Metagenome analyses reveal the influence of the inoculant Lactobacillus buchneri CD034 on the microbial community involved in grass ensiling

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Metagenome analyses reveal the influence of the inoculant Lactobacillus buchneri CD034 on the microbial community involved in grass silaging

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Felix G. Eikmeyer a , Petra Köfinger b , Andrea Poschenel e , Sebastian Jünemann d,f , Martha Zakrzewski d , Stefan Heinl c , Elisabeth Mayrhuber e , Reingard Grabherr c , Alfred Pühler a , Helmut Schwab b , Andreas Schlüter a,∗ a

Institute for Genome Research and Systems Biology, Center for Biotechnology, Bielefeld University, D-33594 Bielefeld, Germany Institute of Molecular Biotechnology, Graz University of Technology, Graz, Austria c CD Laboratory for Genetically Engineered Lactic Acid Bacteria, Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna, Austria d Computational Genomics, Center for Biotechnology (CeBiTec), Bielefeld University, Bielefeld, D-33594 Bielefeld, Germany e Lactosan GmbH & Co. KG, A-8605 Kapfenberg, Austria f Department of Periodontology, University of Münster, D-48419 Münster, Germany b

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Article history: Received 15 May 2013 Received in revised form 10 July 2013 Accepted 10 July 2013 Available online xxx

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Keywords: Ensiling community Metagenome 16S rRNA amplicon sequencing Lactobacillus Taxonomic profiling Fragment recruitment

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1. Introduction

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Silage is green fodder conserved by lactic acid fermentation performed by epiphytic lactic acid bacteria under anaerobic conditions. To improve the ensiling process and the quality of the resulting silage, starter cultures are added to the fresh forage. A detailed analysis of the microbial community playing a role in grass ensiling has been carried out by high throughput sequencing technologies. Moreover, the influence of the inoculant Lactobacillus buchneri CD034 on the microbial community composition was studied. For this purpose, grass was ensiled untreated or inoculated with L. buchneri CD034. The fresh forage as well as silages after 14 and 58 days of fermentation were characterized physicochemically. Characteristic silage conditions such as increased titers of lactic acid bacteria and higher concentrations of acetic acid were observed in the inoculated silage in comparison to the untreated samples. Taxonomic community profiles deduced from 16S rDNA amplicon sequences indicated that the relative abundance of Lactococci diminished in the course of fermentations and that the proportion of bacteria belonging to the phyla Proteobacteria and Bacteroidetes increased during the fermentation of untreated silage. In the inoculated silage, members of these phyla were repressed due to an increased abundance of Lactobacilli. In addition, metagenome analyses of silage samples confirmed taxonomic profiles based on 16S rDNA amplicons. Moreover, Lactobacillus plantarum, Lactobacillus brevis and Lactococcus lactis were found to be dominant species within silages as analyzed by means of fragment recruitments of metagenomic sequence reads on complete reference genome sequences. Fragment recruitments also provided clear evidence for the competitiveness of the inoculant strain L. buchneri CD034 during the fermentation of the inoculated silage. The inoculation strain was able to outcompete other community members and also affected physicochemical characteristics of the silage. © 2013 Published by Elsevier B.V.

Ensiling is a method for conservation of green fodder such as grass or corn by means of lactic acid fermentation by lactic acid bacteria. The resulting silage can afterwards be used as fodder for ruminants or as substrate for biogas production in anaerobic

∗ Corresponding author. Tel.: +49 0 521 106 8757; fax: +49 0 521 106 89041. E-mail address: [email protected] (A. Schlüter).

digesters. Since lactic acid bacteria are present on the green fodder as epiphytic bacteria, ensiling is a spontaneous and natural process as soon as anaerobic conditions prevail. Anaerobiosis and the subsequent formation of lactic and other volatile acids prevent growth of contaminating microorganisms such as yeasts, molds and other bacteria. Known members of microbial ensiling communities involved in lactic acid fermentation belong to the phylum Firmicutes and to the genera Lactobacillus, Lactococcus, Weissella and Leuconostoc (Woolford and Pahlow, 1998) comprising species such as Lactobacillus plantarum, Lactobacillus brevis, Lactococcis

0168-1656/$ – see front matter © 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.jbiotec.2013.07.021

Please cite this article in press as: Eikmeyer, F.G., et al., Metagenome analyses reveal the influence of the inoculant Lactobacillus buchneri CD034 on the microbial community involved in grass silaging. J. Biotechnol. (2013), http://dx.doi.org/10.1016/j.jbiotec.2013.07.021

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Table 1 Physico-chemical parameters of the raw material as well as of untreated and inoculated silage samples after 14 and 58 days of fermentation.

Dry matter [%] LABb [log cfua /g] Yeast [log cfua /g] pH Lactic acid [% of FMc ] Acetic acid [% of FMc ] Ethanol [% of FMc ] 1,2 Propanediol [% of FMc ] a b c

44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93

Raw material

Untreated silage, 14 d

Untreated silage, 58 d

Inoculated silage, 14 d

Inoculated silage, 58 d

36.82 5.86 5.82 6.28 0.08 0.12 0.00 0.00

30.91 8.72 6.49 4.59 6.02 1.37 0.56 0.00

31.55 8.43 5.58 4.41 7.77 1.88 0.49 0.00

29.51 9.95 4.78 4.81 4.35 2.46 0.58 0.44

28.97 9.79 2.60 4.94 2.63 5.17 0.98 1.77

Colony forming units. Lactic acid bacteria. Fresh matter.

