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Animal Science Journal (2015) 86, 45–50
doi: 10.1111/asj.12234
ORIGINAL ARTICLE Identification of bacteria in total mixed ration silage produced with and without crop silage as an ingredient Naoki NISHINO,1 Yu OGATA,1 Hongyan HAN1 and Yasunari YAMAMOTO2 1
Graduate School of Environmental and Life Science, Okayama University, Okayama and 2Mie Prefecture Livestock Research Institute, Mie, Japan
ABSTRACT As a forage source for total mixed ration (TMR) silage production, locally produced crop silage is now used in addition to imported hay. This type of TMR ensiling is regarded as a two-step fermentation process; hence, a survey was carried out to determine whether the bacteria in crop silage affect the subsequent TMR ensiling. Fermentation product contents and bacterial community were determined for TMR silage and its ingredient silages collected in August, October and November. August product contained corn, sorghum and Italian ryegrass silages, October product had wheat silage exclusively and November product did not include any crop silages. Acetic acid, lactic acid, 2,3-butanediol and ethanol were predominant fermentation products in corn, sorghum, Italian ryegrass and wheat silages, respectively. Robust lactic acid fermentation was seen in TMR silage, even if acetate-type and alcohol-type silages were mixed as ingredients. The finding that bacterial community of the TMR silage appeared unrelated to those of ingredient silage supported this. Silages of various fermentation types can therefore be formulated without interfering with lactate-type fermentation in TMR silage.
Key words: bacteria, denaturing gradient gel electrophoresis, silage, total mixed ration.
INTRODUCTION Production of total mixed ration silage is practiced in Japan as a method to utilize wet by-products as feed for ruminants. Food and beverage by-products have an unbalanced composition of nutrients, and thus mixing prior to feeding a ration is necessary, even when suitable preservation is achieved by storing wet by-products alone. Brewers grains and soybean curd residue are representative by-products; various dry feeds such as hay, corn grains, wheat bran, cotton seed cake, soybean meal and beet pulp are combined with the by-products to create a total mixed ration (TMR) mixture (Wang & Nishino 2008). Typical concentrations of dry matter (DM), crude protein (N × 6.25), and total digestible nutrients are 500–600 g/kg, 160– 180 g/kg DM, and 720–740 g/kg DM, respectively, and thus without other supplements dairy cows can perform a high level of milk production (Wang & Nishino 2010). Because small particles of by-products enable high packing density, lactic acid fermentation often dominates the ensiling process (Weinberg et al. 2011). In addition, aerobic spoilage would not take place, and thus feed intake can be kept at a high level even in hot seasons (Wang & Nishino 2013). Therefore, a number of TMR manufacturers exist in © 2014 Japanese Society of Animal Science
various regions, commercializing TMR silage for local farmers. As a forage source of TMR ingredients, manufacturers have started to use locally produced silage in addition to imported hay. Various crop silages from grass, oat, sorghum, whole crop corn and whole crop rice can be used as a single or a combined forage source; hence, present TMR ensiling may have a two-step fermentation process, where bacteria grown in the first round of crop ensiling could serve as a silage additive for the second round of TMR ensiling. Benefits would be seen if only desirable lactic acid bacteria (LAB) species were transferred from crop silage to the TMR silage. However, various undesirable bacteria may also grow in crop silage, and no previous studies have determined whether the bacteria in crop silage can affect the subsequent ensiling process. In this study, bacterial communities of TMR silage and its
Correspondence: Naoki Nishino, Department of Animal Science, Graduate School of Environmental and Life Science, Okayama University, Okayama 700-8530, Japan. (Email: [email protected]) Received 28 August 2013; accepted for publication 3 March 2014.
46 N. NISHINO et al.
ingredient crop silages were determined in order to understand bacterial transfer from crop silage to TMR silage.
MATERIALS AND METHODS Sampling of silages We collected samples of TMR silages produced at Mie Prefecture Livestock Research Institute on August 23, October 29 and November 2, 2010. For the August product, two corn, two sorghum and four wilted Italian ryegrass silages, which had been stored for 13, 12 and 4 months, respectively, were mixed into a batch with other feeds, and the mixture was then wrapped with six layers of plastic film using a roll baler for chopped material (Shito et al. 2006). For the October product, five wheat silages stored for 6 months and one wet brewers grains silage stored for 1 month were used. For the November product, timothy hay was used instead of crop silages, while the same wet brewer grains silage as used in the October product was mixed in. Since the November product was prepared just 4 days after the October product, there were no detectable changes in the wet brewers grain silage. The proportion of crop silage or hay in the TMR mixture was 0.40 on a DM basis, and 7–10 newly-baled TMR silages were prepared from one TMR mixture batch. Bales for TMR silage were stored outside for 1–2 months. Several grab samples were taken to make a composite, and about 0.5 kg of representative samples were stored at −30°C. Silage samples were shipped frozen to our laboratory at Okayama University.
