The difference between cellulolytic 'culturomes' and microbiomes inhabiting two contrasting soil types.

High-throughput 16S rRNA sequencing was performed to compare the microbiomes inhabiting two contrasting soil types-sod-podzolic soil and chernozem-and the corresponding culturome communities of potentially cellulolytic bacteria cultured on standard Hutchinson media. For each soil type, soil-specific microorganisms have been identified: for sod-podzolic soil-Acidothermus, Devosia, Phenylobacterium and Tumebacillus, and for chernozem soil-Sphingomonas, Bacillus and Blastococcus. The dynamics of differences between soil types for bulk soil samples and culturomes varied depending on the taxonomic level of the corresponding phylotypes. At high taxonomic levels, the number of common taxa between soil types increased more slowly for bulk soil than for culturome. Differences between soil-specific phylotypes were detected in bulk soil at a low taxonomic level (genus, species). A total of 13 phylotypes were represented both in soil and in culturome. No relationship was shown between the abundance of these phylotypes in soil and culturome.

Introduction

Cellulolytic microorganisms are one of the most popular subjects of scientific research for several reasons. On the one hand, they have an understandable pattern of nutritional requirements, which makes the selection of a nutritional medium more convenient. On the other hand, they play a crucial role in the process of soil formation and global carbon cycling. Modern metagenomic techniques provide new opportunities to investigate cellulolytic soil communities.

Five different North American forests were studied by Wilhelm with co-authors, who uncovered the biodiversity of lignocellulose-, hemicellulose- and cellulose-degrading bacteria and fungi in soil [1]. It was shown that bacteria from the Caulobacteraceae family were the most active decomposers and utilised all the proposed substrates. The cellulolytic component of the community was enriched with bacteria belonging to the Burkholderiaceae, Comamonadaceae and Oxalobacteraceae families. Cellulose and hemicellulose together were decomposed mostly by bacteria belonging to the Asticaccaulis, Cellvibrio, Janthinobacterium, Cytophaga and Salinibacterium genera. In addition, many previously uncultivated bacteria from relatively new phyla were detected, particularly bacteria from the genera Chtoniobacter, Opititus (phylum Verrucomicrobia) and Candidatus Saccharibacteria (TM7) [1].

The characteristic genera of relatively cold temperate forests (e.g., pine forests and mountain pine forests) were also found by Štursová and co-authors. Among them were Cytophaga, Pedobacter, Burkholderia, Gp1 and Gp2 acidobacteria, Asticaccaulis, Achromobacter, Mucilaginibacter, Herminiimonas, Collimonas and others [2].

A study by Eichorst and Kuske [3] covered the biodiversity of soil bacteria in five different ecosystems within both temperate and subtropical climatic zones. In agreement with previous studies, a prominent role of the Caulobacteriaceae and Burkholderiaceae families in xylan and cellulose degradation was also discovered in this research. Additionally, the list of cellulolytic bacteria was completed with the families Rhizobiales, Sphingobacteriales, Xanthomonadales and Myxococcales as well as unidentified and still uncultured representatives of Acidobacteria group I [3].

Other aspects of metagenomic studies involve large-insert metagenomic library analysis [4], shotgun targeted metagenomic sequencing [5, 6], or proteomics [7] and aim to study not only the taxonomic structure of the cellulolytic community but also the biodiversity of genes involved in decomposition. However, it is not necessary to dwell on them here, because their goals are beyond the scope of the current study.

The only drawback of metagenomic analysis is its dissociation from conventional microbiological methods, which in turn can provide comprehensive information on the physiology of the mineralisation processes in microbial cells. Sharing of data obtained using these methods simultaneously will update the available information on already cultivated microorganisms and emphasise the promising species that should be cultivated for future research.

Currently, there are plenty of works aimed at the comparative analysis of metagenomic data and various culturing methods. However, soil microbiology is still lacking examples of this kind of study. The application of these methods leads to the revision of the composition of the cultivated part of the soil, or even the worldwide microbiome. For example, Shade with co-authors demonstrated that a nutritional medium usually captures only a tiny part of the rhizosphere microbial community, containing in turn the minor phylotypes from the corresponding metagenomic library. Moreover, some species that were detected on a medium did not have corresponding signatures in the metagenomic analysis [8]. This study was conducted by using a very rich medium (specifically the rhizosphere isolation medium, RIM, which contains glucose, amino-acid mixture and vitamins, [9]), so the presence of many spore-forming copiotrophs could introduce significant inaccuracy in the biodiversity data. In this work, we used a relatively poor medium, in which cellulose filter paper was the only source of available carbon, so this should produce more selective conditions for bacterial growth than those in the aforementioned study.

Despite the apparent simplicity of the culturing procedure for cellulolytic bacteria, there is still a lack of comprehensive studies devoted to the biodiversity of microbiomes inhabiting several climatic zones. Known studies with similar goals focused on microbiomes of the Brazilian mangroves [10], sugarcane plots in Mexico [11], or paddy fields in Hainan [12], which all are very specific biomes. Considering the wide fluctuations in the composition of the soil microbiome depending on climate changes [13], we still have much to explore in terms of the biodiversity of the microbial consortia inhabiting climatically unstable subtropical and temperate regions. Thus, the main goal for the current study is simultaneous analysis of cellulolytic culturomes and the corresponding metagenomes in two contrasting biomes—sod-podzolic and chernozem soils—from the temperate and subtropical grasslands of Russia.