lactis, Leuconostoc mesenteroides and Weissella cibaria (Langston and Bouma, 1960a,b; Pang et al., 2011). Species of the genera Enterococcus and Pediococcus are also described as members of microbial ensiling communities (Pahlow et al., 2003). To improve the ensiling process (e.g. acid formation rate, aerobic stability), silage additives such as chemical substances or starter cultures can be added. Homofermentative lactic acid bacteria were preferably used for this purpose as they allow for a rapid pH decrease. In 1996 Weinberg and Muck proposed that the usage of heterofermentative lactic acid bacteria could be beneficial as they also produce other volatile fatty acids (Weinberg and Muck, 1996) inhibiting growth of yeast and fungi after aeration. The production of acetic acid was supposed to be the biggest advantage because it features enhanced antifungal properties (Danner et al., 2003; Holzer et al., 2003). Since then, several studies have shown that the application of the heterofermentative lactic acid bacterium Lactobacillus buchneri for inoculation of grass, alfalfa, corn and small-grain silages indeed is beneficial regarding acid formation and aerobic stability (Holzer et al., 2003; Kleinschmit and Kung, 2006; Kung et al., 2003; Mari et al., 2009; Reich and Kung, 2010; Schmidt and Kung, 2010). L. buchneri CD034 has been isolated from stable silage and is supposed to positively influence the silaging process and the silage quality (Heinl et al., 2012). Within microbial communities (e.g. ensiling communities) bacteria interact with each other and with their environment. Microbial communities often contain a huge amount of bacteria which cannot be cultivated under laboratory conditions. Moreover, bacteria involved in ensiling such as Lactobacillus and Lactococcus strains are known to enter into the viable but non-culturable state (Oliver, 2005). Hence, such bacteria will be missed in analyses based on cultivation experiments (Singh et al., 2009; Warnecke and Hess, 2009). However, cultivation, identification and enumeration of lactic acid bacteria on MRS agar plates (Man et al., 1960) often are applied in ensiling experiments. Metagenomic approaches by means of direct DNA extraction and subsequent analyses are therefore necessary also to comprise the non-cultivable part of microbial communities. By applying denaturing gradient gel electrophoresis (DGGE) (Nishino and Touno, 2005) or sequencing of 16S rDNA fragments (McGarvey et al., 2013; Pang et al., 2011) of microbial ensiling communities it was possible to track changes within these communities and to identify dominant species. Moreover, specific primers have been developed to analyze the abundance of selected species (Klocke et al., 2006; Schmidt et al., 2008; Stevenson et al., 2006). However, both methods do not give detailed information on the composition of the complete microbial community. Hence, direct total community DNA or 16S rDNA amplicon sequencing and subsequent data analysis provides more comprehensive insights into the composition of the whole community of interest. Metagenome analyses were already applied to analyze microbial communities of agricultural importance in anaerobic digesters fermenting maize

silage as substrate (Jaenicke et al., 2011; Krause et al., 2008; Schlüter et al., 2008; Zakrzewski et al., 2012a,b). Better understanding of processes underlying silage formation and the effects of applied starter cultures, especially L. buchneri strains, might help to improve this process. Hence, we analyzed an ensiling process and corresponding microbial ensiling communities by means of a metagenomic approach (including metagenomic DNA analyses as well as 16S rDNA amplicon sequencing) to (1) follow up changes in taxonomic community profiles in the course of the ensiling process, (2) identify microorganisms that are dominantly involved in ensiling and to (3) elucidate the competitiveness of the inoculation strain L. buchneri CD034 within the epiphytic bacterial community.

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2. Materials & methods

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2.1. Ensiling of grass samples

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Silage was prepared from freshly cut grass (third cut) obtained from local farmers (Styria, Austria) in autumn 2010. The grass had a dry matter content of 36.82% (see Table 1). For inoculation 1 × 106 cells of L. buchneri CD034 per gram of raw material were sprayed onto the grass and thoroughly mixed. L. buchneri CD034 had previously been isolated from stable silage (Heinl et al., 2012). Two aliquots of approximately 4 kg of untreated and inoculated grass were compressed in plastic bags kept in plastic buckets. Afterwards, the bags were sealed and the buckets were closed and left at room temperature for 14 and 58 days respectively. 2.2. Physico-chemical analysis of grass silages Before the inoculation as well as after 14 and 58 days of fermentation physico-chemical properties of silage were determined. The pH values, concentrations of lactic acid bacteria (LAB) and yeasts as well as concentrations of lactic acid, acetic acid, ethanol and 1,2 propanediol were determined in an extract. The extract was prepared by homogenization of 30 g ensiled material with 100 ml of sterile physiological NaCl solution in a Stomacher (Seward Ltd., United Kingdom) and subsequent filtration. The pH was then determined directly in the extract (WTW inoLab 720, WTW GmbH, Germany). For enumeration of LAB and yeast the extract was used for decimal dilution series which were plated out onto MRS agar (Merck KGaA, Germany) for LAB enumeration (Man et al., 1960) as well as on YGC agar (Merck KGaA, Germany) for yeast and mold enumeration. The main products of lactic acid fermentation and pyruvate metabolism (lactic and acetic acid, ethanol, 1,2 propanediol) were determined by high-performance liquid chromatography (HPLC) (Agilent 1100 HPLC system, Agilent Technologies Deutschland GmbH, Germany) applying a cleared extract after Carrez precipitation. The dry matter content (DM) was

Please cite this article in press as: Eikmeyer, F.G., et al., Metagenome analyses reveal the influence of the inoculant Lactobacillus buchneri CD034 on the microbial community involved in grass silaging. J. Biotechnol. (2013), http://dx.doi.org/10.1016/j.jbiotec.2013.07.021

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determined by drying 30 g of fresh grass (105 ◦ C, 24 h) and subsequent calculation of the mass loss. 2.3. Metagenomic DNA isolation of microbial ensiling communities

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For isolation of DNA from ensiling microbial communities, cells were first harvested from the silage and then used for DNA isolation. 10× 100 g of silage were homogenized with 300 ml of physiological NaCl solution with 5 hits in 30 s. The created extracts were then filtered and centrifuged at 8000 rpm and 4 ◦ C for 15 min. The resulting cell pellets were then washed in physiological NaCl solution and again pelleted by centrifugation (15,000 × g, 20 min, 4 ◦ C). These pellets were suspended in 70 ml of physiological NaCl solution and the suspensions were divided into two parts. Cell pellets were again generated by centrifugation (3900 × g, 20 min, 4 ◦ C), frozen in dry ice and ice cold ethanol and kept until DNA isolation at −80 ◦ C. The disruption of cells originating from silage samples was done by the liquid nitrogen method. DNA was isolated by means of the Easy DNA Kit (Invitrogen, Cat. No. K1800-01) following the manufacturer’s protocol with prolonged incubation steps and an additional protein degradation step. 100 ␮l of isolated genomic DNA were mixed with 6.75 ml of extraction buffer (26), 50 ␮l lysozyme (100 mg/ml) and 50 ␮l proteinase K (20 mg/ml). The samples were incubated for 2 h at 37 ◦ C with gentle end-over-end inversion every 15 min. After centrifugation at 1500 × g for 10 min, the supernatant was collected and added to equal volumes of 24:1 chloroform/isoamyl alcohol. After centrifugation at 1500 × g for 20 min the aqueous phase was removed and DNA was precipitated using 0.6 volumes of isopropanol for 1 h at room temperature. The extracted nucleic acids were pelleted by centrifugation at 9300 × g for 20 min, two times ethanol-washed and then resuspended in sterile deionised water. The genomic DNA was additionally purified by anion exchange chromatography using the NucleoBond AX-G20 columns (Macherey und Nagel, Cat. No. 740 544) and then resuspended in TE buffer.

