sign in sign up
<<
Home , Acidobacteria, Deinococcus, Euryarchaeota, Meiothermus, Methanobacteria, Methanobacteriales

Vol. 65: 287–302, 2012 AQUATIC MICROBIAL Published online March 2 doi: 10.3354/ame01549 Aquat Microb Ecol

Sediment and community analysis and nutrient fluxes in a sub-tropical polymictic reservoir

Timothy J. Green1,*, Andrew C. Barnes1, Michael Bartkow2, Deb Gale2, Alistair Grinham3

1School of Biological Sciences, The University of Queensland, Brisbane, Queensland 4072, Australia 2Queensland Bulk Water Supply (trading as Seqwater), 240 Margaret Street, Brisbane, Queensland 4000, Australia 3Centre for Water Studies, School of Civil Engineering, The University of Queensland, Brisbane, Queensland 4072, Australia

ABSTRACT: Lake sediments are important areas for remineralisation of nutrients involved in phytoplankton blooms. This study simultaneously analysed the microbial community structure and measured the sediment fluxes of inorganic nitrogen, phosphorus and silica from sediment cores collected at 2 different locations within a sub-tropical polymictic reservoir, Lake Wivenhoe. The bacterial and archaeal community structure was determined by amplifying and cloning the 16S rRNA gene from co-extracted DNA and RNA samples. A total of 19 phyla or candidate divi- sions of bacteria were identified, with sulphur-reducing bacteria within the of Deltapro- teobacteria being the most abundant ribotypes in DNA-derived clones libraries. In contrast, Acti- nobacteria and were the most abundant ribotypes in RNA-derived clone libraries from the upper and lower sediments, respectively. The archaeal community was dominated by , with methanogenic archaea belonging to subdivisions of and accounting for 69 to 98% of the sequenced clones. Comparison of the 16S rDNA and rRNA clone libraries revealed that bacterial groups highly abundant in the sediments were mostly metabolically inactive, whilst those metabolically active were not very abundant. A higher relative abundance of nitrifying ribotypes ( sp.) was identified at Site B, which corre- sponded to a higher efflux of nitrate from the sediments to the water column at this site. At the time of sampling, Lake Wivenhoe was stratified, and sediment cores were collected from the hypolimnion. Our results suggest that water column depth and delivery of dissolved oxygen to the sediments influenced the sediment microbial community structure and the fluxes and speciation of nutrients, which are reported to influence phytoplankton species composition and bloom dynamics.

KEY WORDS: Microbial community · Sediment · Nutrients · Bacteria · Archaea · 16S rRNA

Resale or republication not permitted without written consent of the publisher

INTRODUCTION by many different microbial groups (Schwarz et al. 2007). Many of the within the sedi- Sediments in shallow water bodies are important ar- ments play key roles in biogeochemical cycling of car- eas of nutrient remineralisation and carbon processing bon, nitrogen, phosphorus and silica. In shallow lakes, and are also depositional zones. The large surface these biogeochemical processes greatly influence area generated by the sediment grains and strong gra- phytoplankton blooms and water quality because of dients in organic substrates and terminal electron ac- the intensive interchange of nutrients between the ceptors provide an ideal environment for exploitation sediments and water column (Ye et al. 2009).

*Email: [email protected] © Inter-Research 2012 · www.int-res.com 288 Aquat Microb Ecol 65: 287–302, 2012

Lake Wivenhoe is the largest water storage reser- enhance the dissolution of biogenic silica by removing voir in south-eastern Queensland and supplies 80% organic coatings from diatom frustules and thereby of the drinking water of the city of Brisbane, Aus- enhancing the amount of ‘clean’ surface area exposed tralia (Douglas et al. 2007). The lake is eutrophic and to under-saturated waters (Libes 2009). suffers from frequent blooms of toxic The identification of the microorganisms within the and periodic episodes of earthy odours (geosmin or 2- sediments of Lake Wivenhoe is a key step towards methylisoborneol), likely produced by either Cyano - understanding how N, P and Si are remineralised bacteria or Actinomycetes (Klausen et al. 2004, within this . Thus, clone libraries of poly- Zaitlin & Watson 2006). Research on phytoplankton merase chain reaction (PCR)-amplified bacterial and bloom formation and sources of nutrients is currently archaeal 16S ribosomal genes from sediment sam- underway in Lake Wivenhoe in an attempt to im - ples were sequenced, and the potential biological prove the quality of the water for drinking (Burford et and metabolic process of each within al. 2012, Kerr et al. 2010, 2011a,b). Nitrogen and the clone library was inferred from the phylogenetic phosphorus are the 2 most important nutrients gov- position of its sequence. Putative metabolic processes erning phytoplankton growth (Correll 1998). How- performed by sediment microbes were then com- ever, differences in the ratios of dissolved N:P:Si can pared to nutrient fluxes of dissolved N, P and Si. change phytoplankton species composition within a Because a large fraction of the microbial cells within lake because the growth of diatom species depends the anoxic sediments may be dormant or dead (see on the presence of dissolved silica, whereas the Miskin et al. 1999), the relative abundances of ribo- growth of non-diatom phytoplankton species (e.g. types in clone libraries prepared from either DNA or Cyanobacteria) does not (Conley et al. 1993). RNA were compared to identify the active microbial Lake Wivenhoe is considered to be a polymictic fraction. Our results provide insight into biogeo- lake because it experiences several periods of thermal chemical processes occurring within the sediments of stratification, separated by periods of overturn and Lake Wivenhoe and the responsible for fully mixed conditions, each year. During thermal remineralisation of nutrients in the sediments. stratification, the hypolimnion becomes anoxic from the biodegradation of organic material in the sedi- ments by oxygen-consuming microorganisms. During MATERIALS AND METHODS anoxic conditions, is one of the first com- pounds to be released from the sediments as the bio- Study sites, sampling, core incubations logical oxidation of ammonia to nitrate () is and nutrient fluxes inhibited (Beutel et al. 2008), and the biological uptake of ammonia in anoxic conditions is relatively Sediment cores were collected from 2 different low compared to aerobic conditions (Beutel 2006). locations (Site A, 27° 19.59 S, 152° 33.10 E, and Site B, The sediments of lakes with oxic hypolimnia contain 27° 20.40 S, 152° 32.46 E) within Lake Wivenhoe, SE 3+ oxidized iron (Fe ) as Fe(PO4) and related hydroxides Queensland, Australia. This slightly alkaline lake is (Murray 1995). As the dissolved oxygen further de- shallow (the average depth is 10 m), with a surface 3+ 2+ 3− creases, Fe is reduced, and Fe and PO4 are re- area of 10 940 ha at full capacity (Douglas et al. 2007). leased into the water column (Murray 1995). Ammonia The 2 sites chosen were known to differ in both phys- and phosphorus released into bottom waters from the ical (e.g. temperature and depth) and chemical (e.g. sediments can exacerbate eutrophication when bot- dissolved inorganic nitrogen and pH) parameters, tom waters mix with surface waters (Beutel et al. based on routine water quality monitoring conducted 2008). Ammonia is the preferred nitrogen source for on the lake by Seqwater. The physicochemical pro- the local toxic Cyanobacteria, Cylindrospermopsis file of each sampling site was determined at the time 3− raciborskii (Burford et al. 2006), and pulses of PO4 of sampling using a multi-parameter YSI 6600 V2 were also shown to promote C. raciborskii dominance data sonde to measure dissolved oxygen, tempera- of the phytoplankton community (Posselt et al. 2009). ture and pH at 1 m increments. Three replicate sedi- Remineralisation rates of silica contained in diatom ment cores were collected from each site using a frustules within the sediments are largely controlled gravity corer fitted with acrylic liners (diameter by physiochemical processes, such as the degree of 65 mm and length 400 mm), and 100 to 150 mm of under-saturation of dissolved silica in the water or the sediment covered by ~300 mm of water was collec - blocking of reactive sites by inhibitors, such as metal ted. Cores were capped with an airtight bung and ions (Conley et al. 1993). However, prokaryotes can transported to the laboratory (< 2 h), where they were Green et al.: Sediment microbial community of Lake Wivenhoe 289

