Accepted Article

Received Date : 21-Jan-2014 Revised Date

: 11-Jul-2014

Accepted Date : 27-Jul-2014 Article type

: Research Paper

Editor

: Patricia Sobecky

Application of a new cultivation technology, I-tip, for studying microbial diversity in freshwater sponges of Lake Baikal, Russia

Dawoon Jung1, Eun-Young Seo1, Slava S. Epstein2, Yochan Joung3, Jaemin Han1, Valentina V. Parfenova4, Olga I. Belykh4, Anna Gladkikh4 and Tae Seok Ahn1*

1

Dept. of Environmental Science, Kangwon National University, Chuncheon, Korea

2

Dept. of Biology, Northeastern University, Boston, USA

3

Dept. of Bioscience and Biotechnology, Hankuk University of Foreign Studies, Yongin,

Korea 4

Limnological Institute, Siberian Branch, Russian Academy of Sciences, Irkutsk, Russia

Correspondence: Tae Seok Ahn, Dept. of Environmental Science, Kangwon National University, Chuncheon, 200-701, Korea. Tel: +82 33 250 8574; fax: +82 33 259 5670; e-mail: [email protected] This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/1574-6941.12399 This article is protected by copyright. All rights reserved.

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Running title: New cultivation technology for microbial diversity

Keywords: culturing device, in situ cultivation, microbial trap, pyrosequencing, unculturable microorganism

Abstract One of the fundamental methods for cultivating bacterial strains is conventional plating on solid media, but this method does not reveal the true diversity of the bacterial community. In this study we develop a new technique and introduce a new device we term, I-tip. The I-tip was developed as an in situ cultivation device that allows microorganisms to enter and natural chemical compounds to diffuse, thereby permitting the microorganisms to grow utilizing chemical compounds in their natural environment. The new method was used to cultivate microorganisms from Baikalian sponges and the results were compared with conventional plating as well as a pyrosequencing-based molecular survey. The I-tip method produced cultures of 34 species from five major phyla, Actinobacteria, Alphaproteobacteria, Betaproteobacteria, Firmicutes, and Gammaproteobacteria, “missing” only two major phyla detected by pyrosequencing. Meanwhile, standard cultivation produced a smaller collection of 16 species from three major phyla, Betaproteobacteria, Firmicutes, and Gammaproteobacteria, failing to detect over half of the major phyla registered by pyrosequencing. We conclude that the I-tip method can narrow the gap between cultivated and uncultivated species, at least for some of the more challenging microbial communities such as those associated with animal hosts.

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Introduction Cultivation and isolation of microorganisms are essential for studying the physiological and metabolic characteristics of individual microbial strains (Zeng et al., 2012); however, the majority of microorganisms from natural environments cannot be grown in the laboratory (Amann et al., 1995). Fluorescence in situ hybridization (FISH), denaturing gradient gel electrophoresis (DGGE), pyrosequencing, and other molecular techniques have demonstrated that a large number of uncultivable microorganisms exist in most environments (Handelsman, 2004). Bringing these species into culture collections requires innovations in cultivation strategies.

One such strategy is based on cultivation of microorganisms in their natural environment (Kaeberlein et al., 2002). Several modifications of the original technique have since been proposed and tested (for a recent review, see Epstein et al., 2010). They all share the same basic rationale: natural milieus contain growth factors necessary and sufficient for microbial growth; however, the specifics of each modification lead to selection of particular species, and the resulting culture collections prove different. Recently we introduced a new device for in situ cultivation, the filter plate microbial trap (FPMT), and successfully used it to isolate formerly uncultivated strains from an alkaline soda lake (Jung et al., 2013). We noted however that FPMT worked better in habitats offering flat surfaces, such as soil or aquatic sediment. Here we introduce a new device more suitable for work with microbial communities associated with aquatic invertebrates. We term it I-tip (in situ cultivation by tip) because its main element is a standard micropipette tip. The tip is filled with microbeads and agar; for cultivation it is positioned such that its narrow end touches the surface of the animal. Microorganisms are expected to proliferate in the agar using nutrients diffusing from the

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environment, while the microbeads act as a barrier to prevent larger organisms from invading the space. To test the efficacy of the method, we applied the I-tip to endemic sponges from Lake Baikal, Russia. Sponges are well known to host a large community of microorganisms (Taylor et al., 2007), some of these microorganisms are probably host-specific (Schmitt et al., 2012; Taylor et al., 2004), and most remain uncultivated (Öztürk et al., 2013; Wang, 2006). We show that this method compares with conventional plating, and recovers a substantial portion of the microbial richness detected by molecular methods.