Chemical and bacterial community analyses DM content was determined by oven drying at 60°C for 48 h. The fermentation products content in the silage was determined from water extracts through an ion-exclusion polymeric high-performance liquid chromatography method with refractive index detection (Han et al. 2012). Denaturing gradient gel electrophoresis (DGGE) was performed as previously described (Li & Nishino 2011). Frozen samples were thawed and added to a 19 × volume of sterilized phosphate buffered saline (pH 7.4), and extraction involved shaking the samples vigorously for 10 min at ambient temperature. Bacterial DNA was purified using a commercial kit (DNeasy Tissue Kit; Qiagen, Germantown, MD, USA). Polymerase chain reaction (PCR) was used to amplify a variable (V3) region of the bacterial 16S rRNA gene, with the forward primer GC357f (5′-CGCCCGCCGCGCGCGGCGGG CGGGGCGGGGGCACGGGGGGCCTACGGGAGGCAGCAG-3′) and the reverse primer 517r (5′-ATTACCGCGGCTGCTGG3′). The PCR protocol involved an initial denaturation step at 95°C for 10 min. This step was followed by 30 cycles of: (i) denaturation at 93°C for 30 s; (ii) annealing at (a) 65°C (first 10 cycles), (b) 60°C (second 10 cycles), and (c) 55°C (last 10 cycles) for 30 s; and (iii) extension at 72°C for 1 min. The final extension step was performed at 72°C for 5 min. The GC-clamp PCR products were separated according to their sequences by using a DCode Universal Mutation Detection System (Bio-Rad Ltd, Tokyo, Japan). The samples were applied directly onto 100 g/L (w/v) polyacrylamide gels that had a denaturing gradient ranging from 0.20 to 0.50, which then was prepared using 7 mol/L urea and 400 mL/L © 2014 Japanese Society of Animal Science
formamide, respectively, as 100% denaturants. Electrophoresis was performed at a constant voltage of 150 V for 12 h at 60°C. After electrophoresis, the gels were stained with SYBR Green (Cambrex Bio Science Inc., Rockland, ME, USA) and photographed under UV illumination. Select bands were excised from DGGE gels, and each band was placed in 10 μL of sterilized water at 4°C overnight to allow diffusion of the DNA. Extracted DNA was amplified by PCR performed using the 357f (without GC clamp) and 517r primers. The PCR products were purified using a commercial clean-up kit (GeneClean Kit; Qbiogene, Carlsbad, CA, USA), and then the purified PCR products were cloned into the pTAC-1 vector. The resulting plasmids were transformed into Escherichia coli strain DH5α competent cells (DynaExpress TA cloning kit; BioDynamics Laboratory Inc., Tokyo, Japan). The sequencing reaction was carried out using a BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems Inc., Foster City, CA, USA) and DNA base sequences were analyzed using an ABI PRISM® 310 sequencer (Applied Biosystems Inc., Foster City, CA, USA).
Data analyses Data for ensiling fermentation were expressed as mean values with standard deviation. Bacterial species were identified on Basic Local Alignment Search Tool (BLAST) searches using the GenBank database, and the closest relatives of partial 16S rRNA gene sequences were determined. Positive identifications of unknown sequences were considered similar when more than 0.99 of the sequence was identical to that in the BLAST database. Cluster analysis with both quantitative (pH and fermentation product contents) and qualitative (DGGE band profiles) data was performed to describe the similarity and differences observed within and between ingredients and TMR silages. The DGGE band profiles were analyzed using an image analysis system (Image J; National Institutes of Health, Bethesda, MD, USA), and the migration patterns of the DNA bands were converted into lists of binary numbers. Dendrograms were constructed using an unweighted pairgroup method with arithmetic averages. Cluster analysis was performed using JMP software (ver. 7; SAS Institute, Tokyo, Japan).
RESULTS Fermentation characteristics Three crop silages used in the August product were variable in fermentation product composition (Table 1). The acetic acid, 1,2-propanediol, and 1-propanol contents were much greater than the lactic acid content in corn silage. Although lactic acid dominated the fermentation, high acetic acid content was also seen in sorghum silage. In wilted grass silage, acid content was rather low and the 2,3-butanediol and ethanol contents were more than the lactic acid and acetic acid contents. Although these diverse silages were used as ingredients, TMR silage showed stable lactate-type fermentation. The lactic acid to acetic acid (L/A) ratio was greater than those of corn, sorghum and wilted grass silages, with small variation within the same batch observed as compared with those in each ingredient silage. Animal Science Journal (2015) 86, 45–50
Animal Science Journal (2015) 86, 45–50
†Mean values of two silages produced on the same day and used to prepare the same batch of total mixed ration (TMR) silage. ‡Mean values of four silages produced on the same day and used to prepare the same batch of TMR silage. §Mean values of three silages prepared from the same batch of TMR mixture. ¶Mean values of five silages produced on the same day and used to prepare the same batch of TMR silage. ††This brewers grains silage was used to prepare the October and November products.
– 0.19 – 8.40 1.60 4.02 0.00 3.18 – 0.36 0.00 2.08 – 0.00 0.00 0.00 – 0.31 2.25 3.98 – 1.48 – 5.28 6.97 14.1 – 3.72 5.69 4.50 375 535
– 44.3 0.03 56.8
0.30 0.10 0.35 1.07 0.71 2.49 0.32 7.41 1.76 0.89 3.11 4.26 6.10 1.03 25.4 28.9 2.83 10.7 7.87 50.2 1.76 32.8 7.44 13.9 2.62 21.4 17.1 4.30 2.52 4.11 288 470
0.23 13.9 0.05 53.1
0.03 0.87 0.96 0.26 0.21 1.42 1.97 2.32 5.05 0.63 0.00 1.84 31.1 1.98 0.00 5.79 30.6 4.55 0.00 0.49 37.7 20.4 0.00 16.3 0.00 0.43 4.78 0.20 0.00 1.59 7.46 1.76 0.23 0.54 2.20 0.20 1.41 2.53 5.72 2.87 0.03 10.5 0.95 50.5 11.2 0.04 57.7 21.9 44.2 11.5 0.25 4.46 2.44 2.18 0.86 0.01 52.4 2.54 22.7 1.88 4.06 3.95 5.17 4.20 16.3 7.07 68.3 4.58
Mean SD Mean SD SD Mean SD Mean Mean SD Mean SD Mean SD Mean SD Mean SD
1,2-Propanediol (g/kg DM) 2,3-Butanediol (g/kg DM) Ethanol (g/kg DM) Acetic acid (g/kg DM) Lactic acid (g/kg DM) pH DM (g/kg)
283 226 459 555
August Whole crop corn silage† Sorghum silage† Italian ryegrass silage‡ TMR silage§ October Whole crop wheat silage¶ TMR silage§ November Brewers grains silage†† TMR silage§
Table 1
Dry matter (DM), pH value and fermentation product concentration of total mixed ration silage and its ingredients silage
1-Propanol (g/kg DM)
L/A ratio
BACTERIA IN TOTAL MIXED RATION SILAGE
47
Wheat silage used exclusively in the October product showed extensive alcoholic fermentation. The ethanol content was greater than 50 g/kg DM and the lactic acid and acetic acid contents were less than onethird and half of the ethanol and 2,3-butanediol contents, respectively. Although lactate-type fermentation was greatly enhanced and the L/A ratio increased, the ethanol content was yet higher than the acetic acid content in TMR silage. Wet brewers grains silage, which was used in both the October and November products, showed desirable lactate-type fermentation. High lactic acid and low alcohol contents were also seen in the November product of the TMR silage. However, the silage pH averaged 4.50 and the value appeared greater than that of the TMR silage containing crop silage. Cluster analysis demonstrated that the TMR silage fermentation appeared stable and resembled each other regardless of the product season (Fig. 1). Also, TMR silages were grouped with other lactate-type wet brewer grains and sorghum silages, whereas distinctive separation was observed from alcohol-type wilted Italian ryegrass and wheat silages. A greater difference was found from corn silage, which contains acetic acid as the major fermentation product.