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2.4. High-throughput sequencing of metagenomic DNA

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Whole genome shotgun libraries were created based on the metagenomic DNA. Sequencing of these libraries was performed at the IIT GmbH (Bielefeld, Germany) by using a quarter pico titer plate applying the FLX Titanium chemistry as previously described by (Jaenicke et al., 2011).

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2.5. 16S rDNA amplicon generation and sequencing

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16S rDNA amplicons were created as described before (Zakrzewski et al., 2012a,b). In short a first PCR was performed with universal primers amplifying the V3 and V4 region of the 16S rRNA gene. Amplicons with the right size (480 bp) were extracted by gel electrophoresis and gel extraction. A second PCR was performed using these amplicons as templates and primers containing the universal primer sequence, multiplex identifier (MIDs) and 454 specific linker sequences. Amplicons were again checked for the right length and gel extracted prior to sequencing. For sequencing 109 molecules per sample were pooled and used for a quarter pico titer plate of 454 sequencing, applying FLX Titanium chemistry (IIT GmbH, Bielefeld, Germany).

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2.6. Bioinformatic analysis of sequencing data

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Metagenomic reads were processed with the MetaSAMS platform (Zakrzewski et al., 2012a,b). For taxonomic assignments of metagenomic reads the BLASTx approach of CARMA3 (Gerlach and Stoye, 2011) integrated in MetaSAMS was applied. Taxonomic

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assignments were visualized by means of Krona (Ondov et al., 2011). Sequencing reads gained by 16S rDNA amplicon sequencing were applied to different quality filtering steps. First, raw reads were screened for sequencing adapter sequences and the degenerated 3 and 5 amplification primers allowing for up to three and two errors. This was done using Cutadapt (Martin, 2011), requiring a minimum primer overlap of 10 bases and penalizing initial gaps in the semi-global alignment. Correctly detected sequences were trimmed off during this procedure while reads not matching the adapter and primer sequences were removed from further analysis. Next, reads containing ambiguously called bases (N) or having an average quality score below 20 were filtered out. Finally, putative chimeric reads were removed utilizing the UCHIME algorithm (Edgar et al., 2011). In order to correct potential sequencing errors all reads passing the quality criteria were applied to the Single Linkage Preclustering (SLP) method (Huse et al., 2010) implemented in mothur (Schloss et al., 2009). All subsequent analyses were performed on high quality reads de-noised by SLP. Clustering of operational taxonomic unit (OTU) was carried out using the complete linkage clustering approach implemented in ESPRIT (Sun et al., 2009) at a three percent edit distance threshold. Singleton OTUs were identified and excluded prior to the computation of diversity metrics. Simpson diversity indices (Magurran, 2010) were computed using the statistical software suite R v 2.9.10 (R Development Core Team, 2010) and the vegan R-package (Dixon, 2003). Finally, reads were taxonomically classified by means of the RDP classifier (Wang et al., 2007). Fragment recruitment was performed as described previously (Rusch et al., 2007). Metagenomic reads were aligned to complete bacterial genome sequences of the NCBI database by means of BLASTn (options “-dust yes–soft masking true–lcase masking–penalty -3–reward 2–xdrop gap 150”) (Altschul et al., 1990; Camacho et al., 2009) including 2500 reference sequences. Reads were used for further evaluation if at least 80% of a read were aligned to a subject sequence and if the identity (recalculated by dividing the number of identical bases by the read length) was at least 55%. Alignments were visualized by plotting the identity of the alignment against the alignment position on the reference sequence. The numbers of alignments plotted were normalized. Moreover, the numbers of hits with a certain identity were presented in a histogram to visualize the distribution of hit identities to overcome problems with samples overlaying each other within the fragment recruitment plot. Again, numbers were normalized. Finally, a best blast hit analysis was performed by determining the percentage of reads causing a best hit with an identity greater than or equal to 99%, with an identity smaller than 99% and greater or equal than 55% as well as for reads causing a hit with an identity less than 55% and reads causing no hits.

3. Results & discussion 3.1. Characterization of untreated and inoculated silage at the physico-chemical level To analyze the influence of L. buchneri CD034 on the microbial ensiling community and on silage formation, grass silages were prepared in lab scale. One batch was left untreated while the other was inoculated with L. buchneri CD034. Subsequently, the grass was air-tightly sealed and incubated for 58 days at room temperature. The starting material as well as silage samples were analyzed by physico-chemical methods after 14 and 58 days of fermentation in order to monitor the ensiling progress itself and changes between untreated and inoculated silage. Moreover, silage samples after 14 and 58 days of fermentation were also investigated by

Please cite this article in press as: Eikmeyer, F.G., et al., Metagenome analyses reveal the influence of the inoculant Lactobacillus buchneri CD034 on the microbial community involved in grass silaging. J. Biotechnol. (2013), http://dx.doi.org/10.1016/j.jbiotec.2013.07.021