Materials and methods

Soil sample collection

Soil samples of sod-podzolic (SP) and chernozem (CZ) soil were collected in summer 2017 during expeditions to the Pskov (Pskov Research Institute of Agriculture, 57°50'44.2"N, 28°12'03.7"E) and Voronezh (Kamennaya Steppe reserve, 51°01'41.6"N, 40°43 '39.3"E) regions, respectively (). The director of Federal State Budget Scientific Institution “Kamennaya Steppe Experimental Forest District” and the director of the Federal State Budget Scientific Institution Pskov Research Institute of Agriculture gave permission for the sample collection. Soil samples were taken from the territories of the formerly sown areas from 10 different equidistant points from the upper soil layer (approximately 10 cm from the top of the soil profile). Finally, the selected samples were mixed and transported for laboratory research. Six replicates for each type of soil were formed.

Bacterial growth on nutritional medium

For cultivation, solid Hutchinson medium [14] with cellulose filters was used (grams/L: NaNO3: 2.5, FeCl3: 0.01, К2НРО4: 1.0, MgSO4·7H2O: 0.3, NaCl: 0.1 and CaCl2: 0.1; рН 7.2). The analysis was performed in six replicates for each type of soil. For both soils, the active growth of various types of bacteria was detected. After two weeks of cultivation, Petri dishes were washed out with sterile water, centrifuged, and subjected to DNA isolation. These samples were named gSP and gCZ accordingly.

DNA extraction and sequencing

The DNA was extracted from 0.2 g of soil using the PowerSoil DNA Isolation Kit (Mobio Laboratories, Solana Beach, CA, USA), which included a bead-beating step, according to the manufacturer’s specifications. Samples were homogenised with a Precellys 24 (Bertin Corp., USA) at 6.5 m/sec, twice for 30 s. The purity and quantity of DNA were tested by electrophoresis in 1% agarose in 0.5 × TAE buffer. DNA concentrations were measured at 260 nm using a SPECTROStar Nano (BMG LABTECH, Ortenberg, Germany).

The same DNA extraction procedure was applied to the culture plates. Microbial colonies were removed and solubilised in the extraction buffer, and DNA was extracted according to the manufacturer’s instructions. The average DNA yield was 2–5 μg DNA, with concentrations between 30 and 50 ng/μl. The purified DNA templates were amplified with the universal multiplex primers F515 5′-GTGCCAGCMGCCGCGGTAA-3′ and R806 5′-GGACTACVSGGGTATCTAAT-3′ [15] targeting the variable region V4 of bacterial and archaeal 16S rRNA genes, flanking an approximately 300-bp fragment of the gene, extended with service sequences containing linkers and barcodes according to Illumina technology. The PCR reactions were assembled in a 15-μl mix containing 1 U of Phusion Hot Start II High-Fidelity polymerase and 1X Phusion buffer (Thermo Fisher Scientific, USA), 5 pM of both primers, 10 ng of DNA, and 2 nM of each dNTP (Life Technologies, USA). The PCR thermal profile used was 94°С for 30 s, 50°С for 30 s, and 72°С for 30 s for 29 cycles. A final extension was performed at 72°С for 3 min. PCR products were purified and size selected with AM Pure XP (Beckman Coulter, USA). Further library preparation was done according to the manufacturer’s protocol with the MiSeq Reagent Kit Preparation Guide (Illumina, USA). Libraries were sequenced on an Illumina Miseq with a MiSeq® Reagent Kit v3 (2x300b) sequencing kit.

Data processing

Amplicon libraries of the 16S rRNA gene were processed using packages in R [16] and QIIME2 [17] software environments. RStudio [18] was used as the development environment for R. Raw sequence reads were trimmed and grouped into amplicon sequence variants (phylotypes) by use of the 'dada2' package [16]. The RDP classifier [19] based on Silva 132 [20] was used to classify assign taxonomic ranks to the phylotypes. The phylogenetic tree was built in the QIIME2 software environment in the SEPP package [21]. Data were normalised by a rarefaction algorithm according to the sample with the smallest number of readings for alpha and beta-diversity analysis. For differential analysis of phylotypes and quantitative metrics, the normalisation was performed by a variance stabilisation algorithm through the ‘DEseq2’ package [22]. To estimate the significance of differences between phylotypes previously normalised data were processed using the Wald test, with Benjamin-Hochberg false discovery rate (FDR) correction in the ‘DEseq2’ package [23]. The UniFrac, unweighted UniFrac [24], Bray-Curtis and MPD [25] algorithms were used as metrics for beta diversity. Beta-diversity data was graphically reproduced using PCoA [26]. Statistical analysis of beta-diversity was done by PERMANOVA [27] in the form of the adonis2 function (‘vegan’ package) [28]. The formula by Apostol and Mnatsakanian [29] in package ‘usedist’ [30] was used as an additional statistical approach to calculate the distance between the centres of mass (centroids) of the sample groups in the beta-diversity space. The function cophenetic.phylo from the ‘ape’ [31] package was used to agglomerate closely related taxa using single-linkage clustering. The reliability of the dependence of the representation of phylotypes in soil and culturomes was obtained through the Fisher test for the generalised linear model (‘glm’) [32]. The R packages ‘phyloseq’ [33], ‘ggpubr’ [34], ‘picante’ [35], ‘ggforce’ [36], ‘tidyverse’ [37], ‘ggtree’ [38], ‘ampvis2’ [39] and ‘rnaturalearth’ [40] were used for post-processing and visualisation of the obtained data.

Data deposition

All sequences were deposited to the SRA (NCBI) within the dataset: Submission ID: SUB5714186 and BioProject ID: PRJNA549392.

Results

Alpha diversity of soil microbiomes and culturomes

An amplicon library was obtained for bulk soil samples (246,527 sequences), and culturomes (397,307 sequences). Phylotype richness was higher in bulk soils compared to culturomes in both sample sets; sod-podzolic (SP) soil was more diverse than chernozem (CZ) soil (bulk soils: SP—1505, CZ—1286 and culturomes: SP—274, CZ—239, ). These tendencies can be clearly seen on the rarefaction curves, where culturome samples reached plateaus much earlier than bulk soil samples. The alpha diversity indices (evenness and richness) were similar for bulk soils as well as for culturomes (SP—424, CZ—358 and SP—65, CZ—54 correspondingly). The complete description of vertical soil structure as well as the agrochemical analysis can be seen in Table 1.