immediately placed in incubators filled with water parallel 25 µl PCR reactions using Platinum® Taq from each site that had been passed through a 60 µm DNA Polymerase High Fidelity (Invitrogen) and mesh screen. The incubator water was maintained at 300 nM of each of the universal bacterial primers the measured ambient temperature (Site A 19 ± 1°C 27F (5’-AGA GTT TGA TCC TGG CTC AG-3’) and and Site B 20 ± 1°C), and dark conditions were 1492R (5’-GTT ACC TTG TTA CGA CTT-3’) or uni- imposed because the sediment cores were collected versal archaeal primers 109aF (5’-ACK GCT CAG from the aphotic zone. The water column within each TAA CAC GT-3’) and 1119aR (5’-GGY RSG GGT core was gently mixed using a Teflon-coated mag- CTC GCT CGT T-3’). Replicate amplification prod- netic stirrer positioned below the core cap at a rate ucts were pooled to account for PCR bias, purified that avoided sediment re-suspension. The cores were using a NucleoSpin® Extract II kit (Macherey-Nagel) left overnight to settle, and incubations began when and cloned using the pCR4® TOPO cloning kit (Invit- the dissolved oxygen returned to ambient levels (0.35 rogen). The PCR inserts from 95 positive clones from and 0.55 mg l−1 at Site A and B, respectively). The each library were amplified using PCR and forward sediment fluxes of dissolved inorganic nitrogen spe- sequenced using gene specific primers (27F or + − − cies (NH4 , NO2 and NO3 ), filterable reactive phos- 109aF) by the Australian Genome Research Facility. phorus (FRP), total phosphorus (TP) and reactive High quality partial sequences were aligned using silica were estimated by withdrawing water using a ClustalW to allow the identification of identical or plastic syringe at 6 h intervals over a 24 h period from nearly identical sequences, and 114 bacterial and 92 the sample port located in the incubator core cap. archaeal clones were selected for complete sequenc- Water samples were filtered (0.45 µm), frozen at ing using M13F and M13R primers as above. −20°C and subsequently assayed for the above para- meters colourimetrically using a flow injector ana ly - ser by Queensland Health, Forensic & Scientific Ser- Phylogenetic analysis vices. Dissolved methane fluxes were estimated by withdrawing 6 ml of each water sample from the The obtained sequences were quality scored and sample port and analysing the methane content visually checked for possible sequencing errors using a gas chromatograph as described by O’Sulli- before compilation using Sequencher version 4.9 van et al. (2005). Fluxes of methane, silica, nitrogen (Gene Codes Corporation). The sequences were then and phosphorus species across the sediment–water compared to the available databases using the basic interface were calculated by linear regression of the local alignment search tool (BLAST) network service concentration data as a function of time, core water (Altschul et al. 1997) to determine their approximate volume and surface area (Eyre & Ferguson 2002). phylogenetic affiliations. Chimeric sequences were At the end of the incubations, sediment samples identified and removed from further analysis using from the upper (0 to 50 mm) and lower sediments (100 Bellerophon (Huber et al. 2004) and Mallard (Ashel - to 150 mm) were taken using a sterile serological ford et al. 2006). Sequences were then aligned with pipette from one core per site. Sediment samples selected reference 16S rRNA sequences from Gen- were immediately frozen in liquid nitrogen and stored Bank using the ClustalW algorithm in Mega version at −80°C for subsequent nucleic acid extraction. 4.0 (Tamura et al. 2007), and phylogenetic trees were constructed using the neighbour-joining distance method (Saitou & Nei 1987). The statistical signifi- Co-extraction of RNA/DNA and cance of interior nodes was determined by perform- PCR amplification ing bootstrap analyses based on 1000 re-samplings of the data. Sequences described in this study were Total genomic DNA and total RNA were co- submitted to GenBank and assigned accession num- extracted from sediment core samples using the RNA bers from HQ330530 to HQ330736. PowerSoilTM Total RNA Isolation kit and the DNA Elution Accessory kit (Mo Bio Laboratories). The purified total RNA was DNAse treated using the Identification of ammonia-oxidising archaea RNeasy mini kit with the RNase-Free DNase set and bacteria (Qiagen), and 200 ng of total RNA per sample was reverse-transcribed using the Superscript® III First- The alpha-subunit of the ammonia monooxyge- Strand Synthesis System (Invitrogen). Community nase (amoA) gene was PCR amplified from genomic ribosomal DNAs or cDNAs were PCR amplified in 3 DNA and cDNA in an attempt to determine the bac- 290 Aquat Microb Ecol 65: 287–302, 2012

teria and archaea responsible for ammonia oxidation (Fig. 2). Ammonia fluxes differed between sampling within the sediments. The archaeal amoA gene was sites, with a strong efflux of ammonia observed in amplified using the primers Arch-amoAF (5’-STA cores collected from Site A, whilst a slight influx of ATG GTC TGG CTT AGA CG-3’) and Arch-amoAR ammonia was observed in sediment cores from Site B (5’-GCG GCC ATC CAT CTG TAT GT-3’) following (Fig. 2). Patterns of nitrate and fluxes were sim- the PCR conditions described by Francis et al. (2005). ilar between sites; however, the flux rates differed The bacterial amoA gene was amplified using the greatly, with the nitrate efflux almost 3-fold greater at primers amoA-1F (5’-GGG GTT TCT ACT GGT Site B, whilst the nitrite influx was over 5-fold lower. + – GGT-3’) and amoA-2R (5’-CCC CTC KGS AAA GCC The concentration of NH4 , NO3 and FRP measured TTC TTC-3’) following similar PCR conditions in the bottom waters was in accordance with differ- described by Rotthauwe et al. (1997). Duplicate PCR ences in flux rates from sediment cores collected from products were pooled, purified (QIAquick PCR Sites A and B (data not shown). A strong efflux of Purification kit, Qiagen) and visualised on a 1% methane was also measured from sediment cores agarose/ethidium bromide gel alongside a 1 kb DNA (Fig. 3), with the methane flux almost 4-fold greater ladder (Fermentas). from sediment cores collected from Site A.

RESULTS Bacterial community analysis

Water column profile and sediment flux A total of 570 bacterial clones were randomly of nutrients selected for sequencing from 4 bacterial 16S rDNA and 2 bacterial 16S rRNA clone libraries (95 clones Strong gradients in water temperature, dissolved per library), which resulted in 476 high quality par- oxygen concentration and pH were observed at both tial sequences for further analysis. These partial Sites A and B (Fig. 1). The depth at Site A and B was sequences were clustered into 114 groups, and 29 and 15 m, respectively. The concentration of dis- represen tatives from each group were forward- and solved oxygen, pH and temperature was measured reverse-sequenced, yielding 87 identified opera- directly (0.5 m) above the water/sediment interface, tional tax o nomic units (OTUs), based on a 97% cut- and these parameters were found to be marginally off. A total of 14 chimeric sequences were detected lower at Site A (Fig. 1). and deleted from analysis. The number of clones The daily flux of nutrients from sediment cores is analysed per library is presented in Table 1. Rarefac- presented in Fig. 2. A greater efflux of silica was ob- tion analysis of the 6 samples (Fig. 4) indicated that served from sediment cores collected from Site A sampling did not reach a diversity asymptote based on 97% similarity clustering. Less than 40% of the 16S rRNA gene sequences in the current study were affiliated with already existing sequences in the public database with 97% identity (see Table S1 in the supplement at www. int-res. com/articles/suppl/a065 p287 _supp. pdf). These 16S rRNA gene se - quences had high similarities to those of uncultured environmental prokary- otes retrieved from a phreatic sinkhole (Sahl et al. 2010), wetlands (D’Auria et al. 2010) and lake (Briée et al. 2007, Schwarz et al. 2007, Ye et al. 2009) and estuarine sediment (Jiang et al. 2009). The remaining sequences were remo - tely related to 16S rRNA gene se- Fig. 1. Physiochemical profile of the water column measured at 1.0 m incre- ments prior to core collection at Site A (continuous line) and Site B (dashed quences of known bacteria and repre- line). Sediment depths (thick horizontal grey lines) were 29 m (Site A) and sent novel phylotypes not de scribed in 15 m (Site B) previous studies. Green et al.: Sediment microbial community of Lake Wivenhoe 291

Fig. 3. Methane flux rates from sediment cores collected at Sites A and B. Means ±SE are shown

Fig. 4. Rarefaction curves based on 97% similarity of (A) bac terial and (B) archaeal clone libraries. Libraries were generated from either genomic DNA (16S rDNA) or total Fig. 2. Sediment nutrient fluxes of dissolved inorganic silica, RNA (16S rRNA) at Site A, whereas libraries at Site B were nitrogen and phosphorus species from sediment cores only generated from genomic DNA. OTU: operational collected at Sites A and B. Means ±SE are shown taxonomic unit

