Yu-Chen Ling and John W. Moreau

Home , Acidimicrobiia, Acidobacteria, Actinobacteria, Alphaproteobacteria, Betaproteobacteria, Chloroflexi (class)

Microbial Distribution and Activity in a Coastal Acid Sulfate Soil System Introduction: Bioremediation in Yu-Chen Ling and John W. Moreau coastal acid sulfate soil systems Method A Coastal acid sulfate soil (CASS) systems were School of Earth Sciences, University of Melbourne, Melbourne, VIC 3010, Australia formed when people drained the coastal area Microbial distribution controlled by environmental parameters Microbial activity showed two patterns exposing the soil to the air. Drainage makes iron Microbial structures can be grouped into three zones based on the highest similarity between samples (Fig. 4). Abundant populations, such as Deltaproteobacteria, kept constant activity across tidal cycling, whereas rare sulfides oxidize and release acidity to the These three zones were consistent with their geological background (Fig. 5). Zone 1: Organic horizon, had the populations changed activity response to environmental variations. Activity = cDNA/DNA environment, low pH pore water further dissolved lowest pH value. Zone 2: surface tidal zone, was influenced the most by tidal activity. Zone 3: Sulfuric zone, Abundant populations: the heavy metals. The acidity and toxic metals then Method A Deltaproteobacteria Deltaproteobacteria this area got neutralized the most. contaminate coastal and nearby ecosystems and Method B

1.5 cause environmental problems, such as fish kills, 1.5 decreased rice yields, release of greenhouse gases, Chloroﬂexi and construction damage. In Australia, there is Gammaproteobacteria Gammaproteobacteria about a $10 billion “legacy” from acid sulfate soils, Chloroﬂexi even though Australia is only occupied by around 1.0 1.0 Cyanobacteria,@ Acidobacteria Acidobacteria Alphaproteobacteria 18% of the global acid sulfate soils. Chloroplast Zetaproteobacteria Rare populations: Alphaproteobacteria

Method A log(RNA(%)+1) Zetaproteobacteria log(RNA(%)+1) Method C Method B

0.5 0.5 Cyanobacteria,@ Bacteroidetes Chloroplast

Firmicutes Firmicutes Bacteroidetes Planctomycetes Planctomycetes Ac8nobacteria

Fig. 4 (Ling et al., 2015) Fig. 5 (Ling et al., 2015) Ac8nobacteria 0.0 0.0 Verrucomicrobia Te Tl 0.4 0.8 1.2 Verrucomicrobia0.4 0.8 1.2 These three zones showed similar microbial members but in different abundances, which were controlled by log(DNA(%)+1) Deltaproteobacteria Th Tf log(DNA(%)+1) Deltaproteobacteria environmental parameters. Method D Deltaproteobacteria Fig. 1 Zone 1 Zone 2 Zone 3 Thermoprotei

Thermoprotei Method BAlphaproteobacteria Halobacteria Thermoprotei Methanomicrobia Archaeoglobi 1.5 1.5 Ignavibacteria Thermoplasmata Spirochaetes Deferribacteres Gammaproteobacteria Tidal inundation provides a potential economic Deltaproteobacteria Method C Planctomycetacia Halobacteria Deltaproteobacteria Ignavibacteria Spirochaetes Zetaproteobacteria 1.5 Gammaproteobacteria treatment for the CASS issue. Microbial reduction Phycisphaerae Deltaproteobacteria Chloroﬂexi Chloroﬂexi Acidobacteria Actinobacteria Crenarchaeota Verrucomicrobia Planctomycetacia Crenarchaeota Gammaproteobacteria Euryarchaeota Chlorobi Methanomicrobia Acidobacteria Spirochaetes contributes to more than 50% of the alkalinity by Actinobacteria Planctomycetes Mollicutes Chlorobi 1.0 1.0 Actinobacteria Proteobacteria Ac2nobacteria generating iron(II) and sulfide, which will combine Planctomycetes 1.0 Acidobacteria Acidobacteria_Gp3 Euryarchaeota Alphaproteobacteria Actinobacteria Archaea Thermotogae Acidobacteria_Gp1 Tenericutes Acidobacteria_Gp1 Betaproteobacteria Acidobacteria Deferribacteres Archaea Zetaproteobacteria and deposit iron sulfide minerals back to the soils. Gammaproteobacteria Epsilonproteobacteria Firmicutes Zetaproteobacteria Alphaproteobacteria Acidobacteria Clostridia log(RNA(%)+1) Firmicutes log(RNA(%)+1)

