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Preservation and cultivation of -Oxidizing (AOB)

Yoshihito Uchino, Ken-ichiro Suzuki

NBRC, NITE Biological Resource Center, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba, 292-0818 JAPAN Tips for Mainteinance of AOB

1. Preservation of Ammonia-Oxidizing Bacteria (AOB) L-drying and freezing method

2. Purity check of culture of AOB Introduction of one way ~ PCR-DGGE

3. Cultivation of AOB ~ bicarbonate contained in media of AOB ~ Background

Nitrification, the oxidation of ammonia to by chemolitotrophic , is key process in the global cycling of nitrogen.

Nitrifying bacteria

Ammonia-Oxidizing Bacteria (AOB) - Ammonia (NH3) (NO2 )

Nitrite-Oxidizing Bacteria (NOB) - - Nitrite (NO2 ) Nitrate (NO3 ) Many strains have been isolated from ecosystem all over the world. However Background

Only a few strains available in culture collections

Type strains

ATCC NCIMB BCRC ICMP Univ. Hamburg Nitrosococcus nitrosus no culture isolated Nitrosococcus oceani ATCC 19707 NCIMB 11848 BCRC 17464 Nitrosospira briensis no culture available Nitrosospira multiformis ATCC 25196 NCIMB 11849 Nitrosospira tenuis Nv-1 Nm 36 Nm 2 ATCC 25978 NCIMB 11850 ICMP 13139 Nm 57 Nitrosomonas halophila Nm 1 Nm 22 Nitrosomonas nitrosa Nm 90 Nm 45 Nitrosomonas ureae Nm 10 Non-type strains ATCC: 5 strains NBRC: 1 strains We were unable to find other strains

Allmost all the type strain are not in two different collections in two different countries Background

Only a few strains available in culture collections Difficulty of handling and maintenance of pure culture of AOB

Type strains Growth monitoring of AOB ATCC NCIMB BCRC ICMP Univ. Hamburg Nitrosococcus nitrosus no culture isolated NitrosococcusWe oceanihave toATCC measure19707 NCIMB 11848the BCRCamount 17464 of nitrite Nitrosospira briensis no culture available Nitrosospiraproduction multiformis ATCC of25196 AOBNCIMB by 11849 colorimetric method. Nitrosospira tenuis Nv-1 Nitrosomonas aestuarii Nm 36 ContaminationNitrosomonas communis detection of culture Nm 2 Nitrosomonas europaea ATCC 25978 NCIMB 11850 ICMP 13139 NitrosomonasSome eutropha strains can form colony, but the Nmcolony 57 Nitrosomonas halophila Nm 1 Nitrosomonasformations marina require several months. Nm 22 Nitrosomonas nitrosa Nm 90 NitrosomonasColony oligotropha size of AOB is very small. Nm 45 Nitrosomonas ureae Nm 10 As forNon -manytype researchersstrains RecognitionATCC: that 5 strains freezing NBRC:and vacuum 1 strains-drying are not suitable for AOB We were unable to find other strains Keeping isolates by serial transfer Background 49 AOB strains possessed in NBRC