Material and methods I-tip design The I-tip method allows invertebrate-associated microorganisms to grow on solid support while being supplied with naturally occurring nutrients, signal molecules, and other growth factors from the environment. We used yellow pipette tips (Eppendorf, No 022493022, Germany) as the basic element of the device (Fig. 1.). The bottom part of the tip was filled with acid-washed glass beads to prevent invasion of larger organisms, and 70 μl of sterilized medium containing 0.7% (wt vol-1) agar was added to create space for microbial proliferation. Two sizes of glass beads were used individually for selective cultivation: smaller 60 to 100 μm in diameter (Sigma-Aldrich, No G4649, USA) and larger 150 to 212 μm in diameter (Sigma-Aldrich, No G1145, USA). After autoclaving the assembly, the wide end of the tip was aseptically sealed with a waterproof adhesive (3M, No 05260, USA) to prevent contamination from air and water.

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Media R2A (Difco, USA) medium was used for all cultivation experiments. The pH of the medium was adjusted to 8 by adding a few drops of 0.5 N NaOH. For the I-tip and standard cultivation method, 0.7 and 1.5% (wt vol-1) agar was added respectively.

Sample collection Endemic sponges Baicalospongia sp. and Lubomirskia baicalensis were collected by scuba diver in Lake Baikal at 10 m depth near Listvyanka (51°53'55.71"N, 105°03'52.44"E) in August 2012. The water temperature and pH at the sampling site was 10.2°C and 8.2 respectively. Sponges were kept in fresh lake water and transported to the laboratory and aquarium separately for molecular analyses and microbial cultivation within one hour of sampling.

Isolation of microorganisms with a standard cultivation method Upon transferring to the laboratory, a total of three and five individual Baicalospongia sp. and Lubomirskia baicalensis, respectively, were washed with sterile distilled water and subsampled for standard microbial cultivation. Subsamples of sponge tissue (20 g) from each specimen were homogenized using a Stomacher (Seward Medical, UK) in a sterilized plastic bag and then serially diluted. Aliquots (100 μL) from the serial dilutions were inoculated on R2A agar plates and incubated at 10°C. After 4 weeks, 43 colonies of different shape and color were selected for microbial identification; the remaining colonies

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were indistinguishable from these 43 morphotypes and were assumed to represent one of those types.

In situ cultivation with I-tip Sponges were kept in aquariums in the Baikal Museum, Russian Academy of Science, Listvyanka for in situ cultivation. Lake water was supplied continuously to the aquariums and the water temperature was controlled automatically at 10°C. For I-tip cultivation, three and five individuals of Baicalospongia sp. and Lubomirskia baicalensis, respectively, were placed separately in 30 (W) × 50 (L) × 40 (D) cm aquaria. Twenty I-tips were inserted into various parts of each specimen as a depth of 1.5 cm, and their position was checked every 48 hours. After 4 weeks of incubation, the sponges were taken out from each aquarium and then the I-tips were retrieved from the sponges. The collected I-tips were transported to the laboratory, the wide end of each I-tip was cut aseptically with a blade, and then the agar with the microorganisms was removed using a sterile loop. The agar material was added to 1 ml of sterile PBS buffer solution, mixed vigorously, and plated on R2A agar plates for incubation at 10°C for 3 weeks. After colony formation, a total of 103 colonies of different shape and color were used for microbial identification.

Identification of isolates Taxonomic identification was carried out by sequencing the 16S or 18S rRNA genes. The colony material was used directly as a template for PCR. The rRNA gene was amplified using the universal primers 27F (5’-AGAGTTTGATCCTGGCTCAG-3’) and 1492R (5’-

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GGTTACCTTGTTACGACTT-3’) for bacteria (Lane, 1991) and ITS1F (5’TCCGTAGGTGAACCTGCGCGG-3’) and ITS4 (5’-TCCTCCGCTTATTGATATGC-3’) for fungi (Nikolcheva et al., 2003) with a Hot Start Taq system (Takara, Japan). The PCR conditions for bacteria were an initial denaturing time of 10 min at 94°C, followed by 25 cycles of 30 s at 94°C, 30 s at 55°C, and 1 min at 72°C, with a final extension time of 5 min at 72°C. The conditions for fungi were an initial denaturing time of 10 min at 95°C, followed by 35 cycles of 30 s at 94°C, 45 s at 60°C, and 1 min at 72°C, and a final extension time of 7 min at 72°C. The PCR products were purified and sequenced commercially (Macrogen, Korea) by fluorescent dye terminator sequencing. The sequences were compared to databases in GenBank (http:// blast.ncbi.nih.gov/) using the EzTaxon-e (http://eztaxon-e.ezbiocloud.net, Korea) to identify the closest matching sequences.