Fusobacteria Fusobacteria log(cDNA(%)+1) Acidobacteria_Gp2 Bacteria However, there is limited knowledge about Bacteria Proteobacteria Bacilli Bacteria Proteobacteria 25 Chloroﬂexi Gammaproteobacteria Ignavibacteria MethodChloroflexi E 0.5 Bacteroidetes 20 Planctomycetes Anaerolineae Bacteroidetes Sphingobacteria Firmicutes Flavobacteria Spirochaetes Gammaproteobacteria Actinobacteria Bacteroidetes Alphaproteobacteria microbial dynamics in long-term efficiency in the Planctomycetacia 20 Clostridia Bacteroidia Bacteroidetes 0.5 Tenericutes 0.5 Bacilli Chloroflexi 15 Actinobacteria Dehalococcoidetes Actinobacteria Bacteroidetes Bacteroidetes_incertae_sedis Planctomycetes Cyanobacteria Actinobacteria_Acidimicrobiia

CASS system. 15 Cyanobacteria,@ Acidobacteria Cyanobacteria_Chloroplast Cyanobacteria,@ Firmicutes_Clostridia Nitrospirae Flavobacteria Firmicutes Chloroplast Ktedonobacteria Bacteroidia Chloroflexi Sphingobacteria Method C 0.0 Chloroplast Acidobacteria 10 Mollicutes 0.4 0.8 1.2 Euryarchaeota Chloroflexi

Motivation: Providing detailed information 10 Firmicutes Betaproteobacteria Alphaproteobacteria Sphingobacteria Deltaproteobacteria Crenarchaeota Betaproteobacteria Verrucomicrobia log(DNA(%)+1) Method D Ac8nobacteria Bacteroidetes_incertae_sedis Deltaproteobacteria 5

about microbial distribution and activity patterns Alphaproteobacteria 5 Epsilonproteobacteria Bacteroidia Flavobacteria Planctomycetes Dehalococcoidetes Relative Abundance (%) Relative Abundance (%) Planctomycetes Firmicutes Spirochaetes Firmicutes_Clostridia Sphingobacteria Anaerolineae Alphaproteobacteria Ac8nobacteria 0.0 Acidobacteria

for people who want to build up a complete 0 0 Negativicutes 0.0 Dehalococcoidetes Fig. 6 Anaerolineae Clostridia Others Verrucomicrobia Verrucomicrobia Bacteroidetes Zetaproteobacteria Epsilonproteobacteria Bacilli biogeochemical model, which can be used to Epsilonproteobacteria Zone 2 Zone 3 Abundant Zone(Ling 2 etRareZone al., 32015) 0.4 0.8 Gammaproteobacteria1.2 0.4 0.8 1.2 log(DNA(%)+1) Taxa log(DNA(%)+1) Deltaproteobacteria Site C Zone 1, pH < 5.5 Site B Alphaproteobacteria Zone 1, pH < 5.5 Deltaproteobacteria Gammaproteobacteria Fig. 9 Site A predict and evaluate bioremediation long-term For example, Zone 1 contained microorganisms that favor aZone lower 1, pH = 5.5-6.5 pH condition, Zone Zone 2 1, contained pH = 5.5-6.5 the most Early Zetaproteobacteria Deltaproteobacteria efficiency or potential land management. sulfate-reducing bacteria. Microbial compositions Fig. 10 Microbial activity (cDNA:DNA) across a tidal_Syntrophobacterales cycle Delayed Color Key

