a584dc880c5e640566fb2eCAS |

Francis CA, Roberts KJ, Beman JM, Santoro AE, Oakley BB (2005) Ubiquity and diversity of ammonia-oxidizing archaea in water columns and sediments of the ocean. Proceedings of the National Academy of Sciences of the United States of America 102, 14 683–14 688.
Ubiquity and diversity of ammonia-oxidizing archaea in water columns and sediments of the ocean.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2MXhtFKjsb7J&md5=eac4abff0da894972ea96b93e254fa1bCAS |

Geets J, Boon N, Verstraete W (2006) Strategies of aerobic ammonia-oxidizing bacteria for coping with nutrient and oxygen fluctuations. FEMS Microbiology Ecology 58, 1–13.
Strategies of aerobic ammonia-oxidizing bacteria for coping with nutrient and oxygen fluctuations.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD28XhtVOmtrbF&md5=4811a5dd324769a4b5d9f562c88311a7CAS | 16958903PubMed |

Hao YJ, Wu SW, Wu WX, Chen YX (2007) Research progress on the microbial ecology of aerobic ammonia-oxidizing bacteria. Acta Ecologica Sinica 27, 1573–1582.

He JZ, Shen JP, Zhang LM, Zhu YG, Zheng YM, Xu MG, Di HG (2007) Quantitative analyses of the abundance and composition of ammonia-oxidizing bacteria and ammonia-oxidizing archaea of a Chinese upland red soil under long-term fertilization practices. Environmental Microbiology 9, 2364–2374.
Quantitative analyses of the abundance and composition of ammonia-oxidizing bacteria and ammonia-oxidizing archaea of a Chinese upland red soil under long-term fertilization practices.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2sXhtVyku7rE&md5=8858d4205f407f3bdb4b23df64e367cdCAS | 17686032PubMed |

Huang DF, Li T (2008) Variation of diversity and activity of ammonia-oxidizing bacteria community in the integrated vertical-flow constructed wetlands. Chinese Journal of Environmental Science 29, 2160–2165.

Jia ZJ, Conrad R (2009) Bacteria rather than Archaea dominate microbial ammonia oxidation in an agricultural soil. Environmental Microbiology 11, 1658–1671.
Bacteria rather than Archaea dominate microbial ammonia oxidation in an agricultural soil.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXptlyhurs%3D&md5=41d55b38376570b3b10e6486dbaddbc3CAS |

Könneke M, Bernhard AE, De la Torre JR, Walker CB, Waterbury JB, Stahl DA (2005) Isolation of an autotrophic ammonia-oxidizing marine archaeon. Nature 437, 543–546.
Isolation of an autotrophic ammonia-oxidizing marine archaeon.Crossref | GoogleScholarGoogle Scholar | 16177789PubMed |

Kouki S, Saidi N, M’hiri F, Nasr H, Cherif H, Ouzari H, Hassen A (2011) Isolation and characterization of facultative mixotrophic ammonia-oxidizing bacteria from constructed wetlands. Journal of Environmental Sciences 23, 1699–1708.
Isolation and characterization of facultative mixotrophic ammonia-oxidizing bacteria from constructed wetlands.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3MXhsVCls7bM&md5=669acbd1d8146344d20c614d8c3b65b5CAS |

Kowalchuk GA, Stephen JR (2001) Ammonia-oxidizing bacteria: A model for molecular microbial ecology. Annual Review of Microbiology 55, 485–529.
Ammonia-oxidizing bacteria: A model for molecular microbial ecology.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3MXnslKjtLs%3D&md5=66ce9fdcd9eac45e68282a7b7a503f0cCAS | 11544365PubMed |

Kowalchuk GA, Stienstra AW, Heilig GHJ, Stephen JR, Woldendorp JW (2000a) Molecular analysis of ammonia-oxidising bacteria in soil of successional grasslands of the Drentsche A (The Netherlands). FEMS Microbiology Ecology 31, 207–215.
Molecular analysis of ammonia-oxidising bacteria in soil of successional grasslands of the Drentsche A (The Netherlands).Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3cXhslamtbc%3D&md5=4bae5de1879c83e2b8712a8df9211065CAS | 10719201PubMed |

Kowalchuk GA, Stienstra AW, Heiling GHJ, Stephen JR, Woldendorp JW (2000b) Changes in the community structure of ammonia-oxidizing bacteria during secondary succession of calcareous grasslands. Environmental Microbiology 2, 99–110.
Changes in the community structure of ammonia-oxidizing bacteria during secondary succession of calcareous grasslands.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BD3M3jtlyksw%3D%3D&md5=1920d70847c0e7081f90f72e75464704CAS | 11243267PubMed |