Table 1

The main physical and agrochemical characteristics of the analyzed soil samples.

Parameter	Units	Soil type
		Sod-podzolic	Chernozem
Particle size (mm) distribution
1–0.25	%	15.1	1.8
0.25–0.10	%	16.4	1.1
0.10–0.05	%	23.26	10.86
0.05–0.01	%	28.64	33.36
0.01–0.005	%	1.8	7.68
0.005–0.001	%	3.2	11.08
<0.001	%	11.6	34.12
Agro-chemical analysis
pH	pH units	6.05	7.32
N	%	0.22	0.38
P	mg/kg	85	121
K	mg/kg	60	155
C	%	2.48	8.75
Ca	mmol/100 g	3.25	30.62
Mg	mmol/100 g	2.45	3.82

A. Rarefaction curves for the culturomes (gCZ—Hutchinson medium culturome from the chernozem soil, gSP—Hutchinson medium culturome from the sod-podzolic soil) and bulk soil samples (bsCZ—bulk chernozem soil, bsSP—bulk sod-podzolic soil) B. Dependence of phylotype abundance in the culturome (Y-axis) as compared to bulk soil (X-axis). Color indicates different soil types.

Identification of the core and accessory components of soil microbiomes and culturomes

53 phylotypes for SP and 39 phylotypes for CZ were shared between bulk soil and culturome samples (). This set of phylotypes was dominated by Gammaproteobacteria (Massilia, Pseudoduganella), Actinobacteria (Streptomyces, Glycomyces, Pseudarthrobacter), Alphaproteobacteria (Bradyrhizobium, Devosia, Microvirga), Bacteroidetes (Niastella, Dyadobacter, Chitinophaga (predominated in SP)), Firmicutes (Bacillus(predominated in SP), Paenibacillus).No relationship was found between the representation of the phylotype in the bulk soil and culturome (; P-value for general linear F-test for SP was 0.43, for CZ– 0.5).

Soil specific taxa for bulk soil were phylotypes from the Verrucomicrobia (Candidatus Udaeobacter phylotypes), Actinobacteria (Microlunatus phylotypes, Acidothermus in SP, Blastococcus in CZ), Bacteroidetes (Chitinophagaceae phylotypes, Phylobactius bibliophyllum phylotypes) phylotypes in SP, Bacillus in CZ, Acidobacteria (RB41 phylotypes, Bryobacter phylotypes, Candidatus_Solibacter phylotypes in SP), Entotheonellaeota (specific for CZ) and Alphaproteobacteria (Pseudolabrys phylotypes, Sphingobacteriales phylotypes in CZ; ). Significant increases of 295 phylotypes for SP and 213 for CZ were shown. Differences between soil specific phylotypes were manifested in bulk soil at a low taxonomic level (genus, species). Sod-podzolic soil as compared to the chernozem was characterised by the high values of the species abundance variance within a certain genus.

The comparison of phylotype abundances for sod-podzolic soil samples (SP) vs chernozem samples (CZ) in bulk soil (soil) and culturomes (medium) samples.

Only the significant differences (padj < 0.05) within the dominant phylotypes are shown (baseMean > 60). Heat map legend meaning: increase of the phylotype abundance in CZ—red, SP—blue). The grey colour is for the phylotypes, which are absent either in bulk soil or culturome. Phylotypes not represented either in the bulk soil or in culturome are indicated in grey. CZ—chernozem soil and SP—sod-podzolic soil.

The number of the common phylotypes between the culturomes derived from two soil types was substantially lower compared to bulk soil samples. The culturomes were enriched with Proteobacteria and Actinobacteria and to a lesser extent with Firmicutes and Bacteroidetes. As opposed to bulk soil samples, in culturomes, the presence of the phylum Acidobacteria, Entotheonellaeota was not shown, and archaea were not represented. The representatives of the genus Streptomyces predominated among the detected actinobacteria. Verrucomicrobia was represented only by the Verrucomicrobiaceae family, while the representatives of Chthoniobacteraceae prevailed in the soil samples. The maximal relative abundances were detected for Pseudoduganella, Pseudoxanthomonas, Massilia (Gammaproteobacteria), Streptomyces and Glycomyces (Actinobacteria) in CZ and Streptomyces (Actinobacteria), Chitinophaga (Bacteroidetes), Massilia aerilata, Variovorax paradoxus and Pseudomonas (Gammaproteobacteria) in SP.

Beta-diversity of soil microbiomes and culturomes

In both culturome and bulk soils, chernozem (CZ) and sod-podzolic (SP) soil samples were significantly separated according to beta diversity metrics (PERMANOVA for bulk soils by Bray-Curtis: R2 = 0.75, p-value = 0.003; culturome R2 = 0.39, p-value = 0.004; the significant difference (p-value < 0.05) by weighted/unweighted UniFrac, mean pairwise distance, ).

PCoA ordination plots for various beta diversity metrics.

A. Bray-Curtis B. unweighted UniFrac C. weighted UniFrac. gCZ—Hutchinson medium culturome from the chernozem soil, gSP—Hutchinson medium culturome from the sod-podzolic soil, bsCZ—bulk chernozem soil, bsSP—bulk sod-podzolic soil.