Bacterial communities No great differences were seen in the bacterial community between corn and sorghum silages used in the August product (Fig. 2); Lactobacillus acetotolerans (band 1), L. buchneri (bands 3), and Pediococcus ethanolidurans (band 5) were detected in both crop silages. Wilted grass silage appeared different from corn and sorghum silages with respect to the bacterial community. L. acetotolerans, L. buchneri and P. ethanolidurans found in corn and sorghum silages seemed to survive as bands 12, 14 and 15, respectively, in TMR silage. Moreover, bands of L. acetotolerans (band 10), Lactobacillus parafarraginis (band 9), Pediococcus goldsteinii (band 11) and Pediococcus pentosaceus (band 13) newly appeared. Several bands indicative of Enterobacter amnigenus (bands 17 and 20–23) were found in wheat silage. Unlike wheat silages 3, 4 and 5, which showed similar band patterns, wheat silage 2 had a distinctive band of L. buchneri (band 18). The bacterial community of the TMR silage appeared unrelated to that of brewers grains silage or wheat silage; L. acetotolerans (bands 24) was seen only in TMR silage, and a band in the same migrating position of E. amnigenus (band 23) in wheat silage was identified as Lactobacillus fructivorans (band 27) in TMR silage. Even so, E. amnigenus (band 26) and L. buchneri (band 25) appeared to transfer from wheat silage to TMR silage. Although bands of Bacillus coagulans (band 29) and Bacillus smithii (band 30) were found in brewers grains silage, the corresponding bands in TMR silage were © 2014 Japanese Society of Animal Science
48 N. NISHINO et al.
Based on pH and fermentaon products CS1 (Aug) CS2 (Aug) SG1 (Aug) SG2 (Aug) TMR1 (Aug) TMR2 (Aug) TMR1 (Nov) TMR2 (Nov) TMR1 (Oct) TMR2 (Oct) BG (Oct&Nov) IR1 (Aug) IR2 (Aug) IR4 (Aug) IR3 (Aug) WH1 (Oct) WH5 (Oct) WH3 (Oct) WH4 (Oct) WH2 (Oct)
Based on DGGE band paerns CS1 (Aug) SG1 (Aug) CS2 (Aug) SG2 (Aug) TMR1 (Oct) TMR2 (Oct) TMR1 (Aug) TMR2 (Aug) WH3 (Oct) WH5 (Oct) WH4 (Oct) TMR1 (Nov) TMR2 (Nov) IR1 (Aug) IR2 (Aug) WH2 (Oct) WH1 (Oct) IR3 (Aug) IR4 (Aug) BG (Oct&Nov)
1.0
0.5 Distance-based similarity
1.0
0
0.5 Distance-based similarity
0
Figure 1 Dendrograms constructed using cluster analyses on the basis of pH and fermentation products and the bacterial community are described as denaturing gradient gel electrophoresis (DGGE) profiles. The letters indicate the ingredient and total mixed ration (TMR) silage; CS, corn silage; SG, sorghum silage; IR, wilted Italian ryegrass silage; WH, wheat silage; BG, wet brewers grains silage. The following numbers indicate the sampling batch number, and the abbreviated months in parenthesis describe the August, October and November products, respectively.