Phylogenetic analysis (Figs. 5, 6 & 7) indicated Cyanobacteria, -, , that the bacterial community of the sediments Nitrospira, , , Verruco- within Lake Wivenhoe consisted of Alpha-, Beta-, microbia and the candidate divisions WS3, TM6, Delta- and , Acidobacteria, OP1, OP3, OP8 and OP11. The diversity (Shannon’s , , Chlorobi, , index), number of clones analysed, relative abun- 292 Aquat Microb Ecol 65: 287–302, 2012

Table 1. Distribution of 16S rRNA bacterial ribotypes within each phylum in DNA- and RNA-based analyses between upper (0–50 mm) and lower (100–150 mm) sediments at Sites A and B. The relative abundance (%) of clones for each phylum within a particular sample is based on forward partial sequences. The Shannon diversity index (H ’) and the number of high quality partial sequences analysed (n) from each library is also presented. rDNA (rRNA): ribotypes from sediment-extracted DNA (RNA or cDNA) libraries

Phylum Class Order Site A Site B 0−50 mm 100−150 mm 0−50 mm 100−150 mm rDNA rRNA rDNA rRNA rDNA rDNA

Proteobacteria Alpha 0.0 0.0 0.0 0.0 2.4 1.4 Beta Burkholderiales 0.0 0.0 3.6 0.0 3.7 2.7 Proteobacteria Beta Methylophilales 5.2 0.0 0.0 0.0 1.2 2.7 Proteobacteria Beta Unclassified 0.0 0.0 3.6 1.3 2.4 2.7 Proteobacteria Delta Desulfobacterales 13.0 3.4 18.1 1.3 9.8 13.5 Proteobacteria Delta Desulfuromonadales 2.6 1.1 1.2 0.0 1.2 4.1 Proteobacteria Delta Myxococcales 0.0 2.2 1.2 0.0 1.2 0.0 Proteobacteria Delta Syntrophobacterales 11.7 4.5 8.4 6.5 6.1 6.8 Proteobacteria Delta Unclassified 2.6 1.1 10.8 2.6 7.3 17.6 Proteobacteria Gamma Methylococcales 11.7 6.7 7.2 1.3 7.3 5.4 Proteobacteria Gamma 0.0 0.0 0.0 5.2 0.0 1.4 Proteobacteria Gamma 2.6 2.2 0.0 0.0 0.0 2.7 Proteobacteria Gamma Legionellales 0.0 0.0 0.0 0.0 0.0 1.4 5.2 3.4 3.6 1.3 9.8 4.1 Acidobacteria 7.8 9.0 9.6 20.8 7.3 4.1 Actinobacteria 1.3 20.2 2.4 6.5 1.2 0.0 Firmicutes 5.2 1.1 0.0 1.3 0.0 0.0 Chloroflexi 3.9 11.2 1.2 7.8 2.4 0.0 Cyanobacteria 0.0 2.2 0.0 5.2 1.2 0.0 Nitrospira 0.0 0.0 3.6 1.3 8.5 2.7 Spirochaetes 1.3 0.0 1.2 5.2 1.2 0.0 Planctomycetes 3.9 3.4 2.4 1.3 1.2 8.1 Deinococcus-Thermus 1.3 0.0 0.0 0.0 0.0 0.0 Cholorobi 1.3 0.0 0.0 0.0 0.0 2.7 Bacteroidetes 15.6 1.1 9.6 1.3 11.0 9.5 OP1 0.0 4.5 0.0 13.0 0.0 0.0 OP8 1.3 2.2 1.2 3.9 0.0 0.0 OP11 2.6 6.7 2.4 2.6 2.4 1.4 TM6 0.0 2.2 3.6 0.0 4.9 1.4 WS3 0.0 4.5 1.2 2.6 0.0 1.4 Unknown 0.0 4.7 3.6 3.9 6.1 2.8 H ’ 3.2 3.4 3.5 3.4 3.5 3.3 n = 829287878687

dance and frequency of clones within each major 16S rRNA gene sequences identified in the current phylogenetic division for each clone library are study were affiliated with already existing sequences presented in Table 1. in the public databases with 97% identity (see Table S2 in the supplement at www. int-res. com/ar - ticles/suppl/a065 p287 _supp. pdf). These 16S rRNA Archaeal community analysis

A total of 570 clones from archaeal 16S rDNA and Fig. 5. Unrooted phylogenetic tree of proteobacterial 16S rRNA sequences from the current study and related refer- rRNA clone libraries were randomly sequenced, and ence sequences. Sequences from the current study are des- 458 high quality partial sequences were obtained ignated as Lake Wivenhoe, followed by the clone ID and and grouped into 74 clusters. Representatives from GenBank accession number in brackets. The tree was con- each cluster were forward- and reverse-sequenced, structed using the neighbour-joining method using nearly full-length aligned nucleotide sequences in MEGA version and 51 OTUs were identified, based on a 97% cut- 4. Bootstrap values (%) are based on 1000 replicates and off. A total of 9 chimeras were detected and removed are shown at the nodes with >70% support. Scale bar from the phylogenetic analysis. Almost 70% of the represents 2% sequence divergence Green et al.: Sediment microbial community of Lake Wivenhoe 293

100 DQ067032 Uncultured lake sediment bacterium Lake Wivenhoe-PT23 (HQ330570) CU927575 Uncultured 87 NR_029295 Syntrophus gentianae 99 EU050697 Uncultured Syntrophus sp. FJ484401 Uncultured Deltaproteobacteria bacterium FJ902115 Uncultured Deltaproteobacteria bacterium DQ676358 Uncultured Deltaproteobacteria bacterium 100 Lake Wivenhoe-PT15 (HQ330566) FJ484446 Uncultured Deltaproteobacteria bacterium 100 Lake Wivenhoe-PB44 (HQ330554) 99 FJ484878 Uncultured Deltaproteobacteria bacterium 100 Lake Wivenhoe-PT49 (HQ330578) FJ517036 Uncultured Desulfobacteraceae bacterium 100 AY799901 Uncultured sediment bacterium 75 Lake Wivenhoe-PT50 (HQ330579) FJ517061 Uncultured Desulfobacteraceae bacterium Delta 100 GU208421 Uncultured lake sediment bacterium 100 100 Lake Wivenhoe-PT60 (HQ330582) FJ485074 Uncultured Deltaproteobacteria bacterium 100 Lake Wivenhoe-PT76 (HQ330610) FJ485056 Uncultured Deltaproteobacteria bacterium 100 Lake Wivenhoe-PB52 (HQ330555) 100 EU881241 Uncultured bacterium 100 Lake Wivenhoe-rPB35 (HQ330621) 100 FJ517016 Uncultured Deltaproteobacteria bacterium Lake Wivenhoe-LT45 (HQ330538) EU134319 Uncultured bacterium FJ538126 Uncultured Syntrophorhabdaceae bacterium 100 Lake Wivenhoe-LT70 (HQ330542) 74 EU169605 Uncultured Alcaligenes sp. 100 EU373389 Achromobacter xylosoxidans Lake Wivenhoe-LB09 (HQ330594) FJ391507 Methylophilus sp. 100 HM128980 Uncultured Tibetan Lake bacterium 100 84 Lake Wivenhoe-PT33 (HQ330609) Beta EF626691 Vogesella perlucida 100 Lake Wivenhoe-rPB34 (HQ330620) 100 FJ517105 Uncultured Gammaproteobacteria bacterium 99 100 Lake Wivenhoe-PT42 (HQ330573) AB252884 Uncultured Gammaproteobacteria bacterium 100 FJ493171 monteilii 100 Lake Wivenhoe-rPB62 (HQ330631) FJ379321 Pseudomonas sp. 99 AY007296 Methylosarcina lacus 92 AY546508 Uncultured sediment bacterium 99 D89279 Methylomicrobium japanense 77 99 Lake Wivenhoe-PT14 (HQ330565) GU455112 Uncultured bacterium Gamma 100 FJ484543 Uncultured Methylococcales bacterium Lake Wivenhoe-PT43 (HQ330574) 95 EF203198 Uncultured sediment bacterium NR_026062 Methylocaldum tepidum 82 Lake Wivenhoe-rPT69 (HQ330655) Lake Wivenhoe-LB01 (HQ330593) EU193110 Uncultured Sphingomonadaceae bacterium 99 FJ230844 Erythrobacter sp. 100 DQ811848 Uncultured bacterium

94 Alpha 96 Lake Wivenhoe-LT44 (HQ330537)

0.02 294 Aquat Microb Ecol 65: 287–302, 2012

100 AY225649 Uncultured bacterium Lake Wivenhoe-rPB47 (HQ330626) GU230434 Uncultured Acidobacteria bacterium AB530232 Uncultured bacterium 100 Lake Wivenhoe-PB14 (HQ330548) FJ755753 Uncultured sediment bacterium 100 Lake Wivenhoe-rPB56 (HQ330629) 100 FJ484528 Uncultured Acidobacterium sp. Lake Wivenhoe-PB74 (HQ330560) 100 AY921986 Uncultured Acidobacterium sp. 76 Lake Wivenhoe-PB34 (HQ330550) FJ484557 Uncultured Acidobacterium sp.