Bulk soil samples were generally more diverse in terms of beta-diversity as compared to the corresponding culturomes. The distance between the centroids (the centres of the distributions) belonging to bulk soil samples was higher on the species and genus levels than for culturomes (0.241 for bulk soil samples and 0.155 for culturomes). This tendency reduces and finally turns to the opposite direction when the phylotypes are joined to the higher taxonomic ranks (e.g. on the family level, the numbers of common phylotypes were 72 for bulk soil and 17 for culturomes; the values of the distances between centroids were 0.059 and 0.09 respectively). The dynamics of the discussed changes can be seen more clearly using a graphical representation in Fig 4. The analysis was built on the calculation of the percent of the common tree leaves (tree tips) between the compared samples. The linear trend in the reduction of this value (ANOVA p-value < 0.05) was revealed when moving from the lowest to the highest taxonomic levels and until the number of the tree tips has reached 40–60. The linear regression differences expressed in the values of the line inclination (47.23 for bulk soil samples and 98.27 for culturomes) are statistically significant (p-value < 0.0001).

Fig 4

Percentage of the common phylotypes inhabiting sod-podzolic soil samples (SP) and chernozem samples (CZ) measured by the calculation of the tips (leaves) of the corresponding phylogenetic trees.

A linear trend is shown by the regression line for bulk soil samples as well as for the culturomes.

Discussion

In this work, a mixed culturome and 16S amplicon approach was used to identify the soil microbiome, which allows quick screening of the microbial community from a solid nutrient medium. This combination is quite rare in the literature [41], because usually the studies are aimed at the selection and identification of individual representatives of the microbiome selected on a nutrient medium.

High inconsistency in the NGS profiles for bulk soil samples and culturomes was shown, particularly, significant number of microorganisms from the culturome were absent in the bulk soil. Moreover, no linear relationship was found in phylotypes’ abundances between bulk soil samples and culturomes. This phenomenon was discussed previously by Shade and co-authors for the rhizosphere bacterial communities [8]. Probably, the most feasible source for the culturable part of the metagenome in this case is the microbial “seed bank” [42]. Other authors comparing culturome and NGS-based methods for capturing biodiversity also described a similar phenomenon. In a study of bottom sediment bacteria [43], using the enrichment method, the number of identified phylotypes increased by 16%, while the rarefaction curves almost reached the asymptote, as in our study. In a cultural study aimed at studying the soil microbiome of The Atacama Desert [41], some of the isolates were also not identified with 16S metabarcoding. In a study of the culture of the gut mouse microbiome [44], it was shown that only an insignificant part of the culture and the 16S microbiome coincide, while the culture-specific phylotypes are largely associated precisely with the functional activity of the microbiome.

However, the observed tendencies in phylotypes’ distribution might be caused by the insufficient sequencing depth for bulk soil samples. So, an increase in the sample number will balance the community composition.

The difference in the taxonomic structure is characteristic when comparing the microbiome in different soil types. In our work, it was shown that the differentiation between contrast soil types for a culturome begins to appear at a higher taxonomic level than for bulk soils. This can be attributed to the fact that the selection acting while seeding microorganisms on a solid medium is manifested at a high taxonomic level. At the same time, for many microorganisms in bulk soil, soil specificity was characteristic precisely at a low taxonomic level. It should be noted that there is a significant difference between the microbiomes of SP and CZ, which begins to manifest itself more strongly when growing on a solid nutrient medium that selects cellulolytic microorganisms in the soil. The results obtained may also indicate that part of the community with a low representation, but metabolically specific plays a significant role in the difference between the soil types.

Сulturomes were dominated by bacteria that seemed to have copiotrophic lifestyles, e.g., some groups of beta-, gamma-, and alpha-proteobacteria, belonging to the genera Massilia (also known as root-colonising microbes), Pseudomonas and bacteria from the family Xanthomonadaceae and Rhizobiaceae. Here we should also add actinobacteria from the genus Glycomyces, which were associated with plant roots [45]. These bacteria can play the role of an associated microbiota for the slow-growing and potentially cellulose-degrading bacteria, e.g. from the genus Caulobacter [46].

A noticeable group of bacteria within culturomes could be attributed to the cellulolytic community. Among the candidates for utilisation of complex soil polymers were bacteria belonging to the genera Dyadobacter [47], Chitinophaga, Niastella [48], Flavobacterium [49], Cellvibrio [50], Steroidobacter [51], Stenotrophomonas [52], Myxococcus [53], Variovorax [54], Paenibacillus (particularly P. polymyxa) [55], Cohnella (particularly C. panacarvi, which was observed in the analysed culturomes) [56], Streptomyces [57], Achromobacter [58] and Sphingomonas.

The microbiomes of bulk soil samples showed greater diversity than those of culturomes. Most of the species inhabiting soil microbiomes are unculturable, so little is known about their morphological features and metabolic capacities. Only a few bacteria can be partially characterised in this respect. Particularly, bacteria from the genus Rubrobacter dominated chernozem microbiomes. This bacterium, together with the large group of Gaiellales representatives are reported to be thermophiles, and some of them are involved in the degradation of xylan—a member of the hemicellulose group [59]. The potential for hydrolytic activity, particularly beta-glucosidase activity, was also shown for bacteria belonging to the genera Microlunatus [60] and Kribbella. In particular, K. jejuensis was mentioned for its utilisation of xylan and cellobiose [61].

All other bacteria, as well as archaea, belonged to phylogenetic groups that were discovered in the last two decades, among them were Verrucomicrobia, Thaumarchaeota, and Acidobacteria. There is still a lack of information on their biology because many of them avoid cultivation. The newest publications showed that it might be the consequence of auxotrophy [62] and potentially obligate symbiotic strategies of their lifestyle. Many of them are known to be ubiquitous and widespread soil bacteria, including those from the list, namely the genera RB41, Udaeobacter, and Nitrososphaera. The last one is an example of the unique ecological niche occupied by soil archaea—the indispensable link in the soil nitrogen cycle [63].