identified as L. acetotolerans (band 34) and Lactobacillus casei (band 35), respectively. In addition, Lactobacillus suebicus (band 32) and Weissella paramesenteroides (bands 33 and 34) appeared in the November product of the TMR silage. Cluster analysis indicated that none of the TMR silages formed a close group with its ingredient silage; the October products were grouped with corn and sorghum silages (the August product ingredient), and the August and November products were grouped with wheat silage (October product ingredient). Moreover, two separate groups were formed from each wilted Italian ryegrass and wheat silage; hence, differences within ingredient silage bales appeared greater than that between ingredient and TMR silages. Although wet brewers grains silage was closely clustered with TMR silage based on pH and fermentation product contents, there was a considerable difference between two silages in the clustering based on DGGE band profiles. © 2014 Japanese Society of Animal Science
DISCUSSION Robust lactic acid fermentation was observed in TMR silage despite the fact that diverse fermentation products dominated over ingredient silages. In addition, variation between bales, as indicated by the standard deviation values and by the clustered groups, appeared smaller in TMR silages than in crop silages. The DM contents of TMR silage were 470–550 g/kg in this study; hence, low moisture ensiling could have supported stable lactate-type fermentation. Italian ryegrass silage used in the August product had a large amount of ethanol and 2,3-butanediol, although the DM content (459 g/kg) was close to that of the October product. Alcoholic fermentation can occur when grass is ensiled at a high DM content (Driehuis & van Wikselaar 2000); therefore, low moisture ensiling per se could not account for the robust lactic acid fermentation in the TMR silage. Rather, because many by-products of small particle size are used, TMR silage Animal Science Journal (2015) 86, 45–50
BACTERIA IN TOTAL MIXED RATION SILAGE
11
6
12
1
8 4
20 25 19
16
7
2
10
18
31 24
28
32
9
29
33
13
3
34
14
5 15
17
21 22
26
35 30
CS 1 2 SG 1 2 IR 1 2 3 4 Pre-TMR TMR 1 2 BG WH 1 2 3 4 5 Pre-TMR TMR 1 2 BG Pre-TMR TMR 1 2
23
27
August
October
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35
49
Lactobacillus acetotolerans Lactobacillus brevis Lactobacillus buchneri Lactobacillus buchneri Pediococcus ethanolidurans Uncultured bacterium Weissella cibaria Lactobacillus acetotolerans Lactobacillus parafarraginis Lactobacillus acetotolerans Pediococcus goldsteinii Lactobacillus acetotolerans Pediococcus pentosaceus Lactobacillus buchneri Pediococcus ethanolidurans Lactobacillus delbrueckii Enterobacter amnigenus Lactobacillus buchneri Enterococcus mundi Enterobacter amnigenus Enterobacter amnigenus Enterobacter amnigenus Enterobacter amnigenus Lactobacillus acetotolerans Lactobacillus buchneri Enterobacter amnigenus Lactobacillus frucvorans Lactobacillus acetotolerans Bacillus coagulans Bacillus smithii Lactobacillus suebicus Weissella paramesenteroides Weissella paramesenteroides Lactobacillus acetotolerans Lactobacillus casei
November
Figure 2 Denaturing gradient gel electrophoresis analysis of bacterial communities present in the total mixed ration silage of August, October and November products. Corn (CS), sorghum (SG) and wilted Italian ryegrass (IR) silages were used as ingredient silages for the August product, while wheat (WH) and wet brewers grains (BG) silages were used for the October product, and wet brewers grains silage was used for the November product. Pre-TMR describes a total mixed ration mixture sampled before ensiling fermentation.
has a high packing density of > 200 kg DM·m–3, which is difficult to achieve in low moisture grass silage (Kida et al. 2007). Robust lactic acid fermentation could thus be due to distinctive or nonconventional ensiling conditions of low moisture and high density storage. L. acetotolerans, L. brevis, L. buchneri, P. ethanolidurans, W. cibaria, L. delbrueckii, E. mundtii were the LAB species identified in ingredient silage in this study, and bands showing the same migrating position, except for L. delbrueckii, were found in TMR silage. In contrast, although three non-LAB species (E. amnigenus, B. coagulans and B. smithii) were identified in ingredient silage, corresponding bands were undetectable, faint or replaced by LAB species in TMR silage. These findings suggest that LAB rather than non-LAB species could be transferred more likely from ingredient to TMR silage. However, we did not determine bacterial numbers in this study, and thus, the higher transfer of Animal Science Journal (2015) 86, 45–50
LAB than non-LAB species could be due to differences in the populations in ingredient silage. Meanwhile, cluster analysis indicated that the bacterial community of TMR silage was not closely related with those of ingredient silage. Likewise, there were newly appeared LAB species in TMR silage, such as L. parafarraginis, P. goldsteinii, L. fructivorans, L. suebicus, W. paramesenteroides and L. casei. Overall, the bacterial community of TMR silage is considered largely independent from those of ingredient silage. In the present DGGE analysis, occasionally multiple bands detected at different migrating positions were identified as the same bacterial species. This may have occurred due to the presence of multiple heterogeneous 16S rRNA gene copies in a bacterium, and heteroduplex molecules formed during the PCR amplification process (Muyzer & Smalla 1998). Although DGGE analysis can provide a rapid and © 2014 Japanese Society of Animal Science
50 N. NISHINO et al.
repeatable characterization of the bacterial community, the resolution level is known to be low. Co-migration of DNA from multiple species may also occur (Sekiguchi et al. 2001); hence, we performed cloning to secure pure bacterial DNA for species identification. In TMR silage L. acetotolerans (band 24) was seen even without a detectable corresponding band in ingredient wheat silage. Likewise, although L. buchneri (band 25) was distinctively seen in the October product, only one of five ingredient wheat silages had L. buchneri in their bacterial community. Therefore, TMR silage can be a good habitat for L. acetotolerans and L. buchneri; their growth may have been favored even if the initial population was small. Use of crop silage as a TMR ingredient might have supported inhabitation of L. acetotolerans, because the November product, in which no crop silages were mixed, did not have L. acetotolerans. Likewise, although various commercial products were surveyed before, we did not find L. acetotolerans in the TMR silage with no crop silages (Wang & Nishino 2010). L. buchneri is known to anaerobically metabolize lactic acid into acetic acid and 1,2-propanediol, which can help inhibit aerobic deterioration after silo opening (Oude Elferink et al. 2001). Since corn and sorghum silages had substantial amounts of acetic acid and 1,2-propanediol, detection of the distinctive bands of L. buchneri was reasonable. Also, since L. acetotolerans can tolerate high acetic acid levels such as 30 g/L (Entani et al. 1986), the presence of the bacterium in corn and sorghum silages was plausible. L. acetotolerans has become a normal inhabitant by taking advantage of the culture-independent procedure (Li & Nishino 2011; Wang & Nishino 2013), although isolation of the LAB species from silage has not yet been reported. This study revealed that LAB species can be selected during the ensiling process of TMR silage rather than during transfer and survival of LAB and non-LAB bacteria present in ingredient crop silages. Therefore, it is concluded that silages of various fermentation types can be formulated without interfering with the lactatetype fermentation in TMR silage.