Color Key AcidobacteriaActinobacteria Cyanobacteria Actinobacteria_Acidimicrobiia Firmicutes_Clostridia Nitrospirae

7 Early responders Acidobacteria Microorganisms were divided into Euryarchaeota Chloroflexi 25 20 Delayed responders Crenarchaeota Ac2nobacteria -1.5 -0.5 0.5 1.5

6 -1.5 -0.5 0.5 1.5

Study site and methods 20 Value Value Deltaproteobacteria early and delayed responder based Epsilonproteobacteria WS3 Otu0145 Otu0099Otu0177 Otu0430Otu0414 Otu0145 20 Otu0839Otu0301 Otu0099Otu0177 Otu0442Otu0319 Otu0414 Otu0431Otu0502 Otu0301Otu0430 5 Otu0454 Otu0839 Otu0156Otu0781 Otu0319 Firmicutes_Clostridia Otu0232Otu0531 Otu0502Otu0442 Otu0679Otu0226 Otu0454Otu0431 Spirochaetes Otu0001Otu0782 DeltaproteobacteriaOtu0781 Otu0390 Otu0156 15 Otu0917Otu0780 Otu0531 Otu0307Otu0311 Otu0226Otu0232 Study site: East Trinity wetland, Cairns Otu0332Otu0050 Otu0782Otu0679 Otu0629Otu0089 Otu0001 Otu0233Otu0046 Otu0780Otu0390 Otu0331Otu0224 Otu0311Otu0917 Otu0668Otu0140 Otu0307 Firmicutes Otu0050 15 Method D Otu0383Otu0351 Otu0332 Otu0330Otu0123 Otu0089 Otu0042Otu0334 Otu0046Otu0629 Acidobacteria Otu0192Otu0039 Otu0224Otu0233 Verrucomicrobia Otu0035Otu0157 Otu0140Otu0331 on their activity increasing period Otu0284 Otu0085Otu0547 Otu0351Otu0668 Otu0010Otu0299 Otu0383 Acidobacteria Otu0333Otu0269 Otu0330Otu0123 4 Otu0002Otu0187 Otu0334 Otu0208Otu0628 Otu0039Otu0042 Otu0445Otu0294 Otu0157Otu0192

15 Otu0116 Otu0139Otu0094 Otu0284Otu0035 Otu0211Otu0278 Otu0085Otu0547 Otu0096Otu0129 Otu0299 Otu0095Otu0407 Otu0269Otu0010 Otu0281Otu0276 Otu0187Otu0333 Otu0076Otu0259 Otu0002 Otu0470Otu0154 Otu0208Otu0628 Otu0513Otu0439 Otu0294 Otu0619Otu0072 Otu0116Otu0445 1970s-2001: Drainage; Otu0365Otu0678 Otu0094 Otu0066 Otu0139 Otu0214Otu0023 Otu0211Otu0278 Otu0216Otu0041 Otu0096Otu0129 Otu0079Otu0153 Otu0407 Otu0016Otu0125 Otu0276Otu0095 10 Otu0053 AlphaproteobacteriaOtu0281 Planctomycetes Otu0158Otu0469 Otu0259 Otu0009Otu0197 Otu0154Otu0076

3 Otu0132 Otu0470 Otu0623Otu0228 Otu0439 10 Otu0572 Otu0513 (Fig. 11). Then compared the Otu0389 Otu0072 Otu0379Otu0113 56Otu0678Otu0619% Taxa Otu0036Otu0020 Otu0365 _Planctomycetia Otu0180Otu0044 Otu0023Otu0066 Chloroﬂexi Otu0257 Otu0214 Otu0064Otu0067 Otu0041 Otu0408Otu0115 Otu0153Otu0216 Site C Otu0355Otu0815 Otu0125Otu0079 Deltaproteobacteria