Statistical analysis The Shannon-Weaver diversity and Margalef richness indices of isolated strains were calculated using standard statistical methods. The proportion of known and novel strains (below 97% 16S rRNA similarity to the closest known relative) of culture collections between standard cultivation and I-tip method was compared using Chi-square test.

DNA extraction and pyrosequencing To compare the cultivated bacterial diversity with microbial molecular signatures in the hosts, pyrosequencing was performed based on 16S rRNA genes from two species of sponges, Baicalospongia sp. and Lubomirskia baicalensis. For pyrosequencing analysis, sponges were collected on the same date. After transferring to the laboratory, samples were This article is protected by copyright. All rights reserved.

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washed with DNA-free water and freeze-dried. Genomic DNA from 0.25 g homogenized tissue of three sponges was extracted using a DNA kit (Mobio, USA) according to the manufacturer’s guidelines. The extracted genomic DNA was amplified as previously described (Chun et al., 2010). The DNA sequencing was performed by Chunlab Inc. (Korea) using the 454 GS FLX Titanium Sequencing System (Roche). Each pyrosequencing read was assigned taxonomically using CL community™ version 3.10 program (Chunlab Inc., Korea).

Results Bacterial diversity recovered by culture-dependent methods Using standard cultivation, 10 species from two phyla (Firmicutes and Gammaproteobacteria) were isolated from Baicalospongia sp. and eight species from three phyla (Betaproteobacteria, Firmicutes, and Gammaproteobacteria) were cultivated from Lubomirskia baicalensis (Fig. 2. a). Most isolated strains were representatives of Gammaproteobacteria (80 and 75% of all strains isolated from Baicalospongia sp. and Lubomirskia baicalensis respectively). Note only two species, Aeromonas salmonicida and Enterobacter amnigenus, were common in culture collections between the two sponges. I-tip cultivation led to the isolation of 19 species belonging to five phyla (Actinobacteria, Alphaproteobacteria, Betaproteobacteria, Firmicutes, and Gammaproteobacteria) from Baicalospongia sp. and 22 species from four phyla (Actinobacteria, Alphaproteobacteria, Firmicutes, and Gammaproteobacteria) from Lubomirskia baicalensis (Fig. 2. b). Seven species, Nocardia soli, Rhodococcus erythropolis, Microbacterium hydrocarbonoxydans, Brevundimonas bullata, Sphingomonas panni, Acinetobacter guillouiae, and Pseudomonas trivialis, were isolated from both sponges.

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Higher-diversity bacterial isolates based on the species level were cultivated in the Itip than in standard cultivation, as shown by comparison of two statistical analysis scores. Shannon-Weaver diversity and Margalef richness indices of bacterial species from I-tip were 26.8 and 7.64 while those from standard cultivation method were 13.1 and 4.19. The result of a chi-square analysis for association shows that the proportion of known and novel species differed with the two different isolation methods (x2 = 5.91, p < 0.05). Interestingly, Gammaproteobacteria were three-four times better represented on conventional Petri dishes than in the I-tip derived collection (Figs. 2). In contrast, Actinobacteria and Alphaproteobacteria, while common in the latter, were completely absent from the former. Actinobacteria alone were represented by 36 strains from three genera, Nocadia, Rhodococcus, and Microbacterium (Table 1). Among these, nine isolates were cultivated with I-tips with the larger glass beads (150~212 μm diameter) and 27 isolates with I-tips with the smaller beads (60~100 μm diameter).

Also notable is richness of the fungal collection. A total of 37 fungal stains were isolated from two sponge species; these represented six genera from two fungal phyla, Ascomycota and Basidiomycota. Among these, 22 isolates were cultivated by I-tips with the larger glass beads and eight isolates by I-tips with the smaller beads, while seven isolates were cultivated using the standard cultivation method. In total, two genera, Cladosporium and Rhodotorula, were isolated by standard cultivation and six genera, Cladosporium, Cryptococcus, Meyerozyma, Rhodosporidium, Rhodotorula, and Yarrowia, by the I-tip method. Only one fungal species, Rhodotorula mucilaginosa, was shared by the culture collections obtained by the two culture methods. Higher-diversity fungal and actinobacterial strains were cultivated in the I-tip than in standard cultivation, as shown by comparison of two diversity indices (Table 1). This article is protected by copyright. All rights reserved.