strain source strain source Nitrosovibrio sp. RY6A-2 rhizosphere soil Nitrosomonas sp. IWT310 biological deodorizing equipment Nitrosovibrio sp. TYM9 soil Nitrosomonas sp. BAED410 biological deodorizing equipment Nitrosomonas sp. B2 wastewater Nitrosomonas sp. NIWC6 biological deodorizing equipment Nitrosomonas sp. TK794 soil Nitrosomonas sp. NIWC9 biological deodorizing equipment Nitrosomonas sp. K1 biological deodorizing equipment Nitrosomonas sp. NIWC10 biological deodorizing equipment Nitrosomonas sp. TNO632 marine Nitrosomonas sp. HTA5 rhizoplane Nitrosovibrio sp. RY3C-1 rhizosphere soil Nitrosomonas sp. HTA7 rhizoplane Nitrosospira sp. NRS527 rhizoplane Nitrosolobus sp. WHA23 rhizoplane Nitrosomonas sp. IWT514 biological deodorizing equipment Nitrosomonas sp. HTA10 rhizoplane Nitrosolobus sp. TCH716 soil Nitrosomonas sp. HTA25 rhizoplane Nitrosolobus sp. TCH719 soil Nitrosospira sp. GS832 wastewater Nitrosomonas sp. EGT404 soil Nitrosospira sp. NRS4231 rhizoplane Nitrosomonas sp. CNS326 poultry manure Nitrosospira sp. NRS4262 rhizoplane Nitrosomonas sp. DYS317 biological deodorizing equipment Nitrosolobus sp. TCH711 soil Nitrosospira sp. GS833 wastewater Nitrosolobus sp. KAN8 soil Nitrosolobus sp. PJA1 rhizoplane Nitrosolobus sp. KAN18 soil Nitrosomonas sp. IWT202 biological deodorizing equipment Nitrosovibrio sp. FJI82 Nitrosomonas sp. BAEP210 biological deodorizing equipment Nitrosomonas sp. CNS336 Nitrosomonas sp. CNS332 poultry manure Nitrosomonas sp. NIW311 Nitrosomonas sp. BAED430 biological deodorizing equipment Nitrosomonas sp. OZK11 wastewater Nitrosomonas sp. TNO615 marine Nitrosolobus sp. HBN8222A alkaline soil Nitrosomonas sp. DYS323 biological deodorizing equipment Nitrosolobus sp. HBN8226A Nitrosomonas sp. HTA6 rhizoplane Nitrosolobus sp. CHIC5 Nitrosomonas sp. IWT203 biological deodorizing equipment Nitrosospira sp. FJI425 Nitrosomonas sp. IWT305 biological deodorizing equipment

Our purpose in this study is to make these strains available to the public in our NBRC culture collection. 1. Preservation of AOB

Known methods to maintain AOB 1. Serial transfer 2. Storage under liquid paraffin Nature (1957) 179: p789, Nature (1957) 179: p1200 3. Cold storage 4. Cryopreservation Bull Jpn Soc Microb Ecol (1994) 9: p119-123 5. freeze-drying Soil Sci Plant Nutr (2004) 50: p777-781 6. L-drying NBRC Method

Freezing, freeze-drying & L-drying are regular as long term storage. L-drying We tried some protective media to the strains unable to be preserved by our NBRC method. Freezing We confirmed whether the ordinary method (using glycerol and DMSO as cryoprotectant in deep freezer at -80 degree) was applicable to AOB. 1-1. L-drying preservation

NBRC method 12 kinds of protective media (SM1 – SM12) The media are based on combination are prepared for various microorganisms. of glutamete and phospate buffer. Adonitol and cystein are the mutation SM1 : Standard protective agent. SM8 : For chemolithotroph SM8 is protective medium for SM5 : For Aquaspirillum etc. chemolithotroph.

SM1 SM8 SM5 The growth of AOB is inhibited at high concentration of cystein and phospate. Sodium glutamate 3 g 0.5 g 3 g So, in SM8, the amount of these Adonitol 1.5 g 1.5 g 1.5 g compounds is limited.

L-cystein monohydrate 0.05 g 0.01 g － Therefore, stability of preservation using SM8 is inferior to that of SM1. ED·2HCl － － 0.4 g

Sorbitol － － 1 g

Potassium phospate (100 ml) 0.1 M 0.02 M 0.1 M 1-1. L-drying preservation

Some strains wereStrain unable CNS332 to be preserved using SM8 Growth yieldStrain after NIWC6 rehydration B2 ＋＋ and cultivation for 2 weeks EGT404 Phosphate＋＋ buffer DYS317 ＋＋ CNS332 CNS332 － Thio- We tried01 someSM1 : Standard DYS323 － ED2HCl DYS323 HEPES IWT203 ＋＋ protective NG IWT310 ＋＋ 02 SM8 : For chemolithotroph NIWC6 － NIWC6 media SM1NIWC9SM8 SM5＋ SM1 SM8 SM1 base base base HTA10 ＋ 03 SM5 : For Aquaspirillum HTA25 ＋ ED2HCl in place of cysteine 04 removal of 05 SM5 modified protective media Strain DYS317 06 Ingredients conc. are different