10 Otu0298 Otu0336Otu0325 Otu0053Otu0016 Otu0458Otu0243 Otu0469 Site B Otu0184Otu0485 Otu0197Otu0158 MethodOtu0011Otu0377 E Otu0132Otu0009 Otu0391Otu0287 Otu0228 Alphaproteobacteria Otu0364Otu0777 Otu0572Otu0623 Site A Otu0018Otu0105 Otu0389 Otu0250Otu0199 Otu0379Otu0113 After 2001: Tidal inundation treatment Otu0167Otu0239 Otu0036Otu0020 Gammaproteobacteria Otu0181Otu0783 Otu0044 Otu0321Otu0324 Otu0257Otu0180 2 Otu0477Otu0175 Otu0064Otu0067 Early Otu0005Otu0528 Otu0115 Otu0193Otu0122 Otu0815Otu0408 Otu0809Otu0261 Otu0355 Otu0118Otu0234 Otu0325Otu0298 Otu0003Otu0594 Otu0243Otu0336 Otu0521Otu0416 Otu0458 activity patterns with phylogenetic Otu1008Otu0300 Otu0184Otu0485 Planctomycetes 5 Otu0473 Otu0377 Deltaproteobacteria 5 Otu0317Otu0183 Otu0011 Otu0853Otu0550 Otu0391Otu0287 Otu0616Otu0189 Otu0777 Otu0004Otu0352 Otu0105Otu0364 5 GammaproteobacteriaDelayed Otu0103Otu0006 Otu0199Otu0018 Otu0378Otu0149 Otu0250 _Syntrophobacterales Otu0056Otu0070 Otu0167Otu0239 Relative Abundance (%) Relative Abundance (%) Otu0138Otu0249 Otu0181Otu0783 Otu0701Otu0264 Otu0324 Otu0230Otu0718 Otu0175Otu0321 Relative Abundance (%) Relative Abundance (%) Otu0073 Otu0477 Otu0605Otu0014 Otu0528 Bacteroidetes Otu0569Otu0345 Otu0122Otu0005 1 Otu0225Otu0196 Otu0193 Otu0637Otu0131 Otu0809Otu0261 Otu0755Otu0495 Otu0118Otu0234 Otu0316Otu0347 Otu0594 Otu0182Otu0083 Otu0416Otu0003 Otu0540Otu0357 Otu0521 Otu0429Otu0711 Otu1008Otu0300 Otu0127Otu0240 Otu0183Otu0473 Betaproteobacteria Site A Site Otu0171 Otu0478Otu0034 Otu0550Otu0317 Otu0080Otu0252 Otu0189Otu0853 Otu0128Otu0165 Otu0616 tree (Fig. 12) to find out activity Otu0081 Otu0352 Otu0268Otu0492 Otu0006Otu0004 Otu0338Otu0339 Otu0103 Otu0007Otu0384 Otu0378Otu0149 Otu0534Otu0295 Otu0056Otu0070 Otu0349Otu0106 Otu0249 Otu0110Otu0423 Otu0264Otu0138 0 0 Otu0394 Otu0701 Otu0051Otu0282 Otu0718 Otu0092Otu0065 Otu0073Otu0230 0 0 Color Key Otu0633 Otu0014 Otu0223Otu0260 Otu0605 Firmicutes_Bacilli Otu0082Otu0048 Otu0569Otu0345 Otu0027Otu0296 Otu0196 Chlorobi Otu0460Otu0555 Otu0131Otu0225 Otu0635Otu0496 ZetaproteobacteriaOtu0495Otu0637 Otu0186 Otu0755 Otu0150Otu0069 Otu0347 Otu0068Otu0111 Otu0083Otu0316 Otu0363Otu0074 Otu0182 Bacteroidetes Otu0310Otu0566 Otu0540Otu0357 Otu0209Otu0342 Otu0429Otu0711 Otu0028Otu0466 Otu0240 Otu0086Otu0059 Otu0171Otu0127 Cyanobacteria_Chloroplast Otu0421 Otu0034 Otu0124Otu0600 Otu0478 Bacteroidetes_Flavobacteriia Otu0178Otu0575 