Conclusion

The study of microbiomes together with the cultivated cellulolytic communities of two contrasting soil types was performed by using 16S rRNA phylogenetic typing. Differences in community structure between the studied soil types for bulk soils and culturomes showed that only a small part of the cellulolytic community of the culturome is identified in bulk soil. The soil- and culturome- specific soil microbiome communities were specified. The opportunity to distinguish these groups proved to be very useful in studying the soil microbiome, which tends to be one of the most complex scientific subjects to date. Its complexity obliges the use of many cross-sections of biodiversity, e.g. differential DNA extraction, SIP or transcriptome analyses, which might be used to extend the current study in the future.

Sampling sites visualized on a map.

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Core phylotypes for bulk soil and culturome samples.

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3 Mar 2020

PONE-D-19-26379

The difference between cellulolytic ‘culturomes’ and microbiomes inhabiting two contrasting soil types

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Reviewer #1: In general, both culture independent, e.g. -Omics approaches and culture dependent methods have their own advantages and drawbacks. The intention of the current study is to integrate the advantages from both methods to reveal the difference of microbial community from two distinct types of soil and the functional groups associated with the digestion of cellulose. However, based on the information reported in the manuscript, many important components in a high quality scientific article such as the experimental design of cultivation, the purpose of using cellulose filters as source of cellulose, sampling plan, the association between the degradation of cellulose and types of soil, and methods to visualize beta diversity are not well stated. Instead, some paragraphs in the section of Introduction describing the results of metagenomic studies seem redundant.

The other concern is that the truly important findings are obscure. As stated in the manuscript, the main goal of the current study is simultaneous analysis of cellulolytic culturomes and the corresponding metagenomes in two contrasting biomes from the temperate and sub-tropical grasslands of Russia. However, the description of the results are too restricted to the description of the dominant taxa and the variation of diversity indices. The connection with the types of soils and causes of the differences in community composition is less stated and discussed in the manuscript. If the authors can link or explain the differences of microbial community composition with types of soils or environmental factors, the results will be more interesting. I also wonder the main findings or differences between the current study and published data.

Finally, the procedures and duration of cultivation were not declared in the manuscript. The authors only compared the variation between culturome and bulk soil at one point in time. If the processes associated with cellulose degradation in the cultivation and community variation in time-series could be revealed, the results will become more abundant, raising the scientific importance of this manuscript.

Here are few comments which you might wish to consider.

1. In Line 34: is there a name of genus missed after Candidatus?

2. It will be more understandable, if the style to present number is uniform in the manuscript. For example, in the text, the decimal point is indicated by a period, but in the tables, comma is used to indicate decimal place.

3. In the section of Materials and methods, the description of sampling sites and sampling strategy is unclear. I cannot understand how many samples collected in total and whether both types of soils were sampled from both Pskov and Voronezh. Based on the results present in Fig. 2, it seems that 6 samples were taken from SP and CZ soils, respectively. However, it is unclear whether for each soil type, 3 replicates were taken from Pskov and the other three were from Voronezh, or the 6 replicates were taken from the same region. I also wonder why you said soil samples were taken from 10 different equidistant points in line 115 and in the next sentence you said that “In total, 6 replicates for each type of soil were formed”. Does it mean that each replicate in each type of soil includes 10 samples from the depth of 10 cm? It will be more understandable, if you can provide a sample list which includes the coordination of sampling sites, soil type, and sampling depth, or provide a map indicating sampling sites.

4. In the part of “Bacterial Growth on nutritional medium”, I would like to know about more detail. It is not clear liquid or solid medium used for cultivation. Additionally, it is not clear the amount of soil used to cultivate and the duration of cultivation either. Please provide more information in this part.

5. In Line 175: Does the Dice methods mean “Sørensen–Dice coefficient”?

6. Please use PD whole tree and observed otus instead of the methods “PD_whole_tree” and “observed_otus” of QIIME.

7. Please uniform the usage of indices or indexes.

8. In the section of “Materials and methods”, you mentioned the indices of evenness including Faith’s index and Shannon evenness, but in “Results”, you did not show values of Faith’s index. Does PD_whole_tree means Faith’s index? If so, please uniform the name of index.

9. The authors did not state the methods for the ordination of Bray-curtis and Dice diversity estimates in Fig. 1.

10. For Fig. 2, it is suggested to use percentage instead of number of seqs.

Reviewer #2: The difference between cellulolytic ‘culturomes’ and microbiomes inhabiting two contrasting soil types

In all, attempt is good, isolating and culturing is still an important part of research. Scientific community can be happy with new isolates that can degrade cellulose. I only don’t understand the extensive comparisons of alpha and beta diversity of the culturomics with the total microbiome. It is all so logic these differences, comparing apples with peares. One or two sentences say as much. I would re-focus the results and discussion section, what was the real purpose of the study?

Line 34: genus Candidatus? Wrong..

The authors use a very selective culture media. Why they draw such comparisons between culturome phylotypes and core community? It is logic the communities differ..

Introduction

Line 48-49: a reason that media selection is straightforward, does it makes this a popular research subject? A bit weird reasoning. Sentences 50, 51 do not really connect.

Line 58: entire biodiversity: is a bit exaggerated… ‘entire’, reword. Line 59: they used 13C-SIP?

Line 82 and further: I appreciate the intend for ‘culturomics’, there is no need to defend this approach as being ‘old school’. It serves its purpose and NGS for other purpose so they can perfectly complement each other, depending on the research question. Line 87-88: combining culturing techniques with NGS, I agree it declines, but still I would not call it very few examples.

Table 1: interpret the particle size distribution please, so sandy soil? Which soil classification type? pH in water? N, P, K total or extractable elements? Many information lacking…

Line 145: picoM of primers, is little? Usually 200 nM of primers, and 200 µM of dNTPs instead of 2 nM??

Line 166: which database was used for classification and which version?

Line 164: why de novo, is not recommended by Qiime, open ref-based yes.

