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Applied and Environmental Microbiology, May 2007, p. 3117-3120, Vol. 73, No. 9
0099-2240/07/$08.00+0     doi:10.1128/AEM.02274-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Impacts of Cultivation of Marine Diatoms on the Associated Bacterial Community{triangledown}

Melanie Sapp,* Antje Wichels, and Gunnar Gerdts

Alfred Wegener Institute Foundation for Polar and Marine Research, Biologische Anstalt Helgoland, P.O. Box 180, 27483 Helgoland, Germany

Received 26 September 2006/ Accepted 7 March 2007


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ABSTRACT
 
The composition of bacterial communities associated with four diatom species was monitored during isolation and cultivation of algal cells. Strong shifts in the associated communities, linked with an increase in the numbers of phylotypes belonging to members of the Gammaproteobacteria, were observed during cultivation.


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INTRODUCTION
 
A close linkage of bacterio- and phytoplankton has been assumed in the past, leading to the "phycosphere" concept (1). Strong associations of certain bacterial morphotypes and phytoplankton cells in cultures and natural seawater have been shown previously (12). Nevertheless, identification of these bacteria could not be achieved. Hence, microalgal batch cultures were examined with easy access to microbial biomass for molecular analysis (8, 11, 17, 19).

These experiments are usually conducted with microalgae from culture collections which had been isolated long ago and cultured for years. Three null hypotheses are generally possible: long-term cultures led to selection of bacterial populations because of (i) specific association, (ii) nonspecific cocultivation, or (iii) cocultivation with genetic variants of algal cells over time.

We investigated the bacterial community composition during initial steps of isolation and subsequent cultivation of diatom species to assess possible effects due to cultivation. To our knowledge this is the first time that cultivation shifts have been described in detail for diatom-associated bacteria.


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Algal cultures.
 
Single diatom cells were isolated as described previously (17). These were Guinardia delicatula (Cleve) Hasle, Pseudonitzschia pungens Grunow, Thalassiosira rotula Meunier, and Skeletonema costatum (Greville) Cleve, dominating in the water of Helgoland Roads, Helgoland, Germany (22). Microalgae were incubated in f/2 medium (9) at 16°C with 12-h-light-12-h-dark cycling at 20.0 µmol photons m–2 s–1. After 2 months, single cells were grown to clonal cultures. Afterwards, every month, fresh medium was inoculated with cells of the respective cultures (dilution factor, 0.02). Samples were taken after 2, 4, and 12 months in the early stationary phase of algal growth (steps 1, 3, and 11). Samples of the in situ community retrieved at the date of isolation were used to cover the original bacterial community (16). A 50-ml volume of each sample was filtered through successive membrane filters (17).


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Community analyses.
 
Nucleic acid extraction, amplification of intergenic spacers, ribosomal intergenic spacer analysis (RISA), amplification of 16S rRNA gene fragments, and denaturing gradient gel electrophoresis (DGGE) were carried out as described previously (16). Sequencing of excised DGGE bands and analyses of sequence data were performed as already described (4, 14, 17). These resulted in sequences of approximately 500 bp. Sequences within an ARB database related to sequences retrieved in our study and relatives found by BLAST (http://www.ncbi.nlm.nih.gov) displaying more than 1,300 nucleotides were used to calculate phylogenetic trees (17).

RISA and DGGE profiles were analyzed using BioNumerics 4.5 (Applied Maths BVBA, Belgium). Multivariate analysis of fingerprints was performed using the subroutines ANOSIM and MDS of PRIMER 5 software (3) as described previously (17). The null hypothesis was that no difference between associated communities of different cultivation steps exists. A sample statistic (R; significance level of 0.1%) displayed the degree of separation (17).


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Community shifts.
 
The MDS plot of RISA fingerprints (Fig. 1A) displayed a development of associated communities within three steps. Communities associated with freshly isolated microalgae could be grouped together (group I). These were not similar to the in situ community. The communities displayed a shift after 4 and 12 months (group II, pairwise test, R = 0.561; group III). The communities within group III were strongly separated from the communities associated with freshly isolated microalgae (Fig. 1A) (pairwise test, R = 0.835). General separation of communities was also reflected by ANOSIM analysis (global R = 0.636). A similar development was observed by MDS analysis of DGGE fingerprints (Fig. 1B), displaying a separation of communities (global R = 0.771). A shift occurred after 2 to 4 months (pairwise test, R = 0.787), resulting in more similar communities after 4 to 12 months (group II, pairwise test, R = 0.434). The associated communities in step 1 are completely separated from the communities in step 11 (pairwise test, R = 1.0).


Figure 1
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  		FIG. 1. MDS plots based on Bray-Curtis similarities of community fingerprints of different microalgal cultures (G, Guinardia delicatula; T, Thalassiosira rotula; P, Pseudonitzschia pungens; S, Skeletonema costatum), two sample fractions, and four sampling dates. Cultivation steps are indicated as follows: {triangledown}, in situ; {diamond}, step 1; {circ}, step 3; and {square}, step 11. The arrows indicate the development of the communities. Circles display groups of samples. (A) MDS plot of RISA fingerprints. (B) MDS plot of DGGE fingerprints.


