The distribution of ammonia-oxidizing betaproteobacteria in impounded Black mangroves (Avicennia germinans)
Riks Laanbroek Roos Keijzer Mark Rains Jos Verhoeven Dennis Whigham Biogeochemical oxidation of ammonia: Key process in the nitrogen cycle
ammonium
oxygen-dependent
oxygen-sensitive
nitrous nitric oxide oxide Geochemical process of ammonia oxidation
Oxic conditions • Ammonia oxidation by proteobacteria and thaumarchaea both producing nitrite
Anoxic conditions • Ammonia oxidation with the concomitant reduction of nitrite to dinitrogen gas by planctomycetes (Anammox bacteria) Geochemical process of ammonia oxidation
Oxic conditions • Ammonia oxidation by proteobacteria and thaumarchaea both producing nitrite
Anoxic conditions • Ammonia oxidation with the concomitant reduction of nitrite to dinitrogen gas by planctomycetes (Anammox bacteria) Ammonia-oxidizing betaproteobacteria (β-AOB)
Simple metabolism Steering factors
• Oxidation of ammonia • Ammonia in the presence of concentration oxygen • Oxygen availability • Carbon dioxide fulfills • Carbon dioxide the carbon accessibility requirements • pH • Iron and other metals • Salinity • Temperature
Occurrence of β-AOB clusters in different ecosystems (based on the 16S rRNA gene)
freshwater ecosystems marine ecosystems
0 1 2 3 4 5 0 1 2 3 4 5 6a 6b 7 8 9 6a 6b 7 8 9 The distribution of β-AOB in impounded Black mangroves Location of Impoundments #23 and #24 on North Hutchinson Island, St. Lucie, Fl.
24 Indian River Lagoon
23
North Hutchinson Island Management history of mangroves on North Hutchinson Island, St Lucie, Fl.
Event Impoundment 23 Impoundment 24 Closing of the dike 1966 1970 Dike breach 1974 Placement of one 1985 culvert Placements of four 1987 additional culverts Installing rotational early spring 2009 impoundment management
Partial restoration of the tide movements and subsequently mangrove vegetation Modeled number of inundation days in Impoundments #23 and #24
24 Vegetations: number of inundation days per year Dwarf 350 Sparse 300 Dense 250 200
150
100
number of inundation days per year 50
350 0 300 2007 2008 2009 2010
250
200
150
100
50
0 2007 2008 2009 2010 23 Three different Black Mangrove habitats on North Hutchinson Island, St. Lucie, Fl. dwarf
sparse
dense Steering factors for mangrove growth
• N and P concentrations • Frequency of flooding • pH • Salinity • Predation
Steering factors for mangrove growth and for β-AOB
• N and P • Ammonia concentrations concentration • Frequency of • Oxygen availability flooding • Carbon dioxide • pH accessibility • Salinity • pH • Predation • Salinity • Iron and other metals
Research questions
I. Is there a similarity between the distribution of lineages of β-AOB and the distribution of Black mangrove vegetation types imposed by soil conditions?
II. Is the distribution of lineages of β-AOB affected by the new flooding regime starting spring 2009?
Sampling times and locations of Black mangrove habitats
March 2009 24 March 2010
23
dwarf sparse dense Soil characteristics of sampling locations: Moisture and degradable organic nitrogen
Vegetations: degradable organic nitrogen (mg N / g dry wt) Dwarf 90 80 Sparse 70 Dense 60 50 40 30 20 10 moisture content (%) 0 70 2009 2009 2010 2010 60 23 24 23 24 50 40 30 20 10 0 2009 2009 2010 2010 23 24 23 24 Soil characteristics of sampling locations: Pore water salinity and ammonium
Vegetations: pore water ammonium (mg N / L) Dwarf 3.5 Sparse 3 Dense 2.5 2 1.5 1 0.5 pore water salinity 0 100 2009 2009 2010 2010 23 24 23 24 80
60
40
20
0 2009 2009 2010 2010 23 24 23 24 Potential ammonia-oxidizing activities in the different mangrove habitats
Vegetations: potential ammonia oxidation rate Dwarf (nMol / h x g dry wt) Sparse 18 Dense 16 14 12 10 8 6 4 2 0 2009 2009 2010 2010 23 24 23 24 OTUs of β-AOB based on 97% 16S rRNA similarity (440-460 bp)
OTU Numbers BLAST analysis of OTU’s representatives # Closest type strain β-AOB % Origin closest relative lineage similarity 01 206 Nitrosomonas aestuarii 97 High altitude saline wetland
02 120 Nitrosospira tenuis 96 Deep-water sponge
03 115 Nitrosomonas Nm143 97 Estuarine sediment
04 58 Nitrosomonas aestuarii 98 Prawn farm sediment
05 14 Nitrosomonas europaea 100 Nitrifying bioreactor
06 11 Nitrosomonas aestuarii 97 Estuarine sediment
07 10 Nitrosomonas aestuarii 98 Estuarine sediment
08 6 Nitrosomonas aestuarii 96 Coastal marine sediment Dissimilarities between communities of β-AOB at 97% mutual difference
Groups Comparison R-value P-value Impoundments 23 versus 24 0.058 0.046 * Sampling years 2009 versus 2010 0.063 0.036 * Mangrove habitat type All habitats 0.215 0.001 *** Dwarf versus sparse 0.326 0.001 *** Dwarf versus dense 0.248 0.002 ** Sparse versus dense 0.076 0.069
Of the 24 environmental soil parameters measured, the combination of moisture content, C/N ratio and the amount of magnesium in the pore water explained 15% of the observed variation in community composition. Frequency distribution of OTUs of β-AOB between impoundments at 97% similarity
100% 90% 80% OTU08 70% OTU07 60% OTU06 50% OTU05 40% OTU04 30% OTU03 Distribution in the dwarf 20% OTU02 vegetation samples 10% OTU01 0% 100% dwarf sparse dense 90% 80% OTU08 Overall distribution 70% OTU07 60% OTU06 50% OTU05 40% OTU04 30% OTU03 20% OTU02 10% OTU01 0% 23 dwarf 24 dwarf Niche differentiation among β-AOB?