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16S rRNA gene survey by pyrosequencing From Baicalospongia sp. and Lubomirskia baicalensis, we analyzed respectively 7,496 and 13,964 reads, each typically around 500 nucleotides long. Seven phyla, Actinobacteria, Alphaproteobacteria, Firmicutes, Betaproteobacteria, Cyanobacteria, Planctomycetes, and Verrucomicrobia, were observed in Baicalospongia sp.; all but Verrucomicrobia were also detected in Lubomirskia baicalensis (Fig. 2. c). The best represented taxon was Cyanobacteria, which accounted for 70 to 78% of reads in both sponges; the least represented was Gammaproteobacteria, with less than 1% of reads in either species.

Discussion Molecular surveys show that sponges host a rich community of microorganisms (Hentschel et al., 2006; Imhoff & Stoehr, 2003); however, only a minor component of this richness has been cultivated (Wang, 2006). In this study, the application of conventional plating confirmed its limitations (Fig 2. a). Encouragingly, the results obtained using the I-tip method were quite different (Fig. 2. b). Diversity index of isolated bacterial species from I-tip was much higher than that obtained by standard cultivation (standard cultivation: 13.1, I-tip: 26.8). The main differences was that Gammaproteobacteria strains were dominant on standard Petri dishes (75% of all isolates), but they were a minor component of the I-tipderived collection (20%) (Figs. 2). In general, Gammaproteobacteria such as Pseudomonas are often isolated by conventional methods, even if comprising a small portion of the community under study. This is probably because Gammaproteobacteria usually grow rapidly on rich media typical of standard cultivation conditions. These fast-growing copiotrophic bacteria often have negative effects on the growth of more slowly growing

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oligotrophs, which results in culture collections with skewed species distribution (Jung et al., 2013). The relative lack of Gammaproteobacteria in I-tip-derived culture collection suggests that this method was less affected by this bias. In contrast to Gammaproteobacteria, Actinobacteria and Alphaproteobacteria appear enriched in the library of strains produced by the I-tip method (Fig. 2. b). We obtained 36 actinobacterial strains using the I-tip method, whereas none were obtained using the standard cultivation method even though their presence on or within sponges was indicated by pyrosequencing (Fig. 2. c). Such enrichment was expected as the bead barrier in the tips likely selects for filamentous organisms. Consistent with this, we also isolated more fungal strains using the I-tip method than using standard cultivation. Diversity and richness of the Itip-reared isolates were much higher than those obtained by standard cultivation (Table 1). Interestingly, bead size appears to be important in selecting for filamentous bacteria vs fungi: the majority of Actinobacteria were isolated from I-tips with smaller beads, whereas the opposite was found for fungi. This is not surprising since the smaller (50 to 100 μm in diameter) beads, if packed, produce 0.2 to 0.3 μm interstitial channels, which may be too small for eukaryotes (Gavrish et al., 2008; Polsinelli & Mazza, 1984). The apparent selectivity of I-tips for filamentous forms and identification of parameters favoring Actinobacteria points out to the potential advantages of the I-tip method in accessing the previously unavailable diversity of Actinobacteria, known to be a rich source of bioactive compounds. We noted an absence of Cyanobacteria, Planctomycetes, and Verrucomicrobia in our culture collection. It is possible that these microorganisms require specific growth conditions not met in our study. For example, suitable lighting conditions and specific media may be necessary to cultivate Cyanobacteria. Most of the Planctomycetes group that have been cultivated are slow growing facultative chemoorganotrophs that specialize in carbohydrate This article is protected by copyright. All rights reserved.

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metabolism (Buckley et al., 2006). The Verrucomicrobia phylum contains only a few described species isolated by particular cultivation approaches such as altering the composition of the growth medium (e.g., amending the growth medium with xylan) (Davis et al., 2005), using a substrate concentration that approximates naturally occurring conditions (Hahn et al., 2004), prolonging incubation times (Janssen et al., 2002), and application of repetitive incubation in diffusion chambers (Bollmann et al., 2007).