07 SM1 Thiourea in place cultivation08 SM8 of AdonitolOK HEPES in place 09 SM1 centrifugation of phosphate Rehydration 1-1. L-drying preservation

Strain CNS332 Growth yieldStrain after NIWC6 rehydration and cultivation for 2 weeks Phosphate buffer

Thio- 01 SM1 : Standard ED2HCl urea HEPES 02 SM8 : For chemolithotroph SM1 SM8 SM5 SM1 SM8 SM1 base base base 03 SM5 : For Aquaspirillum ED2HCl in place of cysteine 04 05 StrainSM5 modified DYS317 06 Ingredients conc. are different

07 SM1 Thiourea in place 08 SM8 of Adonitol

HEPES in place 09 SM1 of phosphate 1-1. L-drying preservation

Strain CNS332 Strain NIWC6 Phosphate buffer

Thio- ED2HCl urea HEPES

SM1 SM8 SM5 SM1 SM8 SM1 base base base

Strain DYS317 1-1. L-drying preservation

Strain CNS332 Strain NIWC6 As forPhosphate L-drying buffer preservaion of AOB, Thio- ED2HCl urea HEPES

SM1 SM8 SM5 SM1 SM8 SM1 It is effectivebase forbase baserecovery to remove the protective media.

AOB can be stably preserved using StrainSM8. DYS317

Ethylenediamine dihydrochloride and HEPES are effective. 1-2. Cryopreservation

removal of cryoprotectant

cultivation Cryotube Cryotube Cryotube

Freezed and stored in deep Cryotube freezer (-80C) We can preserve all the strains 5% DMSO (tube) 10% DMSO (tube) we possessed, using this method. 25% DMSO (tube) 5% Glycerol (tube) 10% Glycerol (tube) 25% Glycerol (tube)

Stored in LN vapor phase in the tank Cryotube Cryotube 10% DMSO (straw) 1-2. Cryopreservation

Rapid freezing using straw

Cryotube Cryotube Cryotube

Freezed and stored in deep 0.25 ml Straw 133 mm * 1.6 mm (ID) freezer (-80C) Ionomer resin (cryo bio system) 5% DMSO (tube) 10% DMSO (tube) Rapidly-frozen by plunging Stored in LN25% gas DMSO phase (tube) in into liquid nitrogen the tank 5% Glycerol (tube) 10% Glycerol (tube) 25% Glycerol (tube) The result indicates the importance of dehydration taking place during slow freezing. Stored in LN vapor phase in the tank Cryotube Cryotube 10% DMSO (straw) 1-2. Cryopreservation

Cryotube Cryotube Cryotube

Freezed and stored in deep freezer (-80C) 5% DMSO (tube) 10% DMSO (tube) 25% DMSO (tube) 5% Glycerol (tube) 10% Glycerol (tube) 25% Glycerol (tube)

Stored in LN vapor phase in the tank Cryotube Cryotube 10% DMSO (straw) 1-2. Cryopreservation

As for cryopreservaion of AOB

The method using 5-10% DMSO as cryoprotectant and freezing in -80C is effective.

Cryotube Cryotube Cryotube A The recovery of AOB was improved by Freezedremoval and of storedDMSO in deep before cultivation. freezer (-80C) 5% DMSO (tube) 10% DMSO (tube) 25% DMSO (tube) 5% Glycerol (tube) 10% Glycerol (tube) 25% Glycerol (tube)

Stored in LN vapor phase in the tank Cryotube Cryotube 10% DMSO (straw) 2. Purity check of culture of AOB

Combination of 1 microscopy 2 plate cultivation with some different media 3 sequencing

4 DGGE

Actually, by DGGE we could detect contamination that we can’t detect by other methods.

DGGE condition: Annealing Amplicon DGGE Primer Sequence Target Annealing temp positions length (bp) condition 518R ATTACCGCGGCTGCTGG 518–534 V3 65→55℃, -0.5℃/cycle 194 10%, 30-70%, 1120 V・h - 357F-GC CCT ACG GGA GGC AGC AG 341–357 3. Cultivation of AOB

For growth of AOB, it is important to keep the pH constant.