100 Lake Wivenhoe-LT30 (HQ330534) Acidobacteria 100 FJ713032 Uncultured Acidobacteria bacterium Lake Wivenhoe-PT13 (HQ330564) 100 AM086115 Uncultured lake sediment bacterium 81 Lake Wivenhoe-rPB79 (HQ330636) 86 AY050595 Uncultured groundwater bacterium 100 Lake Wivenhoe-rPB22 (HQ330618) 92 EU266871 Uncultured Rubrobacteraceae bacterium 100 Lake Wivenhoe-rPT64 (HQ330732) 83 DQ676393 Uncultured Actinobacterium sp. GQ406192 Uncultured Actinobacterium sp. 100 Lake Wivenhoe-rPB60 (HQ330630) 98 AF375994 Candidatus Brocadia anammoxidans 96 Lake Wivenhoe-PB79 (HQ330636) Actinobacteria EF032766 Uncultured Planctomycetes bacterium 100 Lake Wivenhoe-PT67 (HQ330584) 96 100 GQ246298 Uncultured sediment bacterium

Lake Wivenhoe-LT35 (HQ330535) Planctomycetes 100 AF465651 Uncultured Verrucomicrobia bacterium 99 Lake Wivenhoe-rPB24 (HQ330619) 100 DQ463705 Uncultured Verrucomicrobium sp. 100 Lake Wivenhoe-rPB07 (HQ330614) 100 L37521 Thermus sp. 100 Y13595 cerbereus Verrucomicrobia Lake Wivenhoe-PT92 (HQ330591) 99 AB252940 Uncultured bacterium Thermus

Y14644 Nitrospira sp. Deinococcus- 100 FJ535101 Uncultured Nitrospirales bacterium 100 Lake Wivenhoe-LB61 (HQ330595) 100 EU266883 Uncultured Chloroflexi bacterium Nitrospira 100 Lake Wivenhoe-PT41 (HQ330572) FJ484552 Uncultured Chloroflexi bacterium Lake Wivenhoe-LT03 (HQ330531) 100 100 DQ811891 Uncultured Chlorofexi bacterium Lake Wivenhoe-rPB90 (HQ330640) 100 DQ811888 Uncultured Chloroflexi bacterium 100

Lake Wivenhoe-rPB87 (HQ330638) Chloroflexi FJ485587 Uncultured Chloroflexi bacterium 100 Lake Wivenhoe-rPT17 (HQ330644) 100 AF067818 Cylindrospermopsis raciborskii Lake Wivenhoe-rPB48 (HQ330627) 100 AB513436 Uncultured Clostridiales bacterium Lake Wivenhoe-PT25 (HQ330608)

AY289504 sp. Cyanobacteria 100 Lake Wivenhoe-PT95 (HQ330592) AY330125 Uncultured sp. Lake Wivenhoe-rPB38 (HQ330623) EU542497 Uncultured sediment bacterium

100 Firmicutes 99 Lake Wivenhoe-rPT06 (HQ330642) FJ484043 Uncultured Spirochaetes bacterium 100 FJ748821 Uncultured estuarine sediment bacterium 100 Lake Wivenhoe-rPB75 (HQ330635) 100 DQ501337 Uncultured Chlorobi bacterium

Lake Wivenhoe-LB78 (HQ330599) Spirochaetes GU454944 Uncultured sludge bacterium

94 100 DQ640709 Uncultured Bacteroidetes bacterium Chlorobi 77 Lake Wivenhoe-LT39 (HQ330536) 100 EU283366 Uncultured Bacteroidetes bacterium 100 Lake Wivenhoe-PT89 (HQ330589) 100 FJ517065 Uncultured Flammovirgaceae bacterium Lake Wivenhoe-PT75 (HQ330585) AM086161 Uncultured lake sediment bacterium 80 100 Lake Wivenhoe-LB72 (HQ330597) AM086137 Uncultured sediment bacterium 100 Lake Wivenhoe-PB25 (HQ330549) 100 Lake Wivenhoe-LB72 (HQ330597) FJ516910 Uncultured bacterium 100 Lake Wivenhoe-PT22 (HQ330569)

GU472718 Uncultured Bacteroidales bacterium Cytophagales Bacteriodetes 0.05

Fig. 6. Unrooted phylogenetic tree of classified bacterial 16S rRNA sequences, except those of Proteobacteria. For details see Fig. 5 legend. Scale bar represents 5% sequence divergence Green et al.: Sediment microbial community of Lake Wivenhoe 295

91 AF280852 Uncultured wastewater bacterium 88 FJ484527 Uncultured candidate division WS3 bacterium 100 Lake Wivenhoe-PB67 (HQ330558) FJ746129 Uncultured marine sediment bacterium WS3 100 AJ390450 Uncultured soil bacterium 83 Lake Wivenhoe-rPB05 (HQ330613) 100 EU245608 Uncultured bacterium Lake Wivenhoe-rPB52 (HQ330628) GQ246427 Uncultured sediment bacterium 100 Lake Wivenhoe-PB40 (HQ330552) 100 GQ427414 Uncultured bacterium

Lake Wivenhoe-LT89 (HQ330546) Unknown 100 GU208344 Uncultured lake sediment bacterium 100 Lake Wivenhoe-PT08 (HQ330606) 100 FN646441 Uncultured candidate division OP3 bacterium Lake Wivenhoe-LT37 (HQ330602) 100 AB294931 Uncultured candidate division OP3 bacterium 99 Lake Wivenhoe-PT30 (HQ330571) 74 EU385826 Uncultured sediment bacterium OP3 FJ437848 Uncultured bacterium 98 100 Lake Wivenhoe-PB80 (HQ330562) 100 GQ397066 Uncultured soil bacterium Lake Wivenhoe-rPT62 (HQ330653)

100 EF688191 Uncultured anaerobic sludge bacterium Unknown 100 Lake Wivenhoe-PB05 (HQ330547) GQ472449 Uncultured sediment bacterium 100 Lake Wivenhoe-PB69 (HQ330559) 100 100 AB426208 Uncultured bacterium Lake Wivenhoe-PB94 (HQ330563) OP8 100 FJ484714 Uncultured candidate division OP8 bacterium AY221615 Uncultured soil bacterium 94 Lake Wivenhoe-rPB46 (HQ330625) 100 AB240236 Uncultured bacterium 100 Lake Wivenhoe-PB35 (HQ330551) 73 AB240511 Uncultured bacterium Lake Wivenhoe-LT56 (HQ330540) 100 Unknown 75 100 AM936568 Uncultured candidate division TM6 Lake Wivenhoe-PB41 (HQ330553) 100 GQ143753 Uncultured candidate division TM6 bacterium 96 Lake Wivenhoe-LT13 (HQ330532) TM6 100 FJ203566 Uncultured coral bacterium 97 Lake Wivenhoe-PB58 (HQ330556) 100 AB300067 Uncultured sediment bacterium 100 Lake Wivenhoe-rPT43 (HQ330648) 100 EU386084 Uncultured sediment bacterium Unknown 100 Lake Wivenhoe-PT17 (HQ330607) 100 FJ484374 Uncultured candidate division OP1 bacterium Lake Wivenhoe-rPB45 (HQ330624) 100 FJ901734 Uncultured candidate division OP1 bacterium 99 Lake Wivenhoe-rPT70 (HQ330656) OP1 EU542522 Uncultured sediment bacterium Lake Wivenhoe-PB87 (HQ330605) 100 FJ615148 Uncultured candidate division OP11 bacterium 99 Lake Wivenhoe-PT19 (HQ330568) GQ355041 Uncultured bacterium 100 Lake Wivenhoe-rPT28 (HQ330645)

100 DQ404853 Uncultured sediment bacterium OP11 Lake Wivenhoe-LT76 (HQ330543) 100 AM712330 Uncultured candidate division OP11 bacterium 100 Lake Wivenhoe-LT02 (HQ330530) DQ676298 Uncultured candidate division OP11 bacterium 100 Lake Wivenhoe-rPB73 (HQ330634) 0.05