We recognize two limitations of our study. First, we did not pick isolates from the plates randomly and instead aimed at collecting as wide a morphological diversity as possible. Second, our samples of grown colonies are of unequal size (103 colonies from the Itip derived Petri dishes vs 43 from conventional plates). As a consequence, while all our data strongly indicate that the I-tip method recovers a larger microbial diversity, this is a semiquantitative observation; however, if we use metrics that are sample size independent, a more direct comparison may be possible. For example, the proportion of strains representing known strains vs novel species, contrasted by method, can indicate which method is likelier to produce more microbiological novelty based on new species level (below 97% 16S rRNA similarity to the closest known relative). This comparison shows that isolates from the I-tip method are more likely to be novel than those from the standard cultivation method, confirmed statistically using the Chi-square test (x2 = 5.91, p < 0.05). To summarize, the I-tip method seems to have produced a richer collection of more novel strains that is a better representation of the natural diversity. This is likely because the first step of the method, in situ cultivation, mimics natural conditions and allows for growth of only those species that are active in the environment. In addition, a low agar concentration (0.7%) would be a potentially positive factor for isolation of more diverse microorganisms. For example, semi-solid medium provides a wide range of oxygen concentrations, which This article is protected by copyright. All rights reserved.

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fulfills microaerophilic bacterial requirements (Ferrara-Guerrero & Bianchi, 1989). A low nutrient medium prevents dominance of fast-growing microorganisms, so it has been successful in isolating novel oligotrophic microorganisms from soil environment (Janssen et al., 2002). Although application of the I-tip method appears to have certain biases as indicated by the absence in the cultures of representatives of some microbial phyla, these biases appear different from those of other methods, making it complimentary with the existing cultivation technologies. Consequently, the I-tip method may be a useful addition to the arsenal of recently introduced novel methods for microbial isolation.

Acknowledgments This work was supported by a grant from Korea Polar Research Institute, KIOST, under project PE12040 and 2013 Research Grant from Kangwon National University (No. 120131195). We thank J. S. Owen for help with editing.

References Amann RI, Ludwig W & Schleifer KH (1995) Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol Rev 59: 143-169. Bollmann A, Lewis K & Epstein SS (2007) Incubation of environmental samples in a diffusion chamber increases the diversity of recovered isolates. Appl Environ Microb 73: 6386-6390.

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Buckley DH, Huangyutitham V, Nelson TA, Rumberger A & Thies JE (2006) Diversity of Planctomycetes in soil in relation to soil history and environmental heterogeneity. Appl Environ Microb 72: 4522-4531. Chun J, Kim KY, Lee JH & Choi Y (2010) The analysis of oral microbial communities of wild-type and toll-like receptor 2-deficient mice using a 454 GS FLX Titanium pyrosequencer. BMC Microbiol 10: 101-108. Davis KER, Joseph SJ & Janssen PH (2005) Effects of growth medium, inoculum size, and incubation time on culturability and isolation of soil bacteria. Appl Environ Microb 71: 826-834. Epstein SS, Lewis K, Nichols D & Gavrish E (2010) New approaches to microbial isolation. Manual of industrial microbiology and biotechnology, Vol. 4 (Baltz RH, Davies JE & Demain A, eds), pp 3–12. ASM, Washington DC. Ferrara-Guerrero MJ & Bianchi A (1989) Comparison of culture methods for enumeration of microaerophilic bacteria in marine sediments. Res Microbiol 140: 255-261. Gavrish E, Bollmann A, Epstein SS & Lewis K (2008) A trap for in situ cultivation of filamentous actinobacteria. J Microbiol Meth 72: 257-262. Hahn MW, Stadler P, Wu QL & Pöckl M (2004) The filtration-acclimatization method for isolation of an important fraction of the not readily cultivable bacteria. J Microbiol Meth 57: 379–390. Handelsman J (2004) Metagenomics: Application of genomics to uncultured microorganisms. Microbiol Mol Biol R 68: 669-685.