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metagenomic approaches including 16S rDNA amplicon sequencing as well as whole metagenome sequencing approach to elucidate changes within the taxonomic composition of the ensiling microbial communities in dependence of fermentation time and inoculation. Previous ensiling experiments applying L. buchneri strains as inoculant showed that inoculation results in higher concentrations of acetic acid, ethanol and 1,2-propanediol as well as lower concentrations of yeast and subsequently to a better aerobic stability compared to untreated silages (Danner et al., 2003; Driehuis et al., 2001; Hu et al., 2009; Kleinschmit and Kung, 2006; Reich and Kung, 2010). Samples of this study therefore were analyzed to determine whether silages inoculated with L. buchneri CD034 also featured these characteristics. Results of these analyses are shown in Table 1. Microbiological methods indicated a higher amount of LAB and a lower amount of yeast in the inoculated silage samples. Moreover, changes in the acid composition were observed. The inoculated silage samples showed higher concentrations of acetic acid and lower concentrations of lactic acid. As the pKa of acetic acid (4.76) is higher than the pKa of lactic acid (3.90), changes in the acid composition were also reflected by a higher pH value within the inoculated samples. Measurements for 1,2propanediol and ethanol also showed higher concentrations for these alcohols in the inoculated silage samples. Hence, the previously reported effects of L. buchneri onto silage characteristics were also observed in this study. Considering the proposed metabolic pathways for lactic acid fermentation in L. buchneri CD034 (Heinl et al., 2012) and L. buchneri LMG6892 (Oude Elferink et al., 2001) observed differences between the silage samples can be related to the proposed metabolic products lactic and acetic acid, ethanol and 1,2-propanediol of L. buchneri strains. 3.2. Taxonomic characterization of the microbial communities of an untreated and an inoculated grass-silage by 16S rDNA analysis 16S rDNA amplicons covering the variable regions 3 and 4 were generated, sequenced and analyzed to trace changes in community structures in the course of ensiling and in dependence on inoculation. For each sample 53,164 to 83,423 sequences were obtained. After stringent filtering, about 30,000 reads per sample were classified by means of the RDP Classifier. More than 99% of the amplicon sequences were assigned to the superkingdom Bacteria while only small proportions were assigned to the superkingdom Archaea 84–90% of these reads assigned to the superkingdom Bacteria could be further assigned to a genus. The main characteristics of these analyses were found to be similar for all four samples (Fig. 1). The phylum Firmicutes was found to be the most abundant assigned phylum. Most of the reads assigned to the phylum Firmicutes were related to the order Lactobacillales comprising the three most prevalent genera Lactobacillus, Lactococcus and Weissella (Fig. 1). Moreover, 16S rDNA sequences were also assigned to 11 other phyla besides the phylum Firmicutes with Proteobacteria being the most abundant phylum (4.4–13.7%) including Enterobacteriaceae (1.3–4.3%) as the most abundant family. In addition to those genera of the phylum Firmicutes about 200 genera of other phyla were assigned within all samples. The abundance of 16S rDNA sequences assigned to the genera Lactobacillus, Lactococcus and Weissella as well as to phyla other than Firmicutes is similar for samples of untreated as well as inoculated silages after 14 days of fermentation (Fig. 1). During the fermentation of the untreated silage the quantity of sequences assigned to the genus Lactobacillus increased clearly while the quantity of sequences assigned to the genus Lactococcus decreased considerably. This observation is accompanied by an increased number of sequences assigned to phyla other than Firmicutes. In contrast, the abundance of assignments to these phyla within the

Fig. 1. Taxonomic profiles of ensiling microbial communities on the taxonomic ranks of phylum as well as genus for selected genera. Profiles shown here are based on 16S rDNA amplicon sequences and were classified applying the RDP Classifier after filtering.

silage sample inoculated with L. buchneri CD034 after 58 days of fermentation is decreased (Fig. 1) while sequences assigned to the genus Lactobacillus feature an increased abundance compared to the sample after 14 days of fermentation. This increase is even more pronounced comparing the samples of untreated and inoculated silage after 58 d of fermentation. Subsequently all 16S rDNA sequences were clustered to calculate the number of OTUs within the samples. Based on these data the species diversity was calculated by means of the Simpson diversity index (Table 2). Numbers of OTUs are quite similar with lower values for inoculated silage samples. This trend is also reflected by the Simpson indices. However, these results still indicate a respectable diversity within the microbial communities. 3.3. Metagenome analysis of the microbial communities residing in an untreated and an inoculated grass silage A whole metagenome shotgun sequencing project was then accomplished to gain deeper insights into the bacterial ensiling communities. Yields of the sequencing runs were found to be similar for all four samples (Table 3), generating about 200,000 sequence reads per sample. These metagenomic sequence reads were classified by applying CARMA3 (Gerlach and Stoye, 2011) to deduce taxonomic profiles of the microbial ensiling communities. The main characteristics of these obtained profiles were similar for all four samples. Fig. 2 exemplarily shows the taxonomic profile for the sample of untreated silage after 58 days of fermentation. This sample was chosen because it featured the greatest diversity (Simpson index 0.943). In total about 80% of all reads were taxonomically assigned by CARMA3. More than 99% of these reads were assigned Table 2 Diversity of ensiling microbial communities as determined by means of 16S rDNA amplicon sequencing and subsequent data analysis.

Untreated silage, 14 d Untreated silage, 58 d Inoculated silage, 14 d Inoculated silage, 58 d a b

OTUsa

Simpson indexb

1387 1387 1099 1111

0.917 0.943 0.915 0.815

Numbers of operational taxonomic units (OTUs) Diversity indices are shown.

Please cite this article in press as: Eikmeyer, F.G., et al., Metagenome analyses reveal the influence of the inoculant Lactobacillus buchneri CD034 on the microbial community involved in grass silaging. J. Biotechnol. (2013), http://dx.doi.org/10.1016/j.jbiotec.2013.07.021

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Table 3 Statistics of metagenome sequencing of untreated and inoculated silage samples after 14 and 58 days.

Untreated silage, 14 d Untreated silage, 58 d Inoculated silage, 14 d Inoculated silage, 58 d

351 352 353 354 355 356 357 358

Sequenced base pairs [bp]

Number of reads

Average read length [bp]

80,367,750 85,468,767 78,057,757 106,601,727

205,880 218,535 198,507 271,875

390.4 391.1 393.2 392.1

to the superkingdom Bacteria while only small proportions were assigned to the superkingdoms Archaea and Eukaryota. The phylum Firmicutes was again found to be the most abundant one comprising the four prevalent genera Lactobacillus, Lactococcus, Weissella and Leuconostoc (Fig. 2). Metagenomic reads were also assigned to 20 phyla (comprising 170 genera) besides the phylum Firmicutes. Assignments of metagenomic reads to the superkingdom Bacteria of the four samples were compared to analyze changes within

the microbial ensiling communities (Fig. 3). Observations based on these taxonomic profiles support the profiles based on 16S rDNA sequences. Lactobacillus, Lactococcus, Weissella and Leuconostoc could be identified as the main genera in the samples analyzed. The abundance of metagenomic sequence reads assigned to the genus Lactococcus decreased between days 14 and 58 of fermentation while the abundance of metagenomic sequence reads assigned to the genus Lactobacillus increased. This increase is more pronounced

Fig. 2. Taxonomic profile of a sample of untreated silage after 58 days of fermentation. Metagenomic reads were taxonomically classified by means of the BLASTx approach of CARMA3 and visualized by Krona.