Fig. 7. Unrooted phylogenetic tree of unclassified bacterial 16S rRNA sequences retrieved in the current study. For details see Fig. 5 legend. Scale bar represents 5% sequence divergence 296 Aquat Microb Ecol 65: 287–302, 2012

99 FJ755723 Uncultured archaeon Lake Wivenhoe-LTA10 (HQ330708) EU155927 Uncultured archaeon Lake Wivenhoe-LBA76 (HQ330667) GU135465 Uncultured archaeon 97 DQ785304 Uncultured archaeon 98 Lake Wivenhoe-LBA66 (HQ330705) AF423187 Methanogenic consortium archaeon 99 Lake Wivenhoe-PBA60 (HQ330687) GU135467 Uncultured Methanomicrobiales archaeon 97 AF481343 Uncultured archaeon Lake Wivenhoe-PTA10 (HQ330714) Lake Wivenhoe-PTA (HQ330719) AB479403 Uncultured Methanomicrobiales archaeon EU155937 Uncultured archaeon 97 Lake Wivenhoe-LBA77 (HQ330668) AB479395 Uncultured Methanolinea sp. 97 Lake Wivenhoe-LBA20 (HQ330661) Lake Wivenhoe-PBA50 (HQ330685) 99 EU155982 Uncultured archeaeon Lake Wivenhoe-rPTA07 (HQ330734) 99 FJ755726 Uncultured archaeon 90 Lake Wivenhoe-PBA38 (HQ330683) 85 99 FJ896280 Uncultured archaeon Lake Wivenhoe-PBA05 (HQ330712) Methanomicrobiales AB479407 Uncultured Methanolinea sp. FJ755727 Uncultured archaeon Lake Wivenhoe-LBA29 (HQ330701) 99 95 90 CU917177 Uncultured Methanomicrobiales archaeon 99 EU591562 Uncultured archaeon CU915060 Uncultured Methanomicrobiales archaeon 99 Lake Wivenhoe-PTA63 (HQ330725) DQ787475 receptaculi Lake Wivenhoe-LTA25 (HQ330677) 71 Lake Wivenhoe-PTA20 (HQ330717) 99 99 DQ301906 Uncultured archaeon 93 Lake Wivenhoe-LTA23 (HQ330676) 99 AY175382 Uncultured archaeon EU155942 Uncultured archaeon 71 FJ705116 Uncultured archaeon Lake Wivenhoe-LTA06 (HQ330707) NR_028157 thermophila 99 FJ755711 Uncultured archaeon 99 Lake Wivenhoe-rPTA65 (HQ330699) 98 EU490305 Uncultured archaeon 88 Lake Wivenhoe-rPBA15 (HQ330689) EU155954 Uncultured archaeon 95 FJ755718 Uncultured archaeon 76 Lake Wivenhoe-PTA17 (HQ330716) 99 AJ937876 Uncultured Euryarchaeota Lake Wivenhoe-LBA34 (HQ330664) 84 EU155903 Uncultured archaeon Lake Wivenhoe-LBA (HQ330703) Euryarchaeota

CU916645 Uncultured Methanosarchinales 99 GU135459 Uncultured Methanosaeta sp. AY780569 Uncultured Methanosaeta sp. FJ755706 Uncultured archaeon Lak e Wivenhoe-PTA70 (HQ330727) 99 EU519277 Uncultured archaeon Lake Wivenhoe-LTA32 (HQ330681) 99 FJ755708 Uncultured arhaeon 99 Lake Wivenhoe-LTA22 (HQ330675) 84 FJ822548 Uncultured archaeon 78 Lake Wivenhoe-LTA53 (HQ330679) 99 FJ755705 Uncultured archaeon 71 Lake Wivenhoe-PTA55 (HQ330723) 99 FJ755710 Uncultured archaeon 99 Lake Wivenhoe-rPBA64 (HQ330732) FJ755717 Uncultured archaeon 99 Lake Wivenhoe-rPBA64 (HQ330732) Unknown 99 FJ755719 Uncultured archaeon 99 Lake Wivenhoe-LTA04 (HQ330670) Lake Wivenhoe-LTA12 (HQ330673) DQ837290 Uncultured Euryarchaeota archaeon 91 Lake Wivenhoe-rPTA33 (HQ330735) 99 Lake Wivenhoe-rPBA17 (HQ330691) Lake Wivenhoe-rPTA31 (HQ330697) Lake Wivenhoe-LTA11 (HQ330672) 98 Lake Wivenhoe-PTA34 (HQ330720) 99 FJ755713 Uncultured archaeon SM1-Group 76 71 EF014531 Uncultured archaeon 99 Lake Wivenhoe-LTA38 (HQ330709) Lake Wivenhoe-LTA38 (HQ330709) Lake Wivenhoe-rPBA70 (HQ330695) Lake Wivenhoe-LTA14 (HQ330674) Lake Wivenhoe-LBA75 (HQ330706) 82 99 DQ310413 Uncultured archaeon Lake Wivenhoe-PTA21 (HQ330718) Lake Wivenhoe-rPBA52 (HQ330693) 99 Lake Wivenhoe-rPBA62 (HQ330694) 99 DQ310408 Uncultured archaeon Lake Wivenhoe-LBA28 (HQ330662) 80 EU731359 Uncultured Euryarchaeota archaeon 99 99 FN432658 Uncultured archaeon Lake Wivenhoe-PBA39 (HQ330684) DQ310460 Uncultured archaeon Unknown 93 DQ417477 Uncultured Euryarchaeota archaeon Lake Wivenhoe-PTA92 (HQ330731) Lake Wivenhoe-rPTA73 (HQ330736) FJ810531 Uncultured archaeon Lake Wivenhoe-LBA36 (HQ330665) 99 EU155996 Uncultured archaeon Lake Wivenhoe-PTA39 (HQ330721) 99 DQ785305 Uncultured archaeon 99 Lake Wivenhoe-rPTA55 (HQ330698) 99 FJ604785 Uncultured archaeon 0.05 Fig. 8. Unrooted phylogenetic tree of archaeal 16S rRNA gene sequences retrieved in the current study and related reference sequences. For details see Fig. 5 legend. The scale bar represents 5% sequence divergence Green et al.: Sediment microbial community of Lake Wivenhoe 297

gene sequences had high similarities to those of tion of autochthonous material (e.g. deceased uncultured methanogenic archaea (Fig. 8) retrieved phyto ) from the above surface waters. This from freshwater lake sediments (Lehours et al. 2007, organic material is utilised by a diverse microbial Ye et al. 2009), wastewater sludge (Rivière et al. community for energy and cellular carbon and in 2009), minerotrophic fen (Cadillo-Quiroz et al. 2008), the process is remineralised back into the water acidic bogs (Chan et al. 2002) and peatlands (Basiliko column. Although the temperature, pH and dis- et al. 2003, Cadillo-Quiroz et al. 2006). The remain- solved oxygen concentrations measured directly ing 24 sequences were remotely related to 16S rRNA (0.5 m) above the water/sediment interface at the 2 gene sequences of known archaea and represent study sites were only marginally different (Fig. 1), novel phylotypes not described in previous studies water column depth clearly influenced both nutrient (see Table S2 in the supplement). The majority of 16S flux rates (Fig. 2) and microbial community struc- rRNA gene sequences in the current study belonged ture (Tables 1 & 2). At Site A, the measured dis- to either Methanomicrobiales or Methanosaeta, and solved oxygen penetrated to a water depth of 18 m, the diversity, number of clones analysed and relative which is approximately 11 m above the water/ abundance of the major phylogenetic divisions in sediment interface. In contrast, Site B is shallower each archaeal 16S rDNA or rRNA clone library is (15 m), and diffused dissolved oxygen would reach presented in Table 2. the water/sediment interface and be rapidly con- sumed by the sediment microbial community (Fig. 1). This supply of dissolved oxygen to the Ammonia-oxidising archaea and bacteria sediments at Site B would alter the and structure of the sediment microbial community. The Numerous attempts to amplify the archaeal and following discussion speculates on the biogeochem- bacterial amoA gene from genomic DNA and cDNA ical processes performed by the different members isolated from Lake Wivenhoe sediment samples were of bacteria and archaea observed in the sediments unsuccessful. In each case, positive controls were cores collected from the 2 different locations and successfully amplified. Additions of PCR enhancers how these microbial communities influence the (3 to 5% dimethyl sulfoxide in master mix) and mod- remineralisation and flux of nutrients known to be ification of PCR parameters, such as annealing tem- involved in phytoplankton blooms. peratures and number of cycles, were also unsuc- cessful for amplifying the amoA gene from Lake Wivenhoe samples. Microbial diversity