Bicarbonate We know by experience that Buffering agent Carbonate the growth of AOB is improved by addition of bicarbonate. HEPES RubisCO formation Growth of AOB IAc IAq IC HCO3- non HCO3- non HTA10 HTA6 NIW IWT305 HTA25C6 HTA7 IWT310 IWT202 16S rDNA HTA5 BAED410 TK794 BAED430 BAEP210 NIWC9 CNS332 CNS326 IWT203 K1 NIW311 CNS336 HPC101 Nm103 Koll-21 ENI-11 NBRC_14298 WH-2 Nitrosomonas europaea ATCC 25978 B2 NRS4262 NRS4231 OZK11 DYS317 DYS323 Nitrosomonas halophila Nm1 Nm93 TNO615 TNO632 Nitrosomonas communis Nm2 Nitrosomonas nitrosa Nm90 FJI425 FJI82 RY3C-1 RY6A-2 Nitrosospira multiformis NL13 KAN18 KAN8 PJA1 HBN8226A WHA23 HBN8222A CHIC5 EGT404 TCH716 TCH711 TCH719 Nitrosospira tenuis Nv1 TYM9 GS833 GS832 NRS527 Nv12 Np39-19 Nsp58 B6 O13 By additionNsp5 of bicarbonate, the growths GM4 Nitrosomonas aestuarii Nm36 of almostNitrosomonas all marinastrains Nm22 were improved. Nitrosomonas ureae Nm10 Nm84 It was no a Nitrosomonasproblem oligotropha Nm45of pH. RubisCO formation Growth of AOB IAc IAq IC HCO3- non HCO3- non HTA10 HTA6 NIW IWT305 HTA25C6 HTA7 IWT310 IWT202 16S rDNA HTA5 BAED410 TK794 BAED430 BAEP210 NIWC9 CNS332 CNS326 IWT203 K1 NIW311 CNS336 HPC101 Nm103 Koll-21 ENI-11 NBRC_14298 WH-2 Nitrosomonas europaea ATCC 25978 B2 NRS4262 NRS4231 OZK11 DYS317 DYS323 Nitrosomonas halophila Nm1 Nm93 TNO615 TNO632 Nitrosomonas communis Nm2 Nitrosomonas nitrosa Nm90 FJI425 FJI82 RY3C-1 RY6A-2 Nitrosospira multiformis NL13 KAN18 KAN8 PJA1 HBN8226A WHA23 HBN8222A CHIC5 EGT404 TCH716 TCH711 TCH719 Nitrosospira tenuis Nv1 TYM9 GS833 GS832 NRS527 Nv12 Np39-19 Nsp58 B6 O13 Nsp5 GM4 For goodNitrosomonas aestuariigrowth Nm36 of AOB, we should Nitrosomonas marina Nm22 Nitrosomonas ureae Nm10 Nm84 add the bicarbonateNitrosomonas oligotropha Nm45 in medium. RubisCO formation Growth of AOB IAc IAq IC HCO3- non HCO3- non HTA10 HTA6 NIW IWT305 HTA25C6 HTA7 The strainsIWT310 in this group can grow well at IWT202 16S rDNA HTA5 low bicarbonate.BAED410 TK794 BAED430 These strainsBAEP210 must have some positive NIWC9 CNS332 CNS326 uptake mechanismIWT203 for CO2. K1 NIW311 CNS336 HPC101 Nm103 Koll-21 ENI-11 NBRC_14298 WH-2 Nitrosomonas europaea ATCC 25978 B2 NRS4262 NRS4231 OZK11 DYS317 DYS323 Nitrosomonas halophila Nm1 Nm93 TNO615 TNO632 Nitrosomonas communis Nm2 Nitrosomonas nitrosa Nm90 FJI425 FJI82 RY3C-1 RY6A-2 Nitrosospira multiformis NL13 KAN18 KAN8 PJA1 HBN8226A WHA23 HBN8222A CHIC5 EGT404 TCH716 TCH711 TCH719 Nitrosospira tenuis Nv1 TYM9 GS833 GS832 NRS527 Nv12 Np39-19 Nsp58 B6 O13 Nsp5 GM4 Nitrosomonas aestuarii Nm36 Nitrosomonas marina Nm22 Nitrosomonas ureae Nm10 Nm84 Nitrosomonas oligotropha Nm45 RubisCO formation Growth of AOB existent IAc CarboxysomesIAq IC are bacterial As for the uptake mechanism, weHCO3 nonchecked- -nonexistent HCO3- non HTA10 