Please cite this article in press as: Eikmeyer, F.G., et al., Metagenome analyses reveal the influence of the inoculant Lactobacillus buchneri CD034 on the microbial community involved in grass silaging. J. Biotechnol. (2013), http://dx.doi.org/10.1016/j.jbiotec.2013.07.021

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Fig. 3. Comparison of taxonomic profiles of ensiling microbial communities of untreated and inoculated silage after 14 and 58 days of fermentation. Metagenomic sequences were evaluated as described for Fig. 2. Profiles shown here only represent those reads assigned to the superkingdom Bacteria. 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386

for the inoculated silage. During the fermentation of the untreated silage the abundance of reads assigned to bacteria not belonging to the phylum Firmicutes also increased. Taxonomic profiles based on 16S rDNA amplicons as well as those based on the metagenomic sequences show the same trends regarding the composition of the microbial ensiling communities and are in accordance with existing knowledge (Woolford and Pahlow, 1998). Bacteria belonging to the phyla Proteobacteria and Bacteroidetes were able to proliferate during the fermentation of the untreated silage, although these silages featured lower pH-values and higher concentrations of lactic acid. Hence, low pH values alone do not necessarily reduce growth of potentially undesired bacteria. Inoculation of silage with L. buchneri CD034 strongly influences the microbial ensiling communities especially in later phases of the fermentation leading to a reduced abundance of Lactococci and bacteria not belonging to the phylum Firmicutes. Lowering or limiting of the amount of putative spoiling organisms in the silage has benefits regarding the quality of the silage. Inoculation of grass silage with L. buchneri CD034 seems to be a convenient treatment to outcompete other bacteria.

3.4. Fragment recruitments indicate the prevalence of L. buchneri CD034 in inoculated silage samples To gain further insight into changes within selected genera of lactic acid bacteria on the species level, BLASTn analyses of metagenomic reads of inoculated and untreated silage samples after 14 and 58 days of fermentation against all NCBI bacterial genomes were performed. 62 to 82% of sequence reads generated a hit against at least one reference sequence, while 36 to 68% of all reads generated hits with at least 95% sequence identity. Table 4 shows those reference genomes of prevalent species identified in the silage samples as determined by BLASTn analyses applying a sequence identity cutoff of 95%. A species was then considered as main species if a chromosomal reference sequence of a strain belonging to this species recruited at least 1% of all reads within at least two samples. All main species represent bacteria belonging to the phylum Firmicutes and to the genera Lactobacillus (L. plantarum, L. brevis, L. buchnerii), Lactococcus (L. lactis subsp. lactis) and Leuconostoc (L. citreum). Moreover, it was evaluated if reads which generate a hit on a reference sequence with at least 95% identity would also generate

Table 4 Prevalent speciesa present in different grass silage samples as determined by BLASTn analyze of metagenomic sequences against sequences of complete genome sequences. Species

Reference strain

Untreated silage 14 d

Untreated silage, 58 d

Inoculated silage, 14 d

Inoculated silage, 58 d

Lactobacillus plantarum Lactobacillus brevis Lactobacillus buchneri

L. plantarum ST-III L. brevis ATCC 367 L. buchneri CD034 L. buchneri NRRL B-30929 L. lactis subsp. lactis KF147 L. citreum KM20

16.3% 7.0% 0.2% 0.3% 16.0% 2.25%

16.1% 7.9% 0.2% 0.3% 4.7% 2.0%

9.7% 3.4% 15.5% 12.0% 14.8% 1.1%

6.3% 1.1% 51.5% 39.4% 1.7% 0.5%

Lactococcus lactis Leuconostoc citreum

a Results were evaluated for the percentage of metagenomic sequence reads generating hits with at least 95% sequence identity. Reference sequences recruiting at least 1% of reads within at least two samples were defined as main species.

Table 5 Differentiation specificity for the assignment of metagenomic reads to prevalent species by means of BLASTn analyses applying a 95% sequence identity cutoff.

Lactobacillus plantarum Lactobacillus brevis Lactobacillus buchneri Lactococcus lactis subsp. lactis Leuconostoc citreum KM20 a b

Untreated silage 14 d

Untreated silage, 58 d

Inoculated silage, 14 d

Inoculated silage, 58 d

Singlea

Singlea

Singlea

Singlea

32,836 14,040 16 32,460 4513

Multipleb 646 355 126 445 132

34,657 16,790 314 10,129 4221

Multipleb 589 420 121 137 95

18,956 6527 30,452 28,953 2089

Multipleb 328 240 326 465 88

16,751 2371 138,531 4517 1384

Multipleb 473 517 1565 86 54

Single hits: Reads showing only sequence identity of at least 95% to reference sequences of this species. Multiple hits. Reads showing also sequence identity of at least 95% to at least one reference sequence of another species.

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hits onto sequences of other species with a corresponding degree of identity (see Table 5). From these calculations it can be concluded that compared to the number of reads generating only a hit onto the selected reference genome or related strains, the number of reads also generating hits on sequences of other species with at least 95% sequence identity is low for L. buchneri CD034, L. plantarum ST-III, L. brevis ATCC 367, L. lactis subsp. lactis KF147 and L. citreum KM20. Hence, a sequence identity cutoff of 95% should be adequate to allow for differentiation of species by applying this method. Hits onto reference sequences were further evaluated by means of fragment recruitment plots, hit distribution plots and best hit per read plots (Fig. 4). The fragment recruitment and the hit distribution plots for the inoculation strain L. buchneri CD034 (Fig. 4A1 and A2) indicate that reads showing a high similarity to its genome are only present in the inoculated silage samples. These reads cover the whole chromosome sequence. Moreover, only a small number of reads from untreated silage samples featured a hit against this reference genome (Fig. 4A3). The abundance of reads causing a hit with an identity higher than or equal to 99% increases from 16 to 52% during the ongoing fermentation of the inoculated silage. Similar results were obtained for fragment recruitments onto the L. buchneri NRRL B-30929 chromosome although these hits show a lower degree of similarity (Fig. 4B3 and B2). The fragment recruitment plot also shows that not all regions of the chromosome (Heinl et al., 2012) could be covered by hits (Figure 4 B1), indicating differences in the genomes of L. buchneri CD034 (inoculation strain) and L. buchneri NRRL B-30929. For example, L. buchneri NRRL B30929 possesses prophage insertions that are missing in L. buchneri CD034 and these were not covered by metagenomic sequences. This result demonstrates that fragment recruitments allow for differentiation also at the strain level at least for the species L. buchneri. Another Lactobacillus strain described to be involved in ensiling is L. plantarum (Weinberg and Muck, 1996) which was also found to be present in the silages analyzed in this study. The best hit per read plot for L. plantarum subsp. plantarum ST-III shows that sequences featuring a high degree of similarity are present in all samples while the abundance of these reads is higher in the untreated silage samples (Fig. 4C3). Taxonomic characterization of ensiling microbial communities also identified Lactococcus as a main genus of ensiling communities. The fragment recruitment for L. lactis subsp. lactis KF147 shows that the abundance of matching reads showing a hit with high identity to this reference decreased during the progress of both fermentations (Figure 4D). Lactobacillus brevis was also described to be a common epiphytic Lactobacillus species. However, highly similar reads as compared to the L. brevis ATCC 367 chromosome (Figure 4E) were mainly present in the untreated silage samples. From these findings it can be concluded that L. buchneri CD034 was only present in the inoculated samples and that no other L. buchneri strain was introduced as a common epiphytic lactic acid bacterium via the grass utilized for ensiling. Further main epiphytic Lactobacilli present within the microbial communities are L. plantarum and L. brevis. L. plantarum features a homofermentative lifestyle (Weinberg and Muck, 1996) being able to also perform heterofermentative lactic acid fermentation (Kleerebezem et al., 2003). In contrast, L. brevis is described to be a heterofermentative lactic acid bacterium (Kim et al., 2009). The dominance of L. plantarum in untreated silage samples correlates with a higher concentration