Otu0080Otu0252 Otu0169Otu0289 Otu0128Otu0165 patterns of certain taxa. Otu0397Otu0141 Otu0081 Otu0033Otu0419 Otu0268Otu0492 Otu0247Otu0061 Otu0339 Zone 2 Zone 3 Otu0329Otu0220 Otu0384Otu0338 Zone 2 Zone 3 Otu0194 Otu0007 Otu0040Otu0241 Otu0534Otu0295 Otu0204Otu0037 44Otu0349Otu0106% Zone 2 Zone 3 Otu0423 Otu0394Otu0110 Otu0051Otu0282 Otu0092Otu0065 Otu0260Otu0633 Otu0048Otu0223 Otu0296Otu0082 Otu0555Otu0027 Zone 2 Zone 3 X.1 X.2 X.3 Otu0460 Otu0496 Otu0186Otu0635 Otu0150Otu0069 Otu0068Otu0111 Acidobacteria Epsilonproteobacteria Otu0363Otu0074 AcidobacteriaOtu0310Otu0566 Zone 1, pH < 5.5 Otu0209Otu0342 Zone 1, pH < 5.5 Otu0466 WS3 Otu0028 Deltaproteobacteria Gammaproteobacteria R_T2_A1 R_T2_A2 R_T2_A3 R_T2_A4 R_T2_A5 R_T2_A6 R_T2_A7 R_T3_A1 R_T3_A2 R_T3_A3 R_T3_A4 R_T3_A5 R_T3_A6 R_T3_A7 R_T4_A1 R_T4_A2 R_T4_A3 R_T4_A4 R_T4_A5 R_T4_A6 R_T4_A7 R_T1_A1 R_T1_A2 R_T1_A3 R_T1_A4 R_T1_A5 R_T1_A6 R_T1_A7 Otu0086Otu0059 Zone 1, pH < 5.5 Otu0600Otu0421 Alphaproteobacteria Acidobacteria Otu0575Otu0124 -1.5 -0.5 0.5 1.5 Otu0289Otu0178 Verrucomicrobia Actinobacteria (except Acidimicrobiia) Euryarchaeota Otu0141Otu0169 Zone 1, pH = 5.5-6.5 Otu0419Otu0397 Fig. 2 Zone 1, pH = 5.5-6.5 Otu0033 Otu0061 Otu0220Otu0247 Zone 1, pH = 5.5-6.5 Otu0194Otu0329 Zone 1, pH < 5.5 Otu0241 Value Otu0040 Otu0204Otu0037 Actinobacteria_Acidimicrobiia Firmicutes_Bacilli Zone 1, pH = 5.5-6.5 Verrucomicrobia Otu0018Otu0326 Acidobacteria Otu0143Otu0782 Alphaproteobacteria Firmicutes_Clostridia (Ling et al., 2015) X.1 X.2 X.3 Otu0052Otu0001 Otu0242 Otu0542Otu0007 Fig. 7 Relative abundances (%) of certain taxa in 3 zones. Otu0100Otu0215 Ac2nobacteriaOtu0334Otu0563 Otu0377Otu0414 Otu0200Otu0469 Bacteroidetes (except Flavobacteriia) Gammaproteobacteria Otu0177Otu0028 Otu0429Otu0234 R_T2_A1 R_T2_A2 R_T2_A3 R_T2_A4 R_T2_A5 R_T2_A6 R_T2_A7 R_T3_A1 R_T3_A2 R_T3_A3 R_T3_A4 R_T3_A5 R_T3_A6 R_T3_A7 R_T4_A1 R_T4_A2 R_T4_A3 R_T4_A4 R_T4_A5 R_T4_A6 R_T4_A7 R_T1_A1 R_T1_A2 R_T1_A3 R_T1_A4 R_T1_A5 R_T1_A6 R_T1_A7 Otu0098Otu0392 Planctomycetes Sampling: Otu0212Otu0710 Planctomycetes Otu0011 Otu0142Otu0037 Otu0503Otu0536 Bacteroidetes_Flavobacteriia Nitrospirae Otu0004Otu0061 _Planctomycetia Otu0539Otu0562 Otu0055Otu0229 Otu0069Otu0079 Otu0103Otu0329 Otu0014Otu0026 Otu0178Otu0149 Betaproteobacteria Other Otu0679 Otu0236Otu0489 Otu0637Otu0798 Gammaproteobacteria Otu0649Otu0301 Otu0473Otu0368 47Otu0138Otu0359% Otu0378Otu0251