The current study identified a total of 87 bacteria DISCUSSION and 57 archaea in the upper and lower sediments of Lake Wivenhoe. However, the true bacterial and The sediments of Lake Wivenhoe presumably archaeal diversity within the sediments is still likely contain organic material from the likely accumula- to be underestimated, as indicated by rarefaction

Table 2. Distribution of 16S rRNA archaea ribotypes within each phylum in DNA- and RNA-based analyses between upper and lower sediments at Sites A and B. The relative abundance of clones for each group within a particular sample is presented as a percentage. Shannon diversity index (H’) and the number of high quality partial sequences analysed (n) from each library is also presented. rDNA (rRNA): ribotypes from sediment-extracted DNA (RNA or cDNA) libraries

Phylum Class Order Site A Site B 0−50 mm 100−150 mm 0−50 mm 100−150 mm rDNA rRNA rDNA rRNA rDNA rDNA

Crenarchaeota Thermoprotei 1.1 2.1 0.0 1.1 0.0 0.0 Euryarchaeota Methanobacteria Methanobacteriales 2.2 2.1 1.1 0.0 3.2 4.3 Euryarchaeota Methanomicrobia Methanomicrobiales 53.8 48.9 69.0 57.8 50.5 49.5 Euryarchaeota Methanomicrobia 35.2 26.6 27.6 21.1 34.7 41.9 Unknown 7.7 20.2 2.3 20.0 11.6 4.3 H’ 2.9 3.2 2.8 3.2 3.1 3.1 n 919487909593 298 Aquat Microb Ecol 65: 287–302, 2012

curves, which do not reach a diversity plateau and sediment samples (Ventura et al. 2007). Actino - (Fig. 4). Despite this, the bacterial and archaeal bacteria play an important role in carbon cycling diversity (based on the Shannon index) was high and and have been shown to excrete a wide range of ranged from 3.2 to 3.5 and 2.8 to 3.2, respectively, in enzymes that degrade complex organic carbons the sediments of Lake Wivenhoe (Tables 1 & 2). The (lignin) during composting of agricultural waste (Yu obtained diversity values in the current study are et al. 2007). Actinobacteria in the upper sediments similar to those of other studies investigating sedi- of Lake Wivenhoe may play an important role in ment communities using similar techniques (Briée et removing the complex polysaccharide coatings from al. 2007, Schwarz et al. 2007). diatom frustules and exposing biogenic silica to under-saturated waters. Sulphate reduction is thought to outcompete Role of microbes in biogeochemical cycling methanogenesis when sulphate is freely available because cultured strains of sulphate-reducing bac- Dissolved reactive silica fluxes were greater from teria have higher specific affinities than methano - sediment cores collected at Site A (Fig. 2), which was genic bacteria and archaea for the main substrates supported by a significantly higher silica concentra- (acetate and H2) used by both groups (Purdy et al. tion in the bottom waters at this site (Fig. S2 in the 2003). However, available sulphate is usually limit- supplement at www. int-res. com/ar ticles/suppl/ a065 ing in freshwater environments, resulting in the p287 _supp. pdf). The sediments of Lake Wivenhoe dominance of methanogenesis as the main micro- presumably contain biogenic silica from the sedi- bial pathway for organic carbon degradation in mentation of deceased diatoms. Remineralisation of freshwater systems (Purdy et al. 2003). The high biogenic silica is largely controlled by abiotic factors relative abundance of both sulphate-reducing bac- (Conley et al. 1993), but microbes can enhance the teria and methanogenic archaea within the 16S dissolution of biogenic silica by removing organic rRNA clone libraries suggests that both of these coatings from diatom frustules (Libes 2009). Degra- microbial pathways do occur in the sediments of dation of organic matter within the sediments Lake Wivenhoe (Table 1). Sulphate-reducing bac - requires multiple steps performed by different teria (Desulfobacterales, Desulfuromonadales and trophic levels of bacteria and archaea. Syn trophobacterales) were the most frequently ob - are responsible for the initial microbial degradation served ribotypes in 16S rDNA clone libraries from of complex organic material within the sediments Sites A and B (Table 1). However, sulphate-reduc- and therefore fill the lowest trophic level. These het- ing bacteria were likely to be metabolically inactive erotrophs utilize this complex organic material for at the time of sampling based on the ratio of 16S energy and cellular carbon and excrete a range of rRNA to 16S rDNA sequences observed (Table 1). simple organic (e.g. acetate) and inorganic (e.g. Sulphate reduction and methanogenesis can occur and ammonia) molecules, which are then further simultaneously in anoxic, sulphate-containing sedi- catabolised by higher trophic levels of chemo - ments if utilise substrates (e.g. . Acetate can be assimilated into cellular methanol) not required by sulphate reducers carbon and further metabolised to methane and CO2 (Oremland & Polcin 1982), or production of methane by acetoclastic methanogenesis or completely oxi- occurs from metha nogenic CO2 reduction by syn- dised to CO2 by sulphate-reducing bacteria (Winfrey trophic consortia of acetate-oxidising bacteria and & Zeikus 1977). H2/CO2-using metha nogens (Nüsslein et al. 2001). Actinobacteria and Acidobacteria were likely to The majority of archaea identified in the present be the most metabolically active heterotrophic bac- study had 16S rRNA sequences related to Metha- teria within the upper and lower sediments, respec- nomicrobiales (49 to 69%), Methanosaeta (21 to tively, of Lake Wivenhoe based on the frequency of 41%) and Methano bacterium (2 to 4%), suggesting 16S rRNA ribotypes in clone libraries. Other hetero - that methane is produced in the sediments of Lake trophs, including Verrucomicrobia, Firmicutes and Wivenhoe by both methanogenic H2/CO2 reduction Pseudo monas sp., were also identified in the 16S (via Methanomicrobiales and ) rDNA libraries and hence may also participate in and acetoclastic methanogenesis (via Methano- the degradation of organic material. Actinobacteria saeta) (Chan et al. 2002). Complete degradation of constitute a diverse phylum, with members possess- organic carbon in freshwater sediments by a syn- ing highly variable physiological and metabolic trophic partnership of hydrogenotrophic and aceto- properties, and are abundant in environmental soil clastic methanogens was recently suggested by Green et al.: Sediment microbial community of Lake Wivenhoe 299