of lactic acid. L. lactis subsp. lactis is also known to be an epiphytic bacterium (Pang et al., 2011; Siezen et al., 2008). It remains unclear why the abundance of Lactococci decreased during the fermentation. Lactococci are also supposed to be able to tolerate those pH values present in the silages analyzed as they were shown to even cope with lower pH values (Kim et al., 1999). A decreased presence of L. lactis over time has also been observed in alfalfa silage previously (Stevenson et al., 2006). Hence, L. lactis might only play a role within the microbial communities in earlier stages of the ensiling process. An epiphytic lifestyle has also been described for Leuconostoc citreum (Pang et al., 2011). All these findings are in accordance with results obtained for lactic acid bacteria within microbial ensiling communities (Langston and Bouma, 1960a; Pang et al., 2011; Woolford and Pahlow, 1998; Yang et al., 2010). Moreover, these analyses show that inoculation of silage with L. buchneri CD034 not only has an influence on bacteria not belonging to the genus Lactobacillus but also affects the proliferation of other Lactobacilli such as L. plantarum and L. brevis by decreasing their abundance.

4. Conclusion In this study, untreated and inoculated grass silage samples were analyzed physico-chemically for parameters that allow evaluation of the ensiling process. Moreover, the taxonomical composition of microbial ensiling communities was analyzed by means of a metagenomic approach including sequencing and analysis of metagenomic DNA and of 16S rDNA amplicons. The methods applied in this study allow monitoring changes within the microbial communities during fermentation in dependence of inoculation and over time. Previous ensiling experiments and corresponding analyses of ensiling microbial communities were based on cultivations and enumerations of only lactic acid bacteria by growing them on MRS agar (Hu et al., 2009; Kung et al., 2003; Mari et al., 2009; Reich and Kung, 2010; Schmidt and Kung, 2010). Other studies only analyzed a small number of cultivable species (Pang et al., 2011). These methods do not allow monitoring changes within phyla, orders or families and therefore are not able to show the displacement of Lactococci by other bacteria during the fermentation. Moreover, lactic acid bacteria that do not grow well on standard MRS medium would be missed. Hence, metagenomic analyses are more suitable to characterize whole microbial ensiling communities and to track changes within them. Results from the fragment recruitments indicate that no L. buchneri or related strains were introduced as a common epiphytic lactic acid bacterium via the fresh forage utilized for ensiling. Hence, L. buchneri CD034 was only present in the inoculated samples. Further epiphytic Lactobacilli identified to be dominantly involved in ensiling are L. plantarum and L. brevis. Inoculation of silage with L. buchneri CD034 decreased their abundance suggesting that this strain is more competitive than the species mentioned before. Moreover, it seems that L. lactis subsp. lactis is the dominant Lactococcus species involved in the earlier stages of silage fermentation. Physico-chemical analyses of the silage samples in this experiment and the fragment recruitment results demonstrated the pronounced presence of L. buchneri CD034 and its effects within the inoculated silage at the end of the fermentation. Hence, L. buchneri

Fig. 4. Visualization of BLASTn analyses of metagenomic sequence reads against selected reference chromosomes of L. buchneri CD034 (A), L. buchneri NRRL B-30929 (B), L. plantarum subsp. plantarum ST-III (C), L. lactis subsp. lactis KF147 (D), L. brevis ATCC 367 (E)). The results for each reference are displayed in three different diagrams. Sequence identities (>55%) between each hit of a metagenomic sequence read and the chromosomal reference sequence are plotted against the position of the alignment in ‘fragment recruitment plots’ (34) (panel 1). In panel 2 the results are further condensed to hit distribution plots to overcome problems of symbol interference. Here, the normalized number of reads generating hits against the chromosomal reference sequence is plotted in 1% intervals against the sequence identity (>55%). Finally, panel 3 shows best hit analyses. These display the percentage of reads causing a best hit with a specified degree of identity (greater than or equal to 99%, smaller than 99% and greater than or equal to 55%, less than 55% and reads causing no hits).

Please cite this article in press as: Eikmeyer, F.G., et al., Metagenome analyses reveal the influence of the inoculant Lactobacillus buchneri CD034 on the microbial community involved in grass silaging. J. Biotechnol. (2013), http://dx.doi.org/10.1016/j.jbiotec.2013.07.021

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CD034 is competitive within the epiphytic microbial community and is able to positively affect ensiling. However, it remains unclear how L. buchneri CD034 can attain such a dominant presence. Metatranscriptome analyses could provide insights into active genes during different stages of the ensiling process and help to identify genes that are important for its competitiveness and adaptability. Moreover, this study is based on grass silage. Results might be different for other substrates used for ensiling such as maize, alfalfa or cereals and may also be affected by the time of forage harvesting. Further studies should address whether L. buchneri CD034 is also a convenient starter culture for other ensiled forages.