7 Otu0558 Chlorobi Planctomycetes (except Planctomycetia) The abundance of iron-reducing bacteria was proportional to sulfate reducing bacteria in Zone 3, this suggests Otu0483 3 sites x 20 cm depth x 4 tidal times FirmicutesOtu0670 Otu0093Otu0071 Planctomycetes Otu0837Otu0917 Otu0352Otu0091 Otu0194Otu0220 Otu0445Otu0393 Others Otu0176Otu0477 Chloroflexi Planctomycetes_Planctomycetia Otu0006Otu0186

20 Otu0296 Otu0509Otu0394 Otu0169Otu0262 Otu0491Otu0080 Otu0274Otu0198 Otu0337Otu0357 Crenarchaeota Spirochaetes 6 Otu0502 Betaproteobacteria Otu0230Otu0279 Abundant Otu0646Otu0371 Rare Otu0114 that iron sulfides deposition favors both iron and sulfate reduction. Otu0423Otu0204 Otu0081Otu0384 Otu0350Otu0034 Otu0179Otu0029 Cyanobacteria Verrucomicrobia Otu0123Otu0086 Otu0109Otu0050 Firmicutes_Bacilli Otu0062Otu0021 ChloroﬂexiOtu0031Otu0706 Chlorobi Otu0276Otu0425 Fig. 12 Otu0287 Otu0321Otu0391 Bacteroidetes Deltaproteobacteria (except Syntrophobacterales) WS3 Otu0266 5 Otu0571 Otu0732Otu1065 Method E Otu0333Otu0389 Otu0023Otu0102 Bacteroidetes_Flavobacteriia 15 Otu0116 Inland Otu0237Otu0239 Site B Otu0022 sea Otu0002Otu0060 Deltaproteobacteria_Syntrophobacterales Otu0454 Otu0303Otu0458 Otu0366Otu0045 Otu0188Otu0277 Otu0361Otu0796 Otu0208Otu0330 Otu0243Otu0140 Otu0735Otu0442 4 Otu0197 Otu0354Otu0076 Color Key Otu0488Otu0364 Otu0319Otu0347 Acidobacteria Epsilonproteobacteria Otu0094Otu0269 Otu0362Otu0151 BacteroidetesOtu0053Otu0044 Otu0064 Otu0381Otu0041 Otu0144Otu0139 Actinobacteria (except Acidimicrobiia) Euryarchaeota Otu0307Otu0306 Otu0085Otu0281 Otu0598Otu0553 10 53Otu0398Otu0003% Otu0299Otu0424 Site A 3 Otu0137Otu0572 Early Actinobacteria_Acidimicrobiiaresponders Firmicutes_Bacilli Delayed responders Otu0226Otu0036 Site B Site C Otu0035Otu0167 Otu0054 Otu0696Otu0077 Otu0154Otu0543 Otu0008Otu0417 Two parallel organic Otu0222Otu0455 Alphaproteobacteria Firmicutes_Clostridia Otu0205Otu0619 Otu0410Otu0500 Otu0136Otu0157 Otu0012Otu0335 Otu0203Otu0042 Otu0313Otu0030 Bacteroidetes (except Flavobacteriia) Gammaproteobacteria