Leininger S, Urich T, Schloeter M, Schwark L, Qi J, Nicol GW, Prosser JI, Schuster SC, Schleper C (2006) Archaea predominate among ammonia-oxidizing prokaryotes in soils. Nature 442, 806–809.
Archaea predominate among ammonia-oxidizing prokaryotes in soils.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD28XotFKmtrs%3D&md5=ef544b030ce1466f5d34e214a94de235CAS | 16915287PubMed |

Li JY, Li B, Zhou Y, Xu JF, Zhao J (2011) A rapid DNA extraction method for PCR amplification from wetland soils. Letters in Applied Microbiology 52, 626–633.
A rapid DNA extraction method for PCR amplification from wetland soils.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3MXos1Khtbw%3D&md5=1eac96cce43d70d06e4eaed9f6982cc1CAS |

McGeehan SL, Naylor DV (1988) Automated instrumental analysis of carbon and nitrogen in plant and soil samples. Communications in Soil Science and Plant Analysis 19, 493–505.
Automated instrumental analysis of carbon and nitrogen in plant and soil samples.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaL1cXitFygur0%3D&md5=3057e6f1779bd14de0f14f50234f769fCAS |

Mehlich A (1978) New extractant for soil test evaluation of phosphorus, potassium, magnesium, calcium, sodium, manganese and zinc. Communications in Soil Science and Plant Analysis 9, 477–492.
New extractant for soil test evaluation of phosphorus, potassium, magnesium, calcium, sodium, manganese and zinc.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaE1cXlt1ygs7g%3D&md5=5960468ee2555232342cdef1669d7ff1CAS |

Nicolaisen MH, Ramsing NB (2002) Denaturing gradient gel electrophoresis (DGGE) approaches to study the diversity of ammonia-oxidizing bacteria. Journal of Microbiological Methods 50, 189–203.
Denaturing gradient gel electrophoresis (DGGE) approaches to study the diversity of ammonia-oxidizing bacteria.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD38Xjtl2is7Y%3D&md5=f533249846ae4fd5dd287931687ada14CAS | 11997169PubMed |

Nicolaisen MH, Risgaard-Petersen N, Revsbech NP, Reichardt W, Ramsing NB (2004) Nitrification–denitrification dynamics and community structure of ammonia oxidizing bacteria in high yield irrigated Philippine rice filed. FEMS Microbiology Ecology 49, 359–369.
Nitrification–denitrification dynamics and community structure of ammonia oxidizing bacteria in high yield irrigated Philippine rice filed.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2cXmvFWrsLY%3D&md5=f85e0dd8b71d275bc30491e1db206961CAS | 19712286PubMed |

Page AL, Miller RH, Keeney DR (1982) Chemical and microbiological properties. In ‘Methods of soil analysis’. pp. 1179–1237. (American Society of Agronomy Publishing: Madison, WI)

Park HD, Wells GF, Bae H, Criddle CS, Francis CA (2006) Occurrence of ammonia-oxidizing archaea in wastewater treatment plant bioreactors. Applied and Environmental Microbiology 72, 5643–5647.
Occurrence of ammonia-oxidizing archaea in wastewater treatment plant bioreactors.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD28XosVKlt7s%3D&md5=24db71f5a115b8971220a695287aaf42CAS | 16885322PubMed |

Prosser JI (1989) Autotrophic nitrification in bacteria. Advances in Microbial Physiology 30, 125–181.
Autotrophic nitrification in bacteria.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK3cXls1ymt7Y%3D&md5=5de9c6515074202b4e565a517f030855CAS | 2700538PubMed |

Purkhold U, Pommerening-Roser A, Juretschko S, Schmid MC, Koops HP, Wagner M (2000) Phylogeny of all recognized species of ammonia oxidizers based on comparative 16S rRNA and amoA sequence analysis: implications for molecular diversity surveys. Applied and Environmental Microbiology 66, 5368–5382.
Phylogeny of all recognized species of ammonia oxidizers based on comparative 16S rRNA and amoA sequence analysis: implications for molecular diversity surveys.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3MXjsVygsro%3D&md5=762186d03fc0919867652df86f263a6dCAS | 11097916PubMed |

Purvaja R, Ramesh R, Ray AK, Rixen T (2008) Nitrogen cycling: a review of the processes, transformations and fluxes in coastal ecosystems. Current Science 94, 1419–1438.