Entani E, Masai H, Suzuki K-I. 1986. Lactobacillus acetotolerans, a new species from fermented vinegar broth. International Journal of Systematic Bacteriology 36, 544–549. Han H, Takase S, Nishino N. 2012. Survival of silage lactic acid bacteria in the goat gastrointestinal tract as determined by denaturing gradient gel electrophoresis. Letters in Applied Microbiology 55, 384–389. Kida T, Matsuo M, Shigeta K, Matsuyama H, Moriya N. 2007. Production of total mixed ration silage by using a roll baler for chopped material. Grassland Science 53 (Suppl), 114–115. Li Y, Nishino N. 2011. Monitoring the bacterial community of maize silage stored in a bunker silo inoculated with Enterococcus faecium, Lactobacillus plantarum and Lactobacillus buchneri. Journal of Applied Microbiology 110, 1561–1570. Muyzer G, Smalla K. 1998. Application of denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) in microbial ecology. Antonie van Leeuwenhoek 73, 127–141. Oude Elferink SJWH, Krooneman J, Gottschal JC, Spoelstra SF, 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, 125–132. Sekiguchi H, Tomioka N, Nakahara T, Uchiyama H. 2001. A single band does not always represent single bacterial strains in denaturing gradient gel electrophoresis analysis. Biotechnology Letters 23, 1205–1208. Shito H, Yamana N, Shibuya Y, Takahashi K. 2006. Development of the roll baler for chopped materials. Japan Agricultural Research Quarterly (JARQ) 40, 233–237. Wang C, Nishino N. 2010. Presence of sourdough lactic acid bacteria in commercial total mixed ration silage revealed by denaturing gradient gel electrophoresis analysis. Letters in Applied Microbiology 51, 436–442. Wang C, Nishino N. 2013. Effects of storage temperature and ensiling period on fermentation products, aerobic stability and microbial communities of total mixed ration silage. Journal of Applied Microbiology 114, 1687–1695. Wang F, Nishino N. 2008. Ensiling of soybean curd residue and wet brewers grains with or without other feeds as a total mixed ration. Journal of Dairy Science 91, 2380–2387. Weinberg ZG, Chen Y, Miron D, Raviv Y, Nahim E, Bloch A, et al. 2011. Preservation of total mixed rations for dairy cows in bales wrapped with polyethylene stretch film – a commercial scale experiment. Animal Feed Science and Technology 164, 125–129.
REFERENCES Driehuis F, van Wikselaar PG. 2000. The occurrence and prevention of ethanol fermentation in high-dry matter grass silage. Journal of the Science of Food and Agriculture 80, 711–718.
© 2014 Japanese Society of Animal Science
Animal Science Journal (2015) 86, 45–50
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Animal Science Journal (2015) 86, 45–50
doi: 10.1111/asj.12234
ORIGINAL ARTICLE Identification of bacteria in total mixed ration silage produced with and without crop silage as an ingredient Naoki NISHINO,1 Yu OGATA,1 Hongyan HAN1 and Yasunari YAMAMOTO2 1
Graduate School of Environmental and Life Science, Okayama University, Okayama and 2Mie Prefecture Livestock Research Institute, Mie, Japan
ABSTRACT As a forage source for total mixed ration (TMR) silage production, locally produced crop silage is now used in addition to imported hay. This type of TMR ensiling is regarded as a two-step fermentation process; hence, a survey was carried out to determine whether the bacteria in crop silage affect the subsequent TMR ensiling. Fermentation product contents and bacterial community were determined for TMR silage and its ingredient silages collected in August, October and November. August product contained corn, sorghum and Italian ryegrass silages, October product had wheat silage exclusively and November product did not include any crop silages. Acetic acid, lactic acid, 2,3-butanediol and ethanol were predominant fermentation products in corn, sorghum, Italian ryegrass and wheat silages, respectively. Robust lactic acid fermentation was seen in TMR silage, even if acetate-type and alcohol-type silages were mixed as ingredients. The finding that bacterial community of the TMR silage appeared unrelated to those of ingredient silage supported this. Silages of various fermentation types can therefore be formulated without interfering with lactate-type fermentation in TMR silage.
Key words: bacteria, denaturing gradient gel electrophoresis, silage, total mixed ration.
INTRODUCTION Production of total mixed ration silage is practiced in Japan as a method to utilize wet by-products as feed for ruminants. Food and beverage by-products have an unbalanced composition of nutrients, and thus mixing prior to feeding a ration is necessary, even when suitable preservation is achieved by storing wet by-products alone. Brewers grains and soybean curd residue are representative by-products; various dry feeds such as hay, corn grains, wheat bran, cotton seed cake, soybean meal and beet pulp are combined with the by-products to create a total mixed ration (TMR) mixture (Wang & Nishino 2008). Typical concentrations of dry matter (DM), crude protein (N × 6.25), and total digestible nutrients are 500–600 g/kg, 160– 180 g/kg DM, and 720–740 g/kg DM, respectively, and thus without other supplements dairy cows can perform a high level of milk production (Wang & Nishino 2010). Because small particles of by-products enable high packing density, lactic acid fermentation often dominates the ensiling process (Weinberg et al. 2011). In addition, aerobic spoilage would not take place, and thus feed intake can be kept at a high level even in hot seasons (Wang & Nishino 2013). Therefore, a number of TMR manufacturers exist in © 2014 Japanese Society of Animal Science
various regions, commercializing TMR silage for local farmers. As a forage source of TMR ingredients, manufacturers have started to use locally produced silage in addition to imported hay. Various crop silages from grass, oat, sorghum, whole crop corn and whole crop rice can be used as a single or a combined forage source; hence, present TMR ensiling may have a two-step fermentation process, where bacteria grown in the first round of crop ensiling could serve as a silage additive for the second round of TMR ensiling. Benefits would be seen if only desirable lactic acid bacteria (LAB) species were transferred from crop silage to the TMR silage. However, various undesirable bacteria may also grow in crop silage, and no previous studies have determined whether the bacteria in crop silage can affect the subsequent ensiling process. In this study, bacterial communities of TMR silage and its
Correspondence: Naoki Nishino, Department of Animal Science, Graduate School of Environmental and Life Science, Okayama University, Okayama 700-8530, Japan. (Email: [email protected]) Received 28 August 2013; accepted for publication 3 March 2014.
46 N. NISHINO et al.
ingredient crop silages were determined in order to understand bacterial transfer from crop silage to TMR silage.