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This work was supported by the Christian Doppler Research Association. F.G.E. acknowledges the receipt of a scholarship from the CLIB Graduate Cluster ‘Industrial Biotechnology’ co-financed by the Ministry of Innovation of North Rhine-Westphalia. The bioinformatics and technological support of the Bioinformatics and Genomics Platforms at the Center for Biotechnology (CeBiTec, Bielefeld University) is gratefully acknowledged. A.S. acknowledges the METAEXPLORE Grant of the European Commission (KBBE-222625). References Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J., 1990. Basic local alignment search tool. Journal of Molecular Biology 215 (3), 403–410. Camacho, C., Coulouris, G., Avagyan, V., Ma, N., Papadopoulos, J., Bealer, K., Madden, T., 2009. BLAST+: architecture and applications. BMC Bioinformatics 10 (1), 421. Danner, H., Holzer, M., Mayrhuber, E., Braun, R., 2003. Acetic acid increases stability of silage under aerobic conditions. Applied and Environmental Microbiology 69 (1), 562–567. Dixon R P., 2003. VEGAN, a package of R functions for community ecology. Journal of Vegetation Science 14 (6), 927–930. Driehuis, F., Oude Elferink, S.J.W.H., van Wikselaar, P.G., 2001. Fermentation characteristics and aerobic stability of grass silage inoculated with Lactobacillus buchneri, with or without homofermentative lactic acid bacteria. Grass and Forage Science 56 (4), 330–343. Edgar, R.C., Haas, B.J., Clemente, J.C., Quince, C., Knight, R., 2011. UCHIME improves sensitivity and speed of chimera detection. Bioinformatics 27 (16), 2194–2200. Gerlach, W., Stoye, J., 2011. Taxonomic classification of metagenomic shotgun sequences with CARMA3. Nucleic Acids Research 39 (14), e91. Heinl, S., Wibberg, D., Eikmeyer, F., Szczepanowski, R., Blom, J., Linke, B., Goesmann, A., Grabherr, R., Schwab, H., Pühler, A., Schlüter, A., 2012. Insights into the completely annotated genome of Lactobacillus buchneri CD034, a strain isolated from stable grass silage. Journal of Biotechnology 161 (2), 153–166. Holzer, M., Mayrhuber, E., Danner, H., Braun, R., 2003. The role of Lactobacillus buchneri in forage preservation. Trends in Biotechnology 21 (6), 282–287. Huse, S.M., Welch, D.M., Morrison, H.G., Sogin, M.L., 2010. Ironing out the wrinkles in the rare biosphere through improved OTU clustering. Environmental Microbiology 12 (7), 1889–1898. Hu, W., Schmidt, R.J., McDonell, E.E., Klingerman, C.M., Kung, L., 2009. The effect of Lactobacillus buchneri 40788 or Lactobacillus plantarum MTD-1 on the fermentation and aerobic stability of corn silages ensiled at two dry matter contents. Journal of Dairy Science 92 (8), 3907–3914. Jaenicke, S., Ander, C., Bekel, T., Bisdorf, R., Droege, M., Gartemann, K.-H., Juenemann, S., Kaiser, O., Krause, L., Tille, F., Zakrzewski, M., Puehler, A., Schlueter, A., Goesmann, A., 2011. Comparative and joint analysis of two metagenomic datasets from a biogas fermenter obtained by 454-pyrosequencing. PLoS ONE 6 (1), e14519. Kim, J.-H., Shoemaker, S.P., Mills, D.A., 2009. Relaxed control of sugar utilization in Lactobacillus brevis. Microbiology (Reading, Engl.) 155 (Pt 4), 1351–1359. Kim, W.S., Ren, J., Dunn, N.W., 1999. Differentiation of Lactococcus lactis subspecies lactis and subspecies cremoris strains by their adaptive response to stresses. FEMS Microbiology Letters 171 (1), 57–65. Kleerebezem, M., Boekhorst, J., van Kranenburg, R., Molenaar, D., Kuipers, O.P., Leer, R., Tarchini, R., Peters, S.A., Sandbrink, H.M., Fiers, M.W.E.J., Stiekema, W., Lankhorst, R.M.K., Bron, P.A., Hoffer, S.M., Groot, M.N.N., Kerkhoven, R., Vries, M., de Ursing, B., Vos, W.M., de Siezen, R.J., 2003. Complete genome sequence of Lactobacillus plantarum WCFS1. Proceedings of the National Academy of Sciences USA 100 (4), 1990–1995. Kleinschmit, D.H., Kung, L., 2006. A meta-analysis of the effects of Lactobacillus buchneri on the fermentation and aerobic stability of corn and grass and small-grain silages. Journal of Dairy Science 89 (10), 4005–4013. Klocke, M., Mundt, K., Idler, C., McEniry, J., O’Kiely, P., Barth, S., 2006. Monitoring Lactobacillus plantarum in grass silages with the aid of 16S rDNA-based quantitative real-time PCR assays. Systematic and Applied Microbiology 29 (1), 49–58.