2 Otu0305 Otu0211Otu0049 Cyanobacteria_ChloroplastOtu0105Otu0185 Otu0163Otu0122 Otu0233Otu0075 Otu0231Otu0129 x 4 tidal times 5 Otu0010 Bacteroidetes_Flavobacteriia Nitrospirae -1.5 -0.5 0.5 1.5 Otu0213Otu0390 20cm Otu0408Otu0015 Otu0051 Cell degradation Otu0210Otu0063 Otu0087Otu0863 matter degradation series Otu0024Otu0106 Value Otu0196Otu0633 Betaproteobacteria Other Relative Abundance (%) Relative Abundance (%) sediment 1 Chlorobi Planctomycetes (except Planctomycetia) Otu0132 Otu0151Otu0424 X.1 X.2 X.3 Otu0060Otu0036 Otu0104Otu0035 Otu0333Otu0023 Otu0152Otu0172 Chloroflexi Planctomycetes_Planctomycetia Otu0407Otu0064 VerrucomicrobiaOtu0471Otu0417 Otu0482Otu0469 Otu0784Otu0049 were observed (Fig. 13), Otu0305 Flavobacteriia, Otu0015 0 0 Otu0085Otu0094 R_T2_B1 R_T2_B2 R_T2_B3 R_T2_B4 R_T2_B5 R_T2_B6 R_T2_B7 R_T3_B1 R_T3_B2 R_T3_B3 R_T3_B4 R_T3_B5 R_T3_B6 R_T3_B7 R_T4_B1 R_T4_B2 R_T4_B3 R_T4_B4 R_T4_B5 R_T4_B6 R_T4_B7 R_T1_B1 R_T1_B2 R_T1_B3 R_T1_B4 R_T1_B5 R_T1_B6 R_T1_B7 Otu0001 core Otu0157Otu0013 Crenarchaeota Spirochaetes Otu0809 Otu0287Otu0455 Otu0197Otu0042 Otu0185Otu0034 Otu0077Otu0003 Otu0299Otu0145 Otu0454Otu0184 Cyanobacteria Verrucomicrobia Otu0095Otu0156 Otu0191Otu0096 Otu0680Otu0097 Otu0317Otu0824 Gamma-proteobacteria, 35Otu0513Otu0631% Otu0210 Otu0182Otu0028 Otu0046Otu0330 Deltaproteobacteria (except Syntrophobacterales) WS3 Otu0332Otu0347 Spirochaetes Zone 2 Zone 3 Otu0391Otu0500 Otu0171 suggesting that microbial Otu0196Otu0538 Otu0075Otu0093 Fig. 3 Sampling strategy Otu0528Otu0548 Otu0719Otu0239 Deltaproteobacteria_Syntrophobacterales PlanctomycetesOtu0457Otu0123 Otu0370Otu0288 Zone 2 Zone 3 Otu0365Otu0600 Otu0122 certain Alpha-proteobacteria Otu0195 Otu0115Otu0244 Otu0306Otu0072 Otu0063Otu0116 Otu0208Otu0325 Otu1171Otu0192 Otu0113Otu1118 Otu0020Otu0227 Zone 1, pH < 5.5 Otu0431Otu0529 Alphaproteobacteria Acidobacteria Otu0553Otu0803 Otu0656Otu0443 Otu0664Otu0748 Otu0504Otu1030 resuscitation provides a Otu0477 Otu1034Otu0848 Zone 1, pH = 5.5-6.5 Otu0501Otu0787 Zone 1, pH < 5.5 Otu0569Otu0277 Otu0353Otu0590 Otu0212Otu0271 Otu0720Otu0429 Otu0705Otu0935 Otu0463Otu0887 Experiments: Otu0065Otu0764 Zone 1, pH = 5.5-6.5 Otu0037 Otu0459Otu0198 OthersOtu0026Otu0103 Otu0574 Otu0618Otu0289 Otu0291Otu0404 Otu0373Otu0372 Otu0223Otu0480 Otu0183Otu0205 Otu0987Otu0193 Site C Otu0945 Fermentation Otu0280Otu0174 survival advantage by Otu0516Otu0279 Otu0073Otu0702 Otu0338Otu0467 Abundant Rare Otu0855Otu0900 