• OTU01 (Nitrosomonas aestuarii) seems to be negatively affected by the more moist conditions in the dense vegetation and also in 2010 after flooding. Oxygen limitation? • OTU02 (Nitrosospira cluster 1) was most prominent in the sparse vegetation. Adaptation to conditions of starvation? • OTU03 (Nitrosomonas sp. Nm143) seems to be positively affected by more moist and rich conditions. Adapted to lower oxygen concentrations? Research questions
I. Is there an agreement between the distribution of lineages of β-AOB and the distribution of mangrove vegetation types as constraint by soil conditions?
II. Is the distribution of lineages of β-AOB affected by flooding?
III.Is the distribution of lineages of β-AOB affected by the supply of extra ammonium and/or oxygen? Effect of ammonium addition on the frequency distribution of OTUs of β-AOB at 97% similarity
100% 90% 80% OTU08 Comparison R- P- 70% OTU07 OTU06 value value 60% 50% OTU05 Control vs 0.084 0.050 40% OTU04 treatment * 30% OTU03 20% OTU02 10% OTU01 0% control treatment
OTU Average abundance Average Contribution Cumulative # dissimilarity (%) contribution control treatment (%) 01 0.43 0.70 22.90 39 39 02 0.25 0.09 14.00 24 63 04 0.10 0.11 8.62 14 77 03 0.13 0.08 8.02 14 91 Effect of ammonium addition on the frequency distribution of OTUs of β-AOB at 97% similarity
100% 90% 80% OTU08 70% OTU07 60% OTU06 50% OTU05 40% OTU04 30% OTU03 20% OTU02 Sparse vegetation samples 10% OTU01 0% 100% dwarf control dwarf treatment 90% 80% OTU08 Dwarf vegetation samples 70% OTU07 60% OTU06 50% OTU05 40% OTU04 30% OTU03 20% OTU02 10% OTU01 0% sparse control sparse treatment Effect of ammonium plus oxygen addition on the frequency distribution of OTUs of β-AOB
100% 90% 80% OTU08 Comparison R- P- 70% OTU07 OTU06 value value 60% 50% OTU05 Control vs 0.149 0.014 40% OTU04 treatment * 30% OTU03 20% OTU02 10% OTU01 0% control treatment
OTU Average abundance Average Contribution Cumulative # dissimilarity (%) contribution control treatment (%) 01 0.29 0.52 22.27 35 35 03 0.32 0.23 15.07 24 59 02 0.22 0.00 11.03 17 76 04 0.12 0.19 10.47 16 92 Effect of ammonium plus oxygen addition on the frequency distribution of OTUs of β-AOB
100% 90% 80% OTU08 70% OTU07 60% OTU06 50% OTU05 40% OTU04 30% OTU03 20% OTU02 Dense vegetation samples 10% OTU01 0% 100% sparse control sparse treatment 90% 80% OTU08 Sparse vegetation samples 70% OTU07 60% OTU06 50% OTU05 40% OTU04 30% OTU03 20% OTU02 10% OTU01 0% dense control dense treatment Conclusions
• Only OTUs related to the Nitrosomonas aestuarii lineage took advantage of the better growth conditions after supplying ammonium. Aeration stimulated these OTUs even more. • OTUs related to the Nitrosomonas Nm143 lineage and Nitrosospira cluster 1 were not stimulated by the better growth conditions. • Nitrosospira cluster 1 almost disappeared as common species in the presence of ammonium and oxygen.
Overall conclusions
1. The distribution of species of β-AOB was not entirely the same for both impoundments. This was largely due to the predominance of OTU01 (N. aestuarii) in the dwarf habitat of Impound- ment #24 (former salt pans) 2. The distribution of species of β-AOB is hardly affected by flooding; role of the dry winter season and subsequent high pore water salinities in 2009? 3. The distribution of species of β-AOB is affected by Black mangrove habitat conditions; role of moisture and nutrient conditions?
Acknowledgments
Smithsonian Marine Science Network
Royal Netherlands Academy of Arts and Sciences