MATERIALS AND METHODS Sampling of silages We collected samples of TMR silages produced at Mie Prefecture Livestock Research Institute on August 23, October 29 and November 2, 2010. For the August product, two corn, two sorghum and four wilted Italian ryegrass silages, which had been stored for 13, 12 and 4 months, respectively, were mixed into a batch with other feeds, and the mixture was then wrapped with six layers of plastic film using a roll baler for chopped material (Shito et al. 2006). For the October product, five wheat silages stored for 6 months and one wet brewers grains silage stored for 1 month were used. For the November product, timothy hay was used instead of crop silages, while the same wet brewer grains silage as used in the October product was mixed in. Since the November product was prepared just 4 days after the October product, there were no detectable changes in the wet brewers grain silage. The proportion of crop silage or hay in the TMR mixture was 0.40 on a DM basis, and 7–10 newly-baled TMR silages were prepared from one TMR mixture batch. Bales for TMR silage were stored outside for 1–2 months. Several grab samples were taken to make a composite, and about 0.5 kg of representative samples were stored at −30°C. Silage samples were shipped frozen to our laboratory at Okayama University.
Chemical and bacterial community analyses DM content was determined by oven drying at 60°C for 48 h. The fermentation products content in the silage was determined from water extracts through an ion-exclusion polymeric high-performance liquid chromatography method with refractive index detection (Han et al. 2012). Denaturing gradient gel electrophoresis (DGGE) was performed as previously described (Li & Nishino 2011). Frozen samples were thawed and added to a 19 × volume of sterilized phosphate buffered saline (pH 7.4), and extraction involved shaking the samples vigorously for 10 min at ambient temperature. Bacterial DNA was purified using a commercial kit (DNeasy Tissue Kit; Qiagen, Germantown, MD, USA). Polymerase chain reaction (PCR) was used to amplify a variable (V3) region of the bacterial 16S rRNA gene, with the forward primer GC357f (5′-CGCCCGCCGCGCGCGGCGGG CGGGGCGGGGGCACGGGGGGCCTACGGGAGGCAGCAG-3′) and the reverse primer 517r (5′-ATTACCGCGGCTGCTGG3′). The PCR protocol involved an initial denaturation step at 95°C for 10 min. This step was followed by 30 cycles of: (i) denaturation at 93°C for 30 s; (ii) annealing at (a) 65°C (first 10 cycles), (b) 60°C (second 10 cycles), and (c) 55°C (last 10 cycles) for 30 s; and (iii) extension at 72°C for 1 min. The final extension step was performed at 72°C for 5 min. The GC-clamp PCR products were separated according to their sequences by using a DCode Universal Mutation Detection System (Bio-Rad Ltd, Tokyo, Japan). The samples were applied directly onto 100 g/L (w/v) polyacrylamide gels that had a denaturing gradient ranging from 0.20 to 0.50, which then was prepared using 7 mol/L urea and 400 mL/L © 2014 Japanese Society of Animal Science
formamide, respectively, as 100% denaturants. Electrophoresis was performed at a constant voltage of 150 V for 12 h at 60°C. After electrophoresis, the gels were stained with SYBR Green (Cambrex Bio Science Inc., Rockland, ME, USA) and photographed under UV illumination. Select bands were excised from DGGE gels, and each band was placed in 10 μL of sterilized water at 4°C overnight to allow diffusion of the DNA. Extracted DNA was amplified by PCR performed using the 357f (without GC clamp) and 517r primers. The PCR products were purified using a commercial clean-up kit (GeneClean Kit; Qbiogene, Carlsbad, CA, USA), and then the purified PCR products were cloned into the pTAC-1 vector. The resulting plasmids were transformed into Escherichia coli strain DH5α competent cells (DynaExpress TA cloning kit; BioDynamics Laboratory Inc., Tokyo, Japan). The sequencing reaction was carried out using a BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems Inc., Foster City, CA, USA) and DNA base sequences were analyzed using an ABI PRISM® 310 sequencer (Applied Biosystems Inc., Foster City, CA, USA).
Data analyses Data for ensiling fermentation were expressed as mean values with standard deviation. Bacterial species were identified on Basic Local Alignment Search Tool (BLAST) searches using the GenBank database, and the closest relatives of partial 16S rRNA gene sequences were determined. Positive identifications of unknown sequences were considered similar when more than 0.99 of the sequence was identical to that in the BLAST database. Cluster analysis with both quantitative (pH and fermentation product contents) and qualitative (DGGE band profiles) data was performed to describe the similarity and differences observed within and between ingredients and TMR silages. The DGGE band profiles were analyzed using an image analysis system (Image J; National Institutes of Health, Bethesda, MD, USA), and the migration patterns of the DNA bands were converted into lists of binary numbers. Dendrograms were constructed using an unweighted pairgroup method with arithmetic averages. Cluster analysis was performed using JMP software (ver. 7; SAS Institute, Tokyo, Japan).
RESULTS Fermentation characteristics Three crop silages used in the August product were variable in fermentation product composition (Table 1). The acetic acid, 1,2-propanediol, and 1-propanol contents were much greater than the lactic acid content in corn silage. Although lactic acid dominated the fermentation, high acetic acid content was also seen in sorghum silage. In wilted grass silage, acid content was rather low and the 2,3-butanediol and ethanol contents were more than the lactic acid and acetic acid contents. Although these diverse silages were used as ingredients, TMR silage showed stable lactate-type fermentation. The lactic acid to acetic acid (L/A) ratio was greater than those of corn, sorghum and wilted grass silages, with small variation within the same batch observed as compared with those in each ingredient silage. Animal Science Journal (2015) 86, 45–50
Animal Science Journal (2015) 86, 45–50
†Mean values of two silages produced on the same day and used to prepare the same batch of total mixed ration (TMR) silage. ‡Mean values of four silages produced on the same day and used to prepare the same batch of TMR silage. §Mean values of three silages prepared from the same batch of TMR mixture. ¶Mean values of five silages produced on the same day and used to prepare the same batch of TMR silage. ††This brewers grains silage was used to prepare the October and November products.