Otu0083 Otu0074Otu0173 Otu0179Otu0009 Otu0806Otu0324 Otu0954Otu0545 Otu0624Otu0752 Otu0588Otu0520 Otu0524Otu0518 Otu0638Otu0496 Otu0555Otu0368 Otu0385Otu0398 Otu0126Otu0460 Otu0427Otu0378 Otu0776Otu0011 Otu0495Otu0260 Otu0673Otu0575 avoiding activity at same 1) DNA extraction Otu0817 Otu0207Otu0622 65Otu0209Otu0316% Clostridia Otu0302Otu0052 Otu0153Otu0393 Otu0133Otu0795 Otu0202Otu0245 Otu0162Otu0290 Otu0112Otu0371 Otu0658Otu0327 Otu0715Otu0561 Otu0410Otu0540 Otu0214Otu0087 Otu0298Otu0134 Otu0955Otu0148 Otu0503Otu0630 Otu0099Otu0932 Bacilli Otu0337 Otu0328Otu0805 Otu0121Otu0047 Otu0124Otu0505 t i m e a n d t h e r e f o r e ▸ Otu0149Otu0282 2) RNA isolation cDNA synthesis Otu0101 Otu0430Otu0625 Otu0089Otu0189 Syntrophobacteria Otu0889 Otu0051Otu0188 Otu0893Otu0633 Otu0394Otu0774 Otu0027Otu0315 Otu0080Otu0187 Otu0359Otu0296 Otu0247Otu0240 Otu0684Otu0311 Otu0376Otu0357 Otu0845Otu0248 Otu0165Otu0341 Otu0127Otu0268 Otu0068Otu0167 3) 16S rRNA, 16S rRNA gene amplicons Otu0130Otu0131 decreasing microbial X.1 X.2 X.3 0-2 cm 2-4 cm 4-6 cm 6-8 cm 0-2 cm 2-4 cm 4-6 cm 6-8 cm 0-2 cm 2-4 cm 4-6 cm 6-8 cm 0-2 cm 2-4 cm 4-6 cm 6-8 cm 8-10 cm 8-10 cm 8-10 cm 8-10 cm 12-14 cm 12-14 cm 12-14 cm 12-14 cm Fig. 8 18-20 cm 18-20 cm 18-20 cm 18-20 cm R_T3_C1 R_T3_C2 R_T3_C3 R_T3_C4 R_T3_C5 R_T3_C6 R_T3_C7 R_T4_C1 R_T4_C2 R_T4_C3 R_T4_C4 R_T4_C5 R_T4_C6 R_T4_C7 R_T1_C1 R_T1_C2 R_T1_C3 R_T1_C4 R_T1_C5 R_T1_C6 R_T1_C7 (Ling et al., 2015) R_T2_C1 R_T2_C2 R_T2_C3 R_T2_C4 R_T2_C5 R_T2_C6 R_T2_C7 Terminal metabolism competitions. Fig. 11 Low tide Flood tide High tide Ebb tide Fig. 13 Sequencing (454 and Miseq sequencing) Bioinformatic analyses (Mothur software) Conclusions Acknowledgments Microbial distribution differed from typical redox Microbial activity showed that abundant population kept Microbial resuscitation provides a survival advantage for The authors acknowledge zonations and is controlled by environmental parameters constant activity across tidal cycling, whereas rare microorganisms in a dynamic environment. This study funding from Australian Research Microbial distribution (DNA abundances) such as pH, soil layering, water saturation, and iron sulfide populations change activity in response to environmental suggests both abundant and rare populations may play C o u n c i l ( g r a n t n o . Microbial activity (cDNA:DNA ratios) mineralization. changes, and overall had a higher activity. important roles in the ecology. LP110100732).