Biderre-Petit et al. (2011) and also appears to be of aerobic chemolithoautotrophic microbes. One step the case in Lake Wivenhoe. oxidises ammonia to nitrite (conducted by ammonia- The measured flux of methane from the sediments oxidising bacteria or archaea), whereas another of Site A was almost 4-fold greater than at Site B group oxidises nitrite to nitrate (Arp & Stein 2003). (Fig. 3). Increased methane production at Site A may The fully anoxic conditions at Site A did not support be the result of a higher abundance or metabolic rate nitrification, and >97% of the nitrogen efflux at this of methanogenic archaea, but methane is more likely site was ammonia. In contrast, the supply of dissolved being oxidised at the water–sediment interface at oxygen to the water/sediment interface at Site B sup- Site B. In freshwater environments, methane is usu- ported nitrification, with 100% of the nitrogen efflux ally oxidised at the oxic/anoxic interface by aerobic as nitrate (Fig. 2). The frequency of Nitrospira ribo- bacteria (Segers 1998). The diffusion of dissolved types (aerobic nitrite oxidisers) was also highest in oxygen to the water/sediment interface at Site B pre- the upper sediment clone library from Site B, sug- sumably allows aerobic methane oxidation to occur gesting that Nitrospira ribotypes play an important by Methylococcales ribotypes (Type I methano- role in nitrite oxidation in Lake Wivenhoe. However, trophs), which were observed in 16S rRNA clone the microbes responsible for ammonia oxidation at libraries. Methane can also be oxidised by anaerobic Site B could not be determined in the current study. methane-oxidising archaea (ANME), which have Most ammonia-oxidising bacteria (AOB) fall within been identified from the anoxic water body of the the order of the eutrophic freshwater Lake Plußsee (Eller et al. 2005). (Arp & Stein 2003), which were not detected in the In the marine environment, anaerobic methane oxi- 16S rDNA libraries in the present study. Recently, dation is reportedly coupled to sulphate reduction ammonia-oxidising archaea (AOA) were discovered (Thomsen et al. 2001). No evidence exists to suggest and have been identified in the water column and this pathway occurs in freshwater systems (Knittel & sediments of freshwater lakes (Pouliot et al. 2009, Ye Boetius 2009), and anaerobic methane oxidation in et al. 2009). These AOA belong to the Crenarchaeota freshwater environments may instead be coupled phylum and are known to occur within the suboxic with reduction of nitrite to dinitrogen (Ettwig et al. zone (<0.32 mg l−1 of dissolved oxygen) (Coolen et al. 2010). No 16S rRNA sequences related to ANME 2007). Attempts to identify AOA and AOB by ampli- were retrieved in the current study. fying the amoA gene were unsuccessful, suggesting The efflux of FRP was greater from the cores col- that ammonia oxidation is possibly performed 3− lected from Site A. Studies have shown that PO4 instead by ammonia-oxidising heterotrophs (e.g. cycling in freshwater lakes is regulated by the avail- Alcaligenes spp.) or methane oxidisers (e.g. Methylo- ability of and complexation with oxidised iron (Klee- coccales), and further research is warranted to deter- berg 1997). The sediments of lakes with oxic mine if these heterotrophs are responsible for ammo- 3+ hypolimnia contain oxidised iron (Fe ), as FePO4, nia oxidation in the sediments of Lake Wivenhoe. and related hydroxides (Murray 1995). As thermal Ammonia could also be oxidised anaerobically stratification occurs, the hypolimnion becomes with nitrite to dinitrogen (anammox) within the sedi- anoxic, and the reductive dissolution of Fe3+ releases ments of Lake Wivenhoe by bacteria falling in the 2+ 3− ferrous Fe and PO4 into the water column (Murray order of Planctomycetales (Jetten et al. 2003). Planc- 1995). Our study identified a wide range of microor- tomycete phylotypes related to environmental ganisms in the sediments of Lake Wivenhoe that are sequences were identified in the present study, with capable of reducing Fe3+ for energy and growth, their highest relative abundance in the lower sedi- including members within sulphur-reducing bac - ments of Site B. However, because Planctomycetales teria, methanogenic archaea, Acidobacteria and are physiologically diverse, it is not possible to com- Deinococcus-Thermus (Lovley et al. 2004). The ment on whether the planctomycete phylotypes greater efflux of FRP at Site A is most likely the result identified in the sediments of Lake Wivenhoe are of the fully anoxic conditions at this site allowing a indeed involved in anammox. Theoretically, anaero- higher abundance and/or metabolic activity of Fe3+ bic ammonia oxidation can also be coupled to man- reducers. ganese or iron reduction to produce nitrite or dinitro- Decomposition of phytoplankton in the sediments gen (Feammox) (Hulth et al. 1999), but this pathway generates ammonia primarily from the deamination is yet to be quantified in nature. Nitrite production of amino acids and urea (Jones et al. 1982). Ammonia has been observed under Fe-reducing conditions in is converted to nitrate in a 2-step process termed wetland sediments, suggesting that Feammox could ‘nitrification’, which is performed by 2 distinct groups exist in sediments (reviewed by Burgin et al. 2011). 300 Aquat Microb Ecol 65: 287–302, 2012

CONCLUSIONS Beutel MW (2006) Inhibition of ammonia release from anoxic profundal sediments in lakes using hypolimnetic oxygenation. Ecol Eng 28:271−279 Our study has contributed to the overall under- Beutel MW, Leonard TM, Dent SR, Moore BC (2008) Effects standing of the role that microorganisms play in of aerobic and anaerobic conditions of P, N, Fe, Mn, and nutrient remineralisation in freshwater sediments. Hg accumulation in waters overlaying profundal sedi- Although the type of analysis carried out here cannot ments of an oligo-mesotrophic lake. Water Res 42: 1953−1962 quantify the abundance of bacteria and archaea in Biderre-Petit C, Jézéquel D, Dugat-Bony E, Lopes F and the sediments of Lake Wivenhoe, it constitutes the others (2011) Identification of microbial communities basis for assessing the relative importance of specific involved in the methane cycle of a freshwater meromictic groups in nutrient remineralisation and carbon pro- lake. FEMS Microbiol Ecol 77: 533−545 cessing. Our results demonstrated that physicochem- Briée C, Moreira D, Lopez-Garcia P (2007) Archaeal and bacterial community composition of sediment and plank- ical properties of the water column (depth of water ton from a suboxic freshwater pond. Res Microbiol 158: column and diffusion of dissolved oxygen) influenced 213−227 the sediment microbial community structure. Fur- Burford MA, McNeale KL, McKenzie-Smith FJ (2006) The thermore, our study showed that the bacteria present role of nitrogen in promoting the toxic cyanophyte Cylin- drospermopsis raciborskii in a subtropical water reser- in the sediments (based on 16S rDNA ribotypes) voir. Freshw Biol 51: 2143−2153 were not necessarily metabolically active (based on Burford M, Green SA, Cook AJ, Johnson SA, Kerr JG, 16S rRNA ribotypes) at the time of sampling. Differ- O’Brian KR (2012) Sources and fate of nutrients in a sub- ences in the observed sediment flux rates and speci- tropical reservoir. Aquat Sci 74:179–190 ation of nutrients between the 2 study sites was likely Burgin AJ, Yang WH, Hamilton SK, Silver WL (2011) Beyond carbon and nitrogen: how the microbial energy the result of differences in the metabolic processes economy couples elemental cycles in diverse ecosys- performed by the corresponding microbial communi- tems. Front Ecol Environ 9: 44−52 ties. Differences in the flux and speciation of these Cadillo-Quiroz H, Bräuer S, Yashiro E, Sun C, Yavitt J, Zin- nutrients released from the sediments into the der S (2006) Vertical profiles of methanogenesis and methanogens in two contrasting acidic peatlands in hypolimnion could influence phytoplankton species Central New York State, USA. Environ Microbiol 8: composition and bloom dynamics when the 1428−1440 hypolimnion mixes with the epilimnion during over- Cadillo-Quiroz H, Yashiro E, Yavitt JB, Zinder SH (2008) turn. Future research should focus on quantifying Characterization of the archaeal community in a minerotrophic fen and terminal restriction fragment and measuring the metabolic processes performed length polymorphism-directed isolation of a novel by individual members within the microbial commu- hydrogenotrophic . Appl Environ Microbiol nity to assess their individual roles in nutrient bio- 74: 2059−2068 geochemical cycling. Chan OC, Wolf M, Hepperle D, Casper P (2002) Metha- nogenic archaeal community in the sediment of an artifi- cially partitioned acidic bog lake. FEMS Microbiol Ecol Acknowledgements. The authors acknowledge the financial 42: 119−129 and in-kind support provided by Queensland Bulk Water Conley DJ, Schelske CL, Stoermer EF (1993) Modification of Supply Authority, trading as Seqwater. The authors also the biogeochemical cycle of silica with eutrophication. acknowledge the assistance of N. Cutts and members of Mar Ecol Prog Ser 101:179−192 Seqwater’s monitoring team during fieldwork. Coolen MJL, Abbas B, van Bleijswijk J, Hopmans EC, Kuypers MMM, Wakeham SG, Sinninghe Damsté JS (2007) Putative ammonia-oxidising Crenarchaeota in LITERATURE CITED suboxic waters of the black sea: a basin-wide ecological study using 16S ribosomal and functional genes and Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, membrane lipids. Environ Microbiol 9: 1001−1016 Miller W, Lipman DJ (1997) Gapped blast and psi-blast: Correll DL (1998) The role of phosphorus in the eutrophica- A new generation of protein database search programs. tion of receiving waters: a review. J Environ Qual 27: Nucleic Acids Res 25:3389−3402 261−266 Arp DJ, Stein LY (2003) Metabolism of inorganic N com- D’Auria G, Barón-Rodríguez MM, Durbán-Vicente A, Moya pounds by ammonia-oxidizing bacteria. Crit Rev A, Rojo C, Latorre A, Rodrigo MA (2010) Unravelling the Biochem Mol Biol 38: 471−495 bacterial diversity found in the semi-arid Tablas de Ashelford KE, Chuzhanova NA, Fry JC, Jones AJ, Weight- Daimiel National Park wetland (Central Spain). Aquat man AJ (2006) New screening software shows that the Microb Ecol 59: 33−44 most recent large 16S rRNA gene clone libraries contain Douglas G, Palmer M, Caitcheon G, Orr P (2007) Iden - chimeras. Appl Environ Microbiol 72: 5734−5741 tification of sediment sources to Lake Wivenhoe, south- Basiliko N, Yavitt JB, Dees PM, Merkel SM (2003) Methane east Queensland, Australia. Mar Freshw Res 58:793−810 biogeochemistry and methanogen communities in two Eller G, Känel L, Krüger M (2005) Cooccurence of aerobic northern peatland , New York State. Geo- and anaerobic methane oxidation in the water column of microbiol J 20: 563−577 Lake Plußsee. Appl Environ Microbiol 71:8925−8928 Green et al.: Sediment microbial community of Lake Wivenhoe 301