– 0.19 – 8.40 1.60 4.02 0.00 3.18 – 0.36 0.00 2.08 – 0.00 0.00 0.00 – 0.31 2.25 3.98 – 1.48 – 5.28 6.97 14.1 – 3.72 5.69 4.50 375 535
– 44.3 0.03 56.8
0.30 0.10 0.35 1.07 0.71 2.49 0.32 7.41 1.76 0.89 3.11 4.26 6.10 1.03 25.4 28.9 2.83 10.7 7.87 50.2 1.76 32.8 7.44 13.9 2.62 21.4 17.1 4.30 2.52 4.11 288 470
0.23 13.9 0.05 53.1
0.03 0.87 0.96 0.26 0.21 1.42 1.97 2.32 5.05 0.63 0.00 1.84 31.1 1.98 0.00 5.79 30.6 4.55 0.00 0.49 37.7 20.4 0.00 16.3 0.00 0.43 4.78 0.20 0.00 1.59 7.46 1.76 0.23 0.54 2.20 0.20 1.41 2.53 5.72 2.87 0.03 10.5 0.95 50.5 11.2 0.04 57.7 21.9 44.2 11.5 0.25 4.46 2.44 2.18 0.86 0.01 52.4 2.54 22.7 1.88 4.06 3.95 5.17 4.20 16.3 7.07 68.3 4.58
Mean SD Mean SD SD Mean SD Mean Mean SD Mean SD Mean SD Mean SD Mean SD
1,2-Propanediol (g/kg DM) 2,3-Butanediol (g/kg DM) Ethanol (g/kg DM) Acetic acid (g/kg DM) Lactic acid (g/kg DM) pH DM (g/kg)
283 226 459 555
August Whole crop corn silage† Sorghum silage† Italian ryegrass silage‡ TMR silage§ October Whole crop wheat silage¶ TMR silage§ November Brewers grains silage†† TMR silage§
Table 1
Dry matter (DM), pH value and fermentation product concentration of total mixed ration silage and its ingredients silage
1-Propanol (g/kg DM)
L/A ratio
BACTERIA IN TOTAL MIXED RATION SILAGE
47
Wheat silage used exclusively in the October product showed extensive alcoholic fermentation. The ethanol content was greater than 50 g/kg DM and the lactic acid and acetic acid contents were less than onethird and half of the ethanol and 2,3-butanediol contents, respectively. Although lactate-type fermentation was greatly enhanced and the L/A ratio increased, the ethanol content was yet higher than the acetic acid content in TMR silage. Wet brewers grains silage, which was used in both the October and November products, showed desirable lactate-type fermentation. High lactic acid and low alcohol contents were also seen in the November product of the TMR silage. However, the silage pH averaged 4.50 and the value appeared greater than that of the TMR silage containing crop silage. Cluster analysis demonstrated that the TMR silage fermentation appeared stable and resembled each other regardless of the product season (Fig. 1). Also, TMR silages were grouped with other lactate-type wet brewer grains and sorghum silages, whereas distinctive separation was observed from alcohol-type wilted Italian ryegrass and wheat silages. A greater difference was found from corn silage, which contains acetic acid as the major fermentation product.
Bacterial communities No great differences were seen in the bacterial community between corn and sorghum silages used in the August product (Fig. 2); Lactobacillus acetotolerans (band 1), L. buchneri (bands 3), and Pediococcus ethanolidurans (band 5) were detected in both crop silages. Wilted grass silage appeared different from corn and sorghum silages with respect to the bacterial community. L. acetotolerans, L. buchneri and P. ethanolidurans found in corn and sorghum silages seemed to survive as bands 12, 14 and 15, respectively, in TMR silage. Moreover, bands of L. acetotolerans (band 10), Lactobacillus parafarraginis (band 9), Pediococcus goldsteinii (band 11) and Pediococcus pentosaceus (band 13) newly appeared. Several bands indicative of Enterobacter amnigenus (bands 17 and 20–23) were found in wheat silage. Unlike wheat silages 3, 4 and 5, which showed similar band patterns, wheat silage 2 had a distinctive band of L. buchneri (band 18). The bacterial community of the TMR silage appeared unrelated to that of brewers grains silage or wheat silage; L. acetotolerans (bands 24) was seen only in TMR silage, and a band in the same migrating position of E. amnigenus (band 23) in wheat silage was identified as Lactobacillus fructivorans (band 27) in TMR silage. Even so, E. amnigenus (band 26) and L. buchneri (band 25) appeared to transfer from wheat silage to TMR silage. Although bands of Bacillus coagulans (band 29) and Bacillus smithii (band 30) were found in brewers grains silage, the corresponding bands in TMR silage were © 2014 Japanese Society of Animal Science
48 N. NISHINO et al.
Based on pH and fermentaon products CS1 (Aug) CS2 (Aug) SG1 (Aug) SG2 (Aug) TMR1 (Aug) TMR2 (Aug) TMR1 (Nov) TMR2 (Nov) TMR1 (Oct) TMR2 (Oct) BG (Oct&Nov) IR1 (Aug) IR2 (Aug) IR4 (Aug) IR3 (Aug) WH1 (Oct) WH5 (Oct) WH3 (Oct) WH4 (Oct) WH2 (Oct)
Based on DGGE band paerns CS1 (Aug) SG1 (Aug) CS2 (Aug) SG2 (Aug) TMR1 (Oct) TMR2 (Oct) TMR1 (Aug) TMR2 (Aug) WH3 (Oct) WH5 (Oct) WH4 (Oct) TMR1 (Nov) TMR2 (Nov) IR1 (Aug) IR2 (Aug) WH2 (Oct) WH1 (Oct) IR3 (Aug) IR4 (Aug) BG (Oct&Nov)
1.0
0.5 Distance-based similarity
1.0
0
0.5 Distance-based similarity
0
Figure 1 Dendrograms constructed using cluster analyses on the basis of pH and fermentation products and the bacterial community are described as denaturing gradient gel electrophoresis (DGGE) profiles. The letters indicate the ingredient and total mixed ration (TMR) silage; CS, corn silage; SG, sorghum silage; IR, wilted Italian ryegrass silage; WH, wheat silage; BG, wet brewers grains silage. The following numbers indicate the sampling batch number, and the abbreviated months in parenthesis describe the August, October and November products, respectively.