9

Krause, L., Diaz, N.N., Edwards, R.A., Gartemann, K.-H., Kroemeke, H., Neuweger, H., Puehler, A., Runte, K.J., Schlueter, A., Stoye, J., Szczepanowski, R., Tauch, A., Goesmann, A., 2008. Taxonomic composition and gene content of a methaneproducing microbial community isolated from a biogas reactor. Journal of Biotechnology 136 (1–2), 91–101. Kung, L., Taylor, C.C., Lynch, M.P., Neylon, J.M., 2003. The effect of treating alfalfa with Lactobacillus buchneri 40788 on silage fermentation, aerobic stability, and nutritive value for lactating dairy cows. Journal of Dairy Science 2003, 336–343. Langston, C.W., Bouma, C., 1960a. A study of the microorganisms from grass silage II. The Lactobacilli. Applied Microbiology 8, 223–234. Langston, C.W., Bouma, C., 1960b. A study of the microorganisms from grass silage: I. The Cocci. Applied Microbiology 8 (4), 212–222. Magurran, A.E., 2010. Measuring biological diversity, 8th ed. Blackwell, Malden, MA, pp. 256. Man, J.C., Rogosa, deM., Sharpe, M.E., 1960. A medium for the cultivation of Lactobacilli. Journal of Applied Microbiology 23 (1), 130–135. Mari, L.J., Schmidt, R.J., Nussio, L.G., Hallada, C.M., Kung, L., 2009. Short communication: an evaluation of the effectiveness of Lactobacillus buchneri 40788 to alter fermentation and improve the aerobic stability of corn silage in farm silos. Journal of Dairy Science 92 (3), 1174–1176. Martin, M., 2011. Cutadapt removes adapter sequences from high-throughput sequencing reads 17. http://journal.embnet.org/index.php/embnetjournal/ article/view/200 McGarvey, J.A., Franco, R.B., Palumbo, J.D., Hnasko, R., Stanker, L., Mitloehner, F.M., 2013. Bacterial population dynamics during the ensiling of Medicago sativa (alfalfa) and subsequent exposure to air. Journal of Applied Microbiology 114 (6), 1661–1670. Nishino, N., Touno, E., 2005. Ensiling characteristics and aerobic stability of directcut and wilted grass silages inoculated with Lactobacillus casei or Lactobacillus buchneri. Journal of the Science of Food and Agriculture 85 (11), 1882–1888. Oliver, J.D., 2005. The viable but nonculturable state in bacteria. Journal of Microbiology 43, 93–100. Ondov, B.D., Bergman, N.H., Phillippy, A.M., 2011. Interactive metagenomic visualization in a web browser. BMC Bioinformatics 12, 385. Oude Elferink, S.J., Krooneman, J., Gottschal, J.C., Spoelstra, S.F., Faber, F., Driehuis, F., 2001. Anaerobic conversion of lactic acid to acetic acid and 1, 2-propanediol by Lactobacillus buchneri. Applied and Environmental Microbiology 67 (1), 125–132. Pahlow, G., Muck, R.E., Driehuis, F., Oude Elferink, S.J.W.H., Spoelstra, S.F., 2003. Microbiology of ensiling. In: Buxton, Muck, R.E., Harrison, J.H. (Eds.), Silage Science and Technology Agronomy: 42. American Society of Agronomy, pp. 31–93. Pang, H., Qin, G., Tan, Z., Li, Z., Wang, Y., Cai, Y., 2011. Natural populations of lactic acid bacteria associated with silage fermentation as determined by phenotype, 16S ribosomal RNA and recA gene analysis. Systematic and Applied Microbiology 34 (3), 235–241. R Development Core Team, 2010. R: A Language and Environment for Statistical Computing, Vienna, Austria. Reich, L.J., Kung, L., 2010. Effects of combining Lactobacillus buchneri 40788 with various lactic acid bacteria on the fermentation and aerobic stability of corn silage. Animal Feed Science and Technology 159 (3–4), 105–109. Rusch, D.B., Halpern, A.L., Sutton, G., Heidelberg, K.B., Williamson, S., Yooseph, S., Wu, D., Eisen, J.A., Hoffman, J.M., Remington, K., Beeson, K., Tran, B., Smith, H., Baden-Tillson, H., Stewart, C., Thorpe, J., Freeman, J., Andrews-Pfannkoch, C., Venter, J.E., Li, K., Kravitz, S., Heidelberg, J.F., Utterback, T., Rogers, Y.-H., Falcón, L.I., Souza, V., Bonilla-Rosso, G., Eguiarte, L.E., Karl, D.M., Sathyendranath, S., Platt, T., Bermingham, E., Gallardo, V., Tamayo-Castillo, G., Ferrari, M.R., Strausberg, R.L., Nealson, K., Friedman, R., Frazier, M., Venter, J.C., 2007. The Sorcerer II global ocean sampling expedition: Northwest Atlantic through eastern tropical Pacific. PLoS Biol 5 (3), e77. Schloss, P.D., Westcott, S.L., Ryabin, T., Hall, J.R., Hartmann, M., Hollister, E.B., Lesniewski, R.A., Oakley, B.B., Parks, D.H., Robinson, C.J., Sahl, J.W., Stres, B., Thallinger, G.G., van Horn, D.J., Weber, C.F., 2009. Introducing mothur: Opensource, platform-independent community-supported software for describing and comparing microbial communities. Applied and Environmental Microbiology 75 (23), 7537–7541. Schlüter, A., Bekel, T., Diaz, N.N., Dondrup, M., Eichenlaub, R., Gartemann, K.-H., Krahn, I., Krause, L., Krömeke, H., Kruse, O., Mussgnug, J.H., Neuweger, H., Niehaus, K., Pühler, A., Runte, K.J., Szczepanowski, R., Tauch, A., Tilker, A., Viehöver, P., Goesmann, A., 2008. The metagenome of a biogas-producing microbial community of a production-scale biogas plant fermenter analysed by the 454-pyrosequencing technology. Journal of Biotechnology 136 (1–2), 77–90. Schmidt, R.J., Emara, M.G., Kung, L., 2008. The use of a quantitative real-time polymerase chain reaction assay for identification and enumeration of Lactobacillus buchneri in silage. Journal of Applied Microbioliology 105 (3), 920–929. Schmidt, R.J., Kung, L., 2010. The effects of Lactobacillus buchneri with or without a homolactic bacterium on the fermentation and aerobic stability of corn silages made at different locations. Journal of Dairy Science 93 (4), 1616–1624. Siezen, R.J., Starrenburg, M.J.C., Boekhorst, J., Renckens, B., Molenaar, D., van Hylckama Vlieg, J.E.T., 2008. Genome-Scale Genotype-Phenotype Matching of Two Lactococcus lactis Isolates from Plants Identifies Mechanisms of Adaptation to the Plant Niche. Applied Environmental Microbiology 74 (2), 424–436. Singh, J., Behal, A., Singla, N., Joshi, A., Birbian, N., Singh, S., Bali, V., Batra, N., 2009. Metagenomics: concept, methodology, ecological inference and recent advances. Biotechnology Journal 4 (4), 480–494. Stevenson, D., Muck, R., Shinners, K., Weimer, P., 2006. Use of real time PCR to determine population profiles of individual species of lactic acid bacteria in

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ARTICLE IN PRESS F.G. Eikmeyer et al. / Journal of Biotechnology xxx (2013) xxx–xxx

alfalfa silage and stored corn stover. Applied Environmental Microbiology 71 (3), 329–338. Sun, Y., Cai, Y., Liu, L., Yu, F., Farrell, M.L., McKendree, W., Farmerie, W., 2009. ESPRIT: estimating species richness using large collections of 16S rRNA pyrosequences. Nucleic Acids Research 37 (10), e76. Wang, Q., Garrity, G.M., Tiedje, J.M., Cole, J.R., 2007. Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Applied Environmental Microbiology 73 (16), 5261–5267. Warnecke, F., Hess, M., 2009. A perspective: metatranscriptomics as a tool for the discovery of novel biocatalysts. Journal of Biotechnology 142 (1), 91–95. Weinberg, Z.G., Muck, R.E., 1996. New trends and opportunities in the development and use of inoculants for silage. FEMS Microbiology Reviews 19 (1), 53–68. Woolford, M.K., Pahlow, G., 1998. The silage fermentation. In: Wood, B.J.B. (Ed.), Microbiology of fermented foods. , 2 ed. Blackie Academic & Professional, London, pp. 73–102.

Yang, J., Cao, Y., Cai, Y., Terada, F., 2010. Natural populations of lactic acid bacteria isolated from vegetable residues and silage fermentation. Journal of Dairy Science 93 (7), 3136–3145. Zakrzewski, M., Bekel, T., Ander, C., Pühler, A., Rupp, O., Stoye, J., Schlüter, A., Goesmann, A., 2012a. MetaSAMS – A novel software platform for taxonomic classification, functional annotation and comparative analysis of metagenome datasets. Journal of Biotechnology (Epub ahead of print). Zakrzewski, M., Goesmann, A., Jaenicke, S., Jünemann, S., Eikmeyer, F., Szczepanowski, R., Al-Soud, W.A., Sørensen, S., Pühler, A., Schlüter, A., 2012b. Profiling of the metabolically active community from a production-scale biogas plant by means of high-throughput metatranscriptome sequencing. Journal of Biotechnology 158 (4), 248–258.

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