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Phylogeny.
 
Most sequences were related to those of Alphaproteobacteria and Gammaproteobacteria spp. (44% and 31%; Fig. 2). The results revealed matches with 98 to 100% similarity to bacterial 16S rRNA gene sequences. A total of 19% of the phylotypes belonged to Bacteroidetes spp., and 6% (two phylotypes) affiliated to Actinobacteria spp. A neighbor-joining tree revealed that sequences belonging to Alphaproteobacteria spp. clustered mainly with Sulfitobacter spp., and sequences related to those of Gammaproteobacteria spp. were mainly assigned to Alteromonadales spp. (Fig. 2). Some phylotypes are already found in microalgal cultures retrieved from phytoplankton of Helgoland Roads (G001, G009, G062) (17). Phylotypes G021 and G046 clustered with sequences retrieved from the in situ sample (16).


Figure 2
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  		FIG. 2. Phylogenetic tree of Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Actinobacteria, and Bacteroidetes spp. GenBank accession numbers are given in parentheses. Bootstrap values above 50% are displayed.

Several authors identified members of Alphaproteobacteria, Gammaproteobacteria, and Bacteroidetes as being associated with algal cells (2, 7, 8, 10, 11, 12, 17, 19, 21). Members of these bacterial groups might play an important part in the interaction of bacteria and microalgae.

However, in most if not all previous studies concerning phycosphere communities, members of Actinobacteria were not detected. Therefore, it is not clear whether this phylotype truly represents bacterium-phytoplankton associations.


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Succession of phylotypes.
 
A total of 18 phylotypes were identified at different positions on DGGE gels. The succession of phylotypes in the cultures is summarized for the main phylotypes (Table 1). Phylotype F128 (GenBank accession no. DQ289519) retrieved from the in situ sample could not be detected in the cultures after isolation. Phylotype G003 occurred in all cultivation steps. Some phylotypes could be retrieved after isolation but not during further cultivation (G046, G048, G049, G061, G062). Phylotype G037 was detected in all cultures after isolation. Some phylotypes were detected exclusively in steps 3 and 11 (G006, G009, G010, G020), whereas phylotype G372 was detected in some cultures solely in step 11. Some phylotypes are not displayed in Table 1, as they occurred in few samples (G001, G012, G021, G024, G356) (Fig. 1).


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  		TABLE 1. Appearance of phylotypes within cultivation steps

Since microalgae were isolated by the technique of micropipetting, it is likely that identified phylotypes originated from the in situ community. With reference to our study, the coherence of in situ detection and recovery during cultivation could be shown for phylotypes G020 and G046.

If we presume that low abundance led to nondetection of phylotypes, those associated bacteria which were not detected in situ but were detected later must have grown rapidly during cultivation, probably due to high substrate concentrations. This has been suggested for some Gammaproteobacteria spp. of Helgoland Roads (5). In our study, this might be the case for several phylotypes. Relatives of these have been found in microalgal cultures retrieved from Helgoland Roads (Fig. 2) (17). It is assumed that those bacterial groups are important for the interaction of bacteria and phytoplankton. Effects of Alteromonas sp. beneficial to microalgae have already been shown previously (20), supporting our assumption.

It has not yet been possible to successfully identify bacterial species attached directly on algal cells; therefore, the possibility cannot be excluded that the detected phylotypes belong to opportunists. These might profit from cultivation conditions. It is possible that the shifts might also result from confinement (6, 13, 18). Furthermore, the dilution after each recultivation step might have favored shifts in community composition.

In conclusion, the process of cultivation did not lead to species-specific associated bacterial communities. Findings from culture experiments should be scrutinized regarding cultivation impacts, and future analysis of bacterium-phytoplankton associations should comprise experiments and field studies. In particular, a combination of fingerprinting methods and multivariate statistics provides a promising tool to achieve further insights into phytoplankton-bacterioplankton interactions (15, 16).


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Nucleotide sequence accession numbers.
 
Sequences obtained in this study are available from GenBank (DQ376145 to DQ376174).


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ACKNOWLEDGMENTS
 
We thank the crew members of the research vessel Aade from the Biologische Anstalt Helgoland. Special thanks to Anne S. Schwaderer for the isolation of microalgae and to Karen Helen Wiltshire, Nicole Aberle-Malzahn, and Tracy Ann Dinmore Maxwell for providing constructive criticism. Two anonymous reviewers also provided helpful advice.

This work is part of the Helgoland Foodweb project in the Coastal Diversity program of the Alfred Wegener Institute.


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FOOTNOTES
 
* Corresponding author. Present address: Center for Environment, Fisheries, and Aquaculture Science, Pakefield Road, Lowestoft, Suffolk NR 33 0HT, United Kingdom. Phone: 44 1502 52 4518. Fax: 44 1502 51 3865. E-mail: Melanie.Sapp{at}cefas.co.uk Back

{triangledown} Published ahead of print on 16 March 2007. Back


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Applied and Environmental Microbiology, May 2007, p. 3117-3120, Vol. 73, No. 9
0099-2240/07/$08.00+0     doi:10.1128/AEM.02274-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.




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