Ettwig KF, Butler MK, Paslier DL, Pelletier E and others Murray TE (1995) The correlation between iron sulfide pre- (2010) Nitrite-driven anaerobic methane oxidation by cipitation and hypolimnetic phosphorus accumulation oxygenic bacteria. Nature 464: 543−548 during one summer in a softwater lake. Can J Fish Aquat Eyre BD, Ferguson AJP (2002) Comparison of carbon pro- Sci 52:1190−1194 duction and decomposition, benthic nutrient fluxes and Nüsslein B, Chin KJ, Eckert W, Conrad R (2001) Evidence in seagrass, phytoplankton, benthic for anaerobic syntrophic acetate oxidation during microalgae- and macroalgae-dominated warm-temper- methane production in the profundal sediment of sub- ate Australian lagoons. Mar Ecol Prog Ser 229:43−59 tropical Lake Kinneret (Israel). Environ Microbiol 3: Francis CA, Roberts KJ, Bemen MJ, Santoro AE, Oakley BB 460−470 (2005) Ubiquity and diversity of ammonia-oxidizing O’Sullivan CA, Burrell PC, Clarke WP, Blackall LL (2005) archaea in water columns and sediments of the ocean. Structure of a cellulose degrading bacterial community Proc Natl Acad Sci USA 102: 14683−14688 during anaerobic digestion. Biotechnol Bioeng 92: Huber T, Faulkner G, Hugenholtz P (2004) Bellerophon: a 871−878 program to detect chimeric sequences in multiple Oremland RS, Polcin S (1982) Methanogenesis and sulfate sequence alignments. Bioinformatics 20:2317−2319 reduction: competitive and noncompetitive substrates in Hulth S, Aller RC, Gilbert F (1999) Coupled anoxic nitrifica- estuarine sediments. Appl Environ Microbiol 44: tion/manganese reduction in marine sediments. 1270−1276 Geochim Cosmochim Acta 63: 49−66 Posselt AJ, Burford MA, Shaw G (2009) Pulses of phosphate Jetten MSM, Slieker O, Kuypers M, Dalsgaard T and others promote dominance of the toxic cyanophyte Cylindro - (2003) Anaerobic ammonium oxidation by marine and spermopsis raciborskii in a subtropical water reservoir. freshwater planctomycete-like bacteria. Appl Microbiol J Phycol 45: 540−546 Biotechnol 63: 107−114 Pouliot J, Galand PE, Lovejoy C, Vincent WF (2009) Vertical Jiang L, Zheng Y, Peng X, Zhou H, Zhang C, Xiao X, Wang structure of archaeal communities and the distribution of F (2009) Vertical distribution and diversity of sulfate- ammonia monooxygenase a gene variants in two mero - reducing prokaryotes in the Pearl River estuarine sedi- mictic high artic lakes. Environ Microbiol 11: 687−699 ments, Southern China. FEMS Microbiol Ecol 70: Purdy KJ, Nedwell DB, Embley TM (2003) Analysis of the 249−262 sulfate-reducing bacterial and methanogenic archaeal Jones JG, Simon BM, Horsley RW (1982) Microbiological populations in contrasting Antarctic sediments. Appl sources of ammonia in freshwater lake sediments. J Gen Environ Microbiol 69: 3181−3191 Microbiol 128:2823−2831 Rivière D, Desvignes V, Pelletier E, Chaussonnerie S and Kerr JG, Burford M, Olley J, Udy J (2010) Phosphorus sorp- others (2009) Towards the definition of a core of micro - tion in and sediments: implications for phosphate involved in anaerobic digestion of sludge. supply to a subtropical river in Southeast Queensland, ISME J 3: 700−714 Australia. Biogeochemistry 102:73−85 Rotthauwe JH, Witzel KP, Liesack W (1997) The ammonia Kerr JG, Burford M, Olley J, Udy J (2011a) Nitrogen and monooxygenase structural gene amoA as a functional phosphorus storage in contrasting reaches of a sub-trop- marker: molecular fine-scale analysis of natural ammo- ical river system. Water Air Soil Pollut 217:523−534 nia-oxidizing populations. Appl Environ Microbiol 63: Kerr JG, Burford M, Olley J, Bunn S, Udy J (2011b) Examin- 4704−4712 ing the link between terrestrial and aquatic phosphorus Sahl JW, Fairfield N, Harris JK, Wettergreen D, Stone WC, speciation in a subtropical catchment: the role of selec- Spear JR (2010) Novel microbial diversity re trieved by tive erosion and transport of the sediments during storm autonomous robotic exploration of the world’s deepest events. Water Res 45:3331–3340 vertical phreatic sinkhole. Astrobiology 10: 201−213 Klausen C, Jørgensen NOG, Burford M, O’Donohue M Saitou N, Nei M (1987) The neighbour-joining method: a (2004) Actinomycetes may also produce taste and odour. new method for reconstructing phylogenetic trees. Mol Vatten 31: 45−49 Biol Evol 4: 406−425 Kleeberg A (1997) Interactions between benthic phosphorus Schwarz JIK, Eckert W, Conrad R (2007) Community struc- release and sulfur cycling in Lake Scharmutzelsee (Ger- ture of archaea and bacteria in a profundal lake sedi- many). Water Air Soil Pollut 99: 391−399 ment Lake Kinneret (Israel). Syst Appl Microbiol 30: Knittel K, Boetius A (2009) Anaerobic oxidation of methane: 239−254 progress with an unknown process. Annu Rev Microbiol Segers R (1998) Methane production and methane con- 63: 311−334 sumption: a review of processes underlying wetland Lehours AC, Evan P, Bardot C, Joblin K, Gerard F (2007) methane fluxes. Biogeochemistry 41: 23−51 Phylogenetic diversity of archaea and bacteria in the Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: mole- anoxic zone of a meromictic lake (Lake Pavin, France). cular evolutionary genetics analysis (MEGA) software Appl Environ Microbiol 73: 2016−2019 version 4.0. Mol Biol Evol 24: 1596−1599 Libes SM (2009) Biogenic silica. In: Libes SM (ed) Introduc- Thomsen TR, Finster K, Ramsing NB (2001) Biogeochemical tion to marine biogeochemistry. Elsevier, Burlington, and molecular signatures of anaerobic methane oxida- MA, p 403−422 tion in a marine sediment. Appl Environ Microbiol 67: Lovley DR, Holmes DE, Nevin KP (2004) Dissimilatory Fe(III) 1646−1656 and Mn (IV) reduction. Adv Microb Physiol 49: 219−286 Ventura M, Canchaya C, Tauch A, Chandra G, Fitzgerald Miskin IP, Farrimond P, Head IM (1999) Identification of GF, Chater KF, van Sinderen D (2007) Genomics of Acti- novel bacterial lineages as active members of microbial nobacteria: tracing the evolutionary history of an ancient populations in a freshwater sediment using a rapid RNA phylum. Microbiol Mol Biol Rev 71: 495−548 extraction procedure and RT-PCR. Microbiology 145: Winfrey MR, Zeikus JG (1977) Effect of sulfate on carbon and 1977−1987 electron flow during microbial methanogenesis in fresh- 302 Aquat Microb Ecol 65: 287–302, 2012

water sediments. Appl Environ Microbiol 33:275−281 Li J (2007) Microbial community succession and lignocel- Ye W, Liu X, Lin S, Tan J, Pan J, Li D, Yang H (2009) The ver- lulose degradation during agricultural waste compost- tical distribution of bacterial and archaeal communities ing. Biodegradation 18: 793−802 in the water and sediment of Lake Taihu. FEMS Micro- Zaitlin B, Watson SB (2006) Actinomycetes in relation to biol Ecol 70:263−276 taste and odour in drinking water: myths, tenets and Yu H, Zeng G, Huang H, Xi X, Wang R, Huang D, Huang G, truths. Water Res 40: 1741−1753

Editorial responsibility: Rutger de Wit, Submitted: July 20, 2011; Accepted: December 20, 2011 Montpellier, France Proofs received from author(s): February 29, 2012