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Hentschel U, Usher KM & Taylor MW (2006) Marine sponges as microbial fermenters. FEMS Microbiol Ecol 55: 167-177. Imhoff JF & Stoehr R (2003) Sponge-associated bacteria: general overview and special aspects of bacteria associated with Halichondria panicea. Sponges (Porifera). (Mueller WEG, ed), pp. 35–57. Springer, New York. Janssen PH, Yates PS, Grinton BE, Taylor PM & Sait M (2002) Improved culturability of soil bacteria and isolation in pure culture of novel members of the divisions acidobacteria, actinobacteria, proteobacteria, and verrucomicrobia. Appl Environ Microb 68: 2391-2396. Jung D, Seo EY, Epstein SS, Joung Y, Yim JH, Lee H & Ahn TS (2013) A new method for microbial cultivation and its application to bacterial community analysis in Buus Nuur, Mongolia. Fund Appl Limnol 182: 171-181. Kaeberlein T, Lewis K & Epstein SS (2002) Isolating “uncultivable” microorganisms in pure culture in a simulated natural environment. Science 296: 1127–1129. Lane, DJ (1991) 16S/23S rRNA Sequencing. In Nucleic Acid Techniques in Bacterial Systematics. (Stackebrandt, E & Goodfellow, M, eds), pp 115-175. John Wiley & Sons, New York. Nikolcheva LG, Cockshutt AM & Barlocher F (2003) Determining diversity of freshwater fungi on decaying leaves: comparison of traditional and molecular approaches. Appl Environ Microbiol 69: 2548–2554. Öztürk B, de Jaeger L, Smidt H & Sipkema D (2013) Culture-dependent and independent approaches for identifying novel halogenases encoded by Crambe crambe (marine sponge) microbiota. Sci Rep 3: Article 2780.

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Polsinelli, M. & Mazza, PG (1984) Use of membrane filters for selective isolation of actinomycetes from soil. FEMS Microbiol Lett 22: 79–83. Schmitt S, Tsai P, Bell J, et al. (2012) Assessing the complex sponge microbiota: core, variable and species-specific bacterial communities in marine sponges. ISME J 6: 564– 576. Taylor MW, Radax R, Steger D & Wagner M (2007) Sponge-associated microorganisms: evolution, ecology, and biotechnological potential. Microbiol Mol Biol Rev 71: 295-347. Taylor MW, Schupp PJ, Dahllof I, Kjelleberg S & Steinberg PD (2004) Host specificity in marine sponge-associated bacteria, and potential implications for marine microbial diversity. Environ Microbiol 6: 121–130. Wang G (2006) Diversity and biotechnological potential of the sponge-associated microbial consortia. J Ind Microbiol Biot 33: 545-551. Zeng Y, Zou Y, Grebmeier J, He J & Zheng T (2012) Culture-independent and -dependent methods to investigate the diversity of planktonic bacteria in the northern Bering Sea. Polar Biol 35: 117-129.

Table Table 1. Number of isolated actinobacterial and fungal strains according to cultivation method and their diversity.

Genus

Standard

I-tip with

I-tip with

cultivation

big beads

small beads

Actinobacterial strains

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Norcardia

0

3

10

Rhodococcus

0

5

14

Microbacterium

0

1

3

Total strains

0

9

27

Diversity index

a

0

2.58 c

Richness index

b

0

0.56 c

Fungal strains

Cladosporium

3

0

0

Cryptococcus

0

3

1

Meyerozyma

0

8

3

Rhodosporidium

0

1

0

Rhodotorula

4

4

3

Yarrowia

0

6

1

Total strains

7

22

8

Diversity index

a

1.98

4.18 c

Richness index

b

0.51

1.18 c

a

Shannon-Weaver diversity index, calculated as follows: H = -∑ (pi) (lnpi), where pi is the

proportion of each phylogenetic group. b

Margalef richness index, calculated as follows: R = (S-1) ln(n), S is the number of genera

and n is the total number of strains. c

Diversity and richness indices of isolates from I-tip were calculated using combined

numbers from I-tip with large and small beads.

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Figure legends Fig. 1. Schematic diagram of the I-tip. Microorganisms along with nutrients, metabolites, and signal molecules will diffuse freely from the host environment into the agar. Fig. 2. Phylogenetic affiliations of bacterial isolates from Baikalian sponges by different cultivation methods, (a) standard cultivation, (b) I-tip on the basis of 16S rRNA gene sequences. Relative abundance of bacterial communities of Baikalian sponges by (c) pyrosequencing analysis. (L) = Baicalospongia sp., (R) = Lubomirskia baicalensis.

Fig. 1.

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Fig. 2. a (Combined fig. 2 is saved as ppt, tif and pdf files)

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Fig. 2. b

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Fig. 2. c

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Application of a new cultivation technology, I-tip, for studying microbial diversity in freshwater sponges of Lake Baikal, Russia.

One of the fundamental methods for cultivating bacterial strains is conventional plating on solid media, but this method does not reveal the true dive...
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