microcompartments that contain HTA6 NIW theIWT305 formation of RubiSO of AOB by PCR. HTA25C6 RubisCO . not yet determined HTA7 IWT310 IWT202 16S rDNA HTA5 BAED410 TK794 These compartments are thought to RubisCOBAED430 is a key enzyme in CO fixation in Calvin Benson cycle. BAEP210 2 NIWC9 CNS332 concentrate CO2 to overcome the CNS326 IWT203 K1 inefficiency of RubisCo. The NIW311enzyme exists in several forms either singly or in multiple CNS336 HPC101 Nm103 combinationKoll-21 in a bacteria ENI-11 NBRC_14298 WH-2 Nitrosomonas europaea ATCC 25978 B2 carboxysomal RubisCO NRS4262 NRS4231 GreenOZK11 -like RubisCO DYS317 DYS323 Nitrosomonas halophila Nm1 Nm93 Detectable TNO615 TNO632 IAc cbbL cbbS csoS2 csoS3orfA C A B Nitrosomonas communis Nm2 csoS1 By PCR Detectable Nitrosomonas nitrosa Nm90 B FJI425 FJI82 By PCR RY3C-1 RY6A-2 Nitrosospira multiformis NL13 KAN18 IAq non-primers KAN8 cbbL cbbScbbQ cbbO PJA1 HBN8226A WHA23 HBN8222A CHIC5 RedEGT404-like RubisCO TCH716 TCH711 TCH719 Detectable Nitrosospira tenuis Nv1 TYM9 GS833 IC cbbF cbbP cbbA cbbL cbbScbbX cbbY cbbZ GS832 By PCR NRS527 Nv12 Np39-19 Nsp58 B6 O13 Nsp5 GM4 Nitrosomonas aestuarii Nm36 Nitrosomonas marina Nm22 Nitrosomonas ureae Nm10 Nm84 Nitrosomonas oligotropha Nm45 RubisCO formation Growth of AOB IAc IAq IC HCO3- non HCO3- non HTA10 These strainsHTA6 in this group can grow well NIW IWT305 HTA25C6 at low bicarbonateHTA7 condition by function of IWT310 IWT202 16SCO2 rDNA concentratingHTA5 mechanism in carboxysome. BAED410 TK794 BAED430 BAEP210 NIWC9 CNS332 CNS326 IWT203 K1 NIW311 CNS336 HPC101 Nm103 Koll-21 ENI-11 NBRC_14298 WH-2 Nitrosomonas europaea ATCC 25978 B2 NRS4262 NRS4231 OZK11 DYS317 DYS323 Nitrosomonas halophila Nm1 Nm93 TNO615 TNO632 Nitrosomonas communis Nm2 Nitrosomonas nitrosa Nm90 FJI425 FJI82 RY3C-1 RY6A-2 Nitrosospira multiformis NL13 KAN18 KAN8 PJA1 HBN8226A WHA23 HBN8222A CHIC5 EGT404 TCH716 TCH711 TCH719 Nitrosospira tenuis Nv1 TYM9 GS833 GS832 NRS527 Nv12 Np39-19 Nsp58 B6 O13 Nsp5 GM4 Each Nitrosomonasgroup aestuarii is Nm36 characterized by Nitrosomonas marina Nm22 Nitrosomonas ureae Nm10 Nm84 their formationNitrosomonas oligotropha of Nm45 RubisCO. Conclution Preservation:

We could preserve all the AOB strains we possessed by freezing using DMSO and L-drying using SM8 medium.

In both methods, the recovery of AOB was improved by removal of protective media before cultivation.

Purity check:

We should check the purity of culture by combination of some methods.

DGGE is effective as one of purity check methods.

Cultivation:

By addition of bicarbonate, the growths of almost all strains were improved, however the effect of bicarbonate is different among the phylogenetic groups of AOB.

We suspect that the difference among group may be related to their RubisCO formations.