Rotthauwe JH, Witzel KP, Liesack W (1997) The ammonia monooxygenase structural gene amoA as a functional marker: molecular fine-scale analysis of natural ammonia-oxidizing populations. Applied and Environmental Microbiology 63, 4704–4712.

Schleper C, Jurgens G, Jonuscheit M (2005) Genomic studies of uncultivated archaea. Nature Reviews. Microbiology 3, 479–488.
Genomic studies of uncultivated archaea.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2MXksFKmsLw%3D&md5=53ea03d1d4135ffb4948454ebb117b36CAS | 15931166PubMed |

Shen JP, Zhang LM, Zhu YG, Zhang JB, He JZ (2008) Abundance and composition of ammonia-oxidizing bacteria and ammoniaoxidizing archaea communities of an alkaline sandy loam. Environmental Microbiology 10, 1601–1611.
Abundance and composition of ammonia-oxidizing bacteria and ammoniaoxidizing archaea communities of an alkaline sandy loam.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1cXnvVWkurk%3D&md5=d3bf264633986a2ea5d0776ca35e951aCAS | 18336563PubMed |

Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: molecular evolutionary genetic analysis (MEGA) software version 4.0. Molecular Biology and Evolution 24, 1596–1599.
MEGA4: molecular evolutionary genetic analysis (MEGA) software version 4.0.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2sXpsVGrsL8%3D&md5=8ddb2974f8a09a98f33bb1e6868956c1CAS | 17488738PubMed |

Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research 25, 4876–4882.
The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK1cXntFyntQ%3D%3D&md5=494548ce9ced2753babf5357eb0a5adeCAS | 9396791PubMed |

Treusch AH, Leininger S, Kletzin A, Schuster SC, Klenk HP, Schleper C (2005) Novel genes for nitrite reductase and amo-related proteins indicate a role of uncultivated mesophilic crenarchaeota in nitrogen cycling. Environmental Microbiology 7, 1985–1995.
Novel genes for nitrite reductase and amo-related proteins indicate a role of uncultivated mesophilic crenarchaeota in nitrogen cycling.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD28Xks1Gjuw%3D%3D&md5=bfda68b7f82680670b9f2a1f89c00357CAS | 16309395PubMed |

Wang SY, Wang Y, Feng XJ, Zhai LM, Zhu GB (2011) Quantitative analyses of ammonia-oxidizing archaea and bacteria in the sediments of four nitrogen-rich wetlands in China. Applied Microbiology and Biotechnology 90, 779–787.
Quantitative analyses of ammonia-oxidizing archaea and bacteria in the sediments of four nitrogen-rich wetlands in China.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3MXjsFGhs7k%3D&md5=cafb8fcf3d7a6ae19807ca5a5024b54eCAS |

Webster G, Martin Embley T, Freitag TE, Smith Z, Prosser JI (2005) Links between ammonia oxidizer species composition, functional diversity and nitrification kinetics in grassland soils. Environmental Microbiology 7, 676–684.
Links between ammonia oxidizer species composition, functional diversity and nitrification kinetics in grassland soils.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2MXksFCgsbw%3D&md5=759db3b1ef1db21dbfa855376ea0cacbCAS | 15819850PubMed |

Wei FS, Xu XB, Yan JC (2007) pH determination. In ‘Monitoring and analysis method of water and wastewater’. p. 198. (China Environmental Science Publishing: Beijing)

Wuchter C, Abbas B, Coolen MJL, Herfort L, van Bleijswijk J, Timmers P, Strous M, Teira E, Herndl GJ, Middelburg JJ, Schouten S, Sinninghe Damste JS (2006) Archaeal nitrification in the ocean. Proceedings of the National Academy of Sciences of the United States of America 103, 12 317–12 322.
Archaeal nitrification in the ocean.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD28Xos1Klsbg%3D&md5=1b0fd880cdb46d4d297882e6860af087CAS |

Yuan F, Ran W, Shen QR (2005) Nitrification potential of soils under liquid incubation conditions. Pedosphere 15, 379–385.

Zhang LM, Wang M, Prosser JI, Zheng YM, He JZ (2009) Altitude ammonia-oxidizing bacteria and archaea in soils of Mount Everest. FEMS Microbiology Ecology 70, 208–217.
Altitude ammonia-oxidizing bacteria and archaea in soils of Mount Everest.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXhtlOit7bM&md5=66f37cfb52b825d83a347256400dba1bCAS |