Line 174: beta-diversity in PAST3, bray Curtis? What about normalisation? What about other Unifrac based methods?

Line 186: MEGA X needs a reference

Line 197: wrong: the PD whole tree of culturomes cannot exceed those for bulk soils.

Table 2: in header, DP and in text, and legend SP? I cannot follow this.

Line 198: substantial decrease, significant?

Line 200: had lower, statistics done? No..

Line 216-217: so culturomics community is similar to whole bulk soil microbiome, based on presence/absence of OTUs, species, which taxonomic level similar? Hard to believe similar in species…Next line 217: they are again separate compact clusters, which statistics applied?

Line 228: OTUs which amounts…, amounts poor English, proportions, relative abundance?

Line 231: qualitative is usually which OTUs, quantitative: how much, RA of the OTUs

Line 235: explain this sentence again, not clear. In which both cases?

Line 280: I don’t know how useful it is to calculate simper analyses on cultured bacteria versus total soil sample sequencing… actually this remark holds for the whole results description. Apples comparing with pears. Keep culturomics to one paragraph, and total community seq to other. Make comparisons between the total and cultured collection, ok, but not in terms of diversity please.

Line 315-316: very logic cultured collection differs in diversity from total microbiome community composition, and in taxonomic composition and in core microbiome…

Line 318-319: very logic conclusion, no study needed for this

Line 320: might be the consequence, really might, it is obvious the consequence of…

Line 321: tiny portion… subset of…

Line 324-326: or did you sequence artifacts?

Line 328-331: again all very logic. I liked the idea of culturomics, but the results description and discussion is so straightforward and disappointing. A different angle of discussion could have been followed here, what was the real purpose of the study, isolating more cellulolytic bacteria to study their function? There is no use to extensively compare beta and alpha diversity of culturomics with total microbiome sequencing, mention it in one sentence.

Rewrite some paragraphs in the discussion. Find the message you want to give to the readers.

**********

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Reviewer #1: Yes: Tzu-Hsuan Tu

Reviewer #2: Yes: Sofie Thijs

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22 Jul 2020

Reviewer #1:

At the request of reviwer #2, the structure of the work was significantly revised. Data analysis with MiSeq was re-done, the results were rewritten, the discussion was partially rewritten.

1. In Line 34: is there a name of genus missed after Candidatus?

This part of the text was rewritten.

2. It will be more understandable, if the style to present number is uniform in the manuscript. For example, in the text, the decimal point is indicated by a period, but in the tables, comma is used to indicate decimal place.

Fixed

3. In the section of Materials and methods, the description of sampling sites and sampling strategy is unclear. I cannot understand how many samples collected in total and whether both types of soils were sampled from both Pskov and Voronezh. Based on the results present in Fig. 2, it seems that 6 samples were taken from SP and CZ soils, respectively. However, it is unclear whether for each soil type, 3 replicates were taken from Pskov and the other three were from Voronezh, or the 6 replicates were taken from the same region. I also wonder why you said soil samples were taken from 10 different equidistant points in line 115 and in the next sentence you said that “In total, 6 replicates for each type of soil were formed”. Does it mean that each replicate in each type of soil includes 10 samples from the depth of 10 cm? It will be more understandable, if you can provide a sample list which includes the coordination of sampling sites, soil type, and sampling depth, or provide a map indicating sampling sites.

The information in the Materials and methods section was changed.

4. In the part of “Bacterial Growth on nutritional medium”, I would like to know about more detail. It is not clear liquid or solid medium used for cultivation. Additionally, it is not clear the amount of soil used to cultivate and the duration of cultivation either. Please provide more information in this part.

Explanation was added

5. In Line 175: Does the Dice methods mean “Sørensen–Dice coefficient”?

Beta and alpha diversity metrics were recalculated using UniFrac, unweighted UniFrac and Bray-Curtis metrics.

6. Please use PD whole tree and observed otus instead of the methods “PD_whole_tree” and “observed_otus” of QIIME.

Faith Index did not use in the revised version of the article?

7. Please uniform the usage of indices or indexes.

Thank. Fixed

8. In the section of “Materials and methods”, you mentioned the indices of evenness including Faith’s index and Shannon evenness, but in “Results”, you did not show values of Faith’s index. Does PD_whole_tree means Faith’s index? If so, please uniform the name of index.

this part of the work was changed, according to the request of the second reviewer,

9. The authors did not state the methods for the ordination of Bray-curtis and Dice diversity estimates in Fig. 1.

Fixed

10. For Fig. 2, it is suggested to use percentage instead of number of seqs.

This figure have replased.

Reviewer #2:

Introduction

Line 48-49: a reason that media selection is straightforward, does it makes this a popular research subject?

Я не знаю что здесь ответить..

This medium is often used in the institute of agricultural microbiology, where this work was done. We are looking forward to compare the obtained metagenomic data with the previous research. This medium also known to be standard for the culturing of cellulolytic microorganisms, e.g. https://sfamjournals.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1365-2672.1997.tb03299.x

A bit weird reasoning. Sentences 50, 51 do not really connect.

Line 58: entire biodiversity: is a bit exaggerated… ‘entire’, reword. Line 59: they used 13C-SIP?

This part of the introduction has been revised.

Line 82 and further: I appreciate the intend for ‘culturomics’, there is no need to defend this approach as being ‘old school’. It serves its purpose and NGS for other purpose so they can perfectly complement each other, depending on the research question. Line 87-88: combining culturing techniques with NGS, I agree it declines, but still I would not call it very few examples.

Fixed. Added to the discussion are examples of works comparing the cultural approach and the results of NGS

Table 1: interpret the particle size distribution please, so sandy soil? Which soil classification type? pH in water? N, P, K total or extractable elements? Many information lacking…

P2O5 and K2O by Machigin method, N – total. pH – in water.

Line 145: picoM of primers, is little? Usually 200 nM of primers, and 200 µM of dNTPs instead of 2 nM??

5 pM is a сommonly used concentration according to Illumina MiSeq technology standarts a

Line 166: which database was used for classification and which version?

Data analysis with Illumina MiSeq was redone again using a different pipeline, more correct for this work. The taxonomic classification of phylotypes was determined using the RDP classifier based on Silva 132

Line 164: why de novo, is not recommended by Qiime, open ref-based yes.

This part of analysis has been redone

The whole analysis was done again by use of database-independent methods for out-picking

Line 174: beta-diversity in PAST3, bray Curtis? What about normalisation? What about other Unifrac based methods?

Redone. The main emphasis is on phylogenetic metrics.

Line 186: MEGA X needs a reference

This part has been changed

Line 197: wrong: the PD whole tree of culturomes cannot exceed those for bulk soils.

Thank

Table 2: in header, DP and in text, and legend SP? I cannot follow this.

This figure has been replased

Line 198: substantial decrease, significant?

We agree. Less emphasis is placed on alpha diversity indices.

Line 200: had lower, statistics done? No..

In the corrected work, where possible, statistics are added

Because according to the data obtained below, we cannot say with certainty that the depth of sequencing for bulk soils is sufficient; comparisons of alpha diversity indices are removed from the results. In addition, the results have been significantly reworked. Where possible, statistical processing has been added.

Line 216-217: so culturomics community is similar to whole bulk soil microbiome, based on presence/absence of OTUs, species, which taxonomic level similar? Hard to believe similar in species…Next

This part has been rewrited

line 217: they are again separate compact clusters, which statistics applied?

ANOVA-like test for beta-diversity results has been added

Line 228: OTUs which amounts…, amounts poor English, proportions, relative abundance?

In the previous version - relative abundance

Line 231: qualitative is usually which OTUs, quantitative: how much, RA of the OTUs

Thank

Line 235: explain this sentence again, not clear. In which both cases?

This part of analysis has been completely redone

Line 280: I don’t know how useful it is to calculate simper analyses on cultured bacteria versus total soil sample sequencing… actually this remark holds for the whole results description. Apples comparing with pears. Keep culturomics to one paragraph, and total community seq to other. Make comparisons between the total and cultured collection, ok, but not in terms of diversity please.

Direct comparisons of bulk soil and culturome were removed from the article. In addition, the discussion has been revised, comparisons have been removed based on the direct use of diversity indices.

Line 315-316: very logic cultured collection differs in diversity from total microbiome community composition, and in taxonomic composition and in core microbiome…

In the present version of the work, this part is significantly reduced

Line 318-319: very logic conclusion, no study needed for this

Thank

Line 320: might be the consequence, really might, it is obvious the consequence of…

Thank. This part of discussion have been completely rewritten

Line 321: tiny portion… subset of…

Line 324-326: or did you sequence artifacts?

We cannot deny that methodological artifacts could have a significant impact on the results of the work. In a discussion in several places, an attempt was made to point this out.

Line 328-331: again all very logic. I liked the idea of culturomics, but the results description and discussion is so straightforward and disappointing. A different angle of discussion could have been followed here, what was the real purpose of the study, isolating more cellulolytic bacteria to study their function? There is no use to extensively compare beta and alpha diversity of culturomics with total microbiome sequencing, mention it in one sentence.

Rewrite some paragraphs in the discussion. Find the message you want to give to the readers.

A significant part of the analysis has been redone. Your comments have been taken into account, if possible.

Submitted filename: Reviewer_resp.docx

Click here for additional data file.

13 Aug 2020

PONE-D-19-26379R1

The difference between cellulolytic ‘culturomes’ and microbiomes inhabiting two contrasting soil types

PLOS ONE

Dear Dr. Evdokimova,

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PLOS ONE

Additional Editor Comments (if provided):

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Some minor modifications were suggested by the reviewer, please revise accordingly. Best, CH

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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Reviewer #1: All comments have been addressed

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Reviewer #1: Yes

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Reviewer #1: Yes

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PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The manuscript has been major revised based on the comments from the two reviewers. However, there are still some ambiguous places have to be clarified and improved.

Introduction

In general, the introduction provided a complete review of cellulolytic soil communities from distinct climatic zones. The taxonomic information of the dominant cellulolytic decomposers was also described in detail. However, the overall description of these paragraphs seems not compact and little bit redundant. The main purpose of the current study is to connect metagenomic approach and cellulolytic culturomes to reveal whole microbial community composition and diversity of cellulolytic decomposers in the climatic unstable temperate and subtropical grassland. Therefore, it would be better to focus on this topic and not too disperse.

L48, L59, and L82: The way to describe the first author and the remaining coauthors should be unified.

L57: phylum name is not necessary in italic form.

L70: there is a space between comma and sequencing.

Materials and Methods

It is suggested to use map to illustrate the sampling locations. Currently, it is not clear where the samples were taken.

Results

Alpha diversity of soil microbiomes and culturomes

Currently, only number of observed OTUs was used to demonstrate alpha diversity in both soil microbiomes and culturome. However, the number of observed OTUs only reflects species diversity and can not reveal the information of evenness. Therefore, it would be better to demonstrate the pattern of alpha diversity not only in number of observed OTUs, but also in other indices such as Shannon or Simpson. According to the manuscript, the term OTU is not present, but, in Figure 1A, the title of Y axis is “Number of observed OTUs”. It would be better to use phylotypes to replace OTUs.

Identification of the core and accessory components of soil microbiomes and culturomes

It seems that the authors compare two different types of soil and culturomes together to find out the shared phylotypes. It is suggested that the comparison should be separated based on the types of soils. SP and its culturome, and CZ and its culturome should be compared separately because they are different types of soils and originally have their specific core microbiome. The low number of shared phylotypes may be resulted from different types of

Please provide the information of total number of phylotypes in each sample.

L198: It is not necessary to put the family name in italic form.

L198-L200: If you would like to list the family name after each genus, it would be better to add it after all genera and at the same taxonomic level, and not some in the level of family and some in the level of order.

L200: Pactrobactertes --> I have no idea what it is.

L203: Please modify “Verrucomicrobia phylum” to phylum Verrucomicrobia.

L204: Please modify “Candidatus Udaeobacter phylotypes” to Candidatus Udaeobacter phylotypes.

Fig. 3: It is suggested to add the stress values of NMDS analyses.

Discussion

L269-L272: Please add citation.

In the paragraph starts from L273 to L285, similar to the pattern in the section of Introduction, the described examples seem too disperse. It would be better to focus on similar habitats.

L289-L291: I can not fully understand the meaning of “the high degree of composition of culturome data and methodological artefacts” because the microbial composition of culturome seems simpler than bulk community. Therefore, I have no idea about the meaning of high degree of composition.

L289: Could you explain more about the meaning of “high resolution of the classical cultural approach”? Generally, classical cultural only can reflect a small proportion of microorganisms from field sample. Therefore, I have no idea why the classical cultural approach possesses high resolution.

L304: Modify Xanthomonadaceae and Rhizobiaceae family to the family Xanthomonadaceae and Rhizobiaceae

L313: Modify Paenibacillus polymyxa to P. polymyxa

L313: Modify Cohnella panacarvi to C. panacarvi

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

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7 Oct 2020

Below is a list of fixes based on the reviewer's answer:

L48, L59, and L82: The way to describe the first author and the remaining coauthors should be unified.

Done

L57: phylum name is not necessary in italic form.

Done

L70: there is a space between comma and sequencing.

Done

Materials and Methods

It is suggested to use map to illustrate the sampling locations. Currently, it is not clear where the samples were taken.

Map added to supplementary.

Results

Alpha diversity of soil microbiomes and culturomes

Currently, only number of observed OTUs was used to demonstrate alpha diversity in both soil microbiomes and culturome. However, the number of observed OTUs only reflects species diversity and can not reveal the information of evenness. Therefore, it would be better to demonstrate the pattern of alpha diversity not only in number of observed OTUs, but also in other indices such as Shannon or Simpson. According to the manuscript, the term OTU is not present, but, in Figure 1A, the title of Y axis is “Number of observed OTUs”. It would be better to use phylotypes to replace OTUs.

Fixed. Added Shannon Index

Identification of the core and accessory components of soil microbiomes and culturomes

It seems that the authors compare two different types of soil and culturomes together to find out the shared phylotypes. It is suggested that the comparison should be separated based on the types of soils. SP and its culturome, and CZ and its culturome should be compared separately because they are different types of soils and originally have their specific core microbiome. The low number of shared phylotypes may be resulted from different types of

Please provide the information of total number of phylotypes in each sample.

Thank you for noticing this annoying error in the article! The paragraph was rewritten, the picture was corrected, the statistics were redone. Table with general list of core phylotypes added to supplement. Fortunately, the conclusions from this work coincide with the previous(incorrect) paragraph results.

L198: It is not necessary to put the family name in italic form.

Fixed

L198-L200: If you would like to list the family name after each genus, it would be better to add it after all genera and at the same taxonomic level, and not some in the level of family and some in the level of order.

Fixed.

L200: Pactrobactertes --> I have no idea what it is.

Typo – fixed

L203: Please modify “Verrucomicrobia phylum” to phylum Verrucomicrobia.

Done.

L204: Please modify “Candidatus Udaeobacter phylotypes” to Candidatus Udaeobacter phylotypes.

Done.

Fig. 3: It is suggested to add the stress values of NMDS analyses.

Thank you for your comment! Out of habit, I used NMDS for this dataset. Naturally, this is completely wrong - in this work the groups are very different from each other. I changed it to a more suitable PCoA.

Discussion

L269-L272: Please add citation.

In the paragraph starts from L273 to L285, similar to the pattern in the section of Introduction, the described examples seem too disperse. It would be better to focus on similar habitats.

Added. I'm afraid I could not find articles combining both approaches for the soil types studied in the article.

L289-L291: I can not fully understand the meaning of “the high degree of composition of culturome data and methodological artefacts” because the microbial composition of culturome seems simpler than bulk community. Therefore, I have no idea about the meaning of high degree of composition.

I removed the text about a higher degree of compositionality for a culturome data. For this paper, I use the more common normalization approach, without using specialized approaches for compositional data. You are right that for this article it makes no sense to get into this topic.

L289: Could you explain more about the meaning of “high resolution of the classical cultural approach”? Generally, classical cultural only can reflect a small proportion of microorganisms from field sample. Therefore, I have no idea why the classical cultural approach possesses high resolution.

Rewrote, I hope this will make the thought more accessible to the reader.

L304: Modify Xanthomonadaceae and Rhizobiaceae family to the family Xanthomonadaceae and Rhizobiaceae

Fixed

L313: Modify Paenibacillus polymyxa to P. Polymyxa

Fixed

L313: Modify Cohnella panacarvi to C. Panacarvi

Done

Thanks for your time! Your work helped us a lot.

Submitted filename: Response.docx

Click here for additional data file.

27 Oct 2020

The difference between cellulolytic ‘culturomes’ and microbiomes inhabiting two contrasting soil types

PONE-D-19-26379R2

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Reviewer #1: All comments have been addressed

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The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

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**********

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The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

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11 Nov 2020

PONE-D-19-26379R2

The difference between cellulolytic ‘culturomes’ and microbiomes inhabiting two contrasting soil types

Dear Dr. Evdokimova:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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