identified as L. acetotolerans (band 34) and Lactobacillus casei (band 35), respectively. In addition, Lactobacillus suebicus (band 32) and Weissella paramesenteroides (bands 33 and 34) appeared in the November product of the TMR silage. Cluster analysis indicated that none of the TMR silages formed a close group with its ingredient silage; the October products were grouped with corn and sorghum silages (the August product ingredient), and the August and November products were grouped with wheat silage (October product ingredient). Moreover, two separate groups were formed from each wilted Italian ryegrass and wheat silage; hence, differences within ingredient silage bales appeared greater than that between ingredient and TMR silages. Although wet brewers grains silage was closely clustered with TMR silage based on pH and fermentation product contents, there was a considerable difference between two silages in the clustering based on DGGE band profiles. © 2014 Japanese Society of Animal Science
DISCUSSION Robust lactic acid fermentation was observed in TMR silage despite the fact that diverse fermentation products dominated over ingredient silages. In addition, variation between bales, as indicated by the standard deviation values and by the clustered groups, appeared smaller in TMR silages than in crop silages. The DM contents of TMR silage were 470–550 g/kg in this study; hence, low moisture ensiling could have supported stable lactate-type fermentation. Italian ryegrass silage used in the August product had a large amount of ethanol and 2,3-butanediol, although the DM content (459 g/kg) was close to that of the October product. Alcoholic fermentation can occur when grass is ensiled at a high DM content (Driehuis & van Wikselaar 2000); therefore, low moisture ensiling per se could not account for the robust lactic acid fermentation in the TMR silage. Rather, because many by-products of small particle size are used, TMR silage Animal Science Journal (2015) 86, 45–50
BACTERIA IN TOTAL MIXED RATION SILAGE
11
6
12
1
8 4
20 25 19
16
7
2
10
18
31 24
28
32
9
29
33
13
3
34
14
5 15
17
21 22
26
35 30
CS 1 2 SG 1 2 IR 1 2 3 4 Pre-TMR TMR 1 2 BG WH 1 2 3 4 5 Pre-TMR TMR 1 2 BG Pre-TMR TMR 1 2
23
27
August
October
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35
49
Lactobacillus acetotolerans Lactobacillus brevis Lactobacillus buchneri Lactobacillus buchneri Pediococcus ethanolidurans Uncultured bacterium Weissella cibaria Lactobacillus acetotolerans Lactobacillus parafarraginis Lactobacillus acetotolerans Pediococcus goldsteinii Lactobacillus acetotolerans Pediococcus pentosaceus Lactobacillus buchneri Pediococcus ethanolidurans Lactobacillus delbrueckii Enterobacter amnigenus Lactobacillus buchneri Enterococcus mundi Enterobacter amnigenus Enterobacter amnigenus Enterobacter amnigenus Enterobacter amnigenus Lactobacillus acetotolerans Lactobacillus buchneri Enterobacter amnigenus Lactobacillus frucvorans Lactobacillus acetotolerans Bacillus coagulans Bacillus smithii Lactobacillus suebicus Weissella paramesenteroides Weissella paramesenteroides Lactobacillus acetotolerans Lactobacillus casei
November
Figure 2 Denaturing gradient gel electrophoresis analysis of bacterial communities present in the total mixed ration silage of August, October and November products. Corn (CS), sorghum (SG) and wilted Italian ryegrass (IR) silages were used as ingredient silages for the August product, while wheat (WH) and wet brewers grains (BG) silages were used for the October product, and wet brewers grains silage was used for the November product. Pre-TMR describes a total mixed ration mixture sampled before ensiling fermentation.
has a high packing density of > 200 kg DM·m–3, which is difficult to achieve in low moisture grass silage (Kida et al. 2007). Robust lactic acid fermentation could thus be due to distinctive or nonconventional ensiling conditions of low moisture and high density storage. L. acetotolerans, L. brevis, L. buchneri, P. ethanolidurans, W. cibaria, L. delbrueckii, E. mundtii were the LAB species identified in ingredient silage in this study, and bands showing the same migrating position, except for L. delbrueckii, were found in TMR silage. In contrast, although three non-LAB species (E. amnigenus, B. coagulans and B. smithii) were identified in ingredient silage, corresponding bands were undetectable, faint or replaced by LAB species in TMR silage. These findings suggest that LAB rather than non-LAB species could be transferred more likely from ingredient to TMR silage. However, we did not determine bacterial numbers in this study, and thus, the higher transfer of Animal Science Journal (2015) 86, 45–50
LAB than non-LAB species could be due to differences in the populations in ingredient silage. Meanwhile, cluster analysis indicated that the bacterial community of TMR silage was not closely related with those of ingredient silage. Likewise, there were newly appeared LAB species in TMR silage, such as L. parafarraginis, P. goldsteinii, L. fructivorans, L. suebicus, W. paramesenteroides and L. casei. Overall, the bacterial community of TMR silage is considered largely independent from those of ingredient silage. In the present DGGE analysis, occasionally multiple bands detected at different migrating positions were identified as the same bacterial species. This may have occurred due to the presence of multiple heterogeneous 16S rRNA gene copies in a bacterium, and heteroduplex molecules formed during the PCR amplification process (Muyzer & Smalla 1998). Although DGGE analysis can provide a rapid and © 2014 Japanese Society of Animal Science
50 N. NISHINO et al.
repeatable characterization of the bacterial community, the resolution level is known to be low. Co-migration of DNA from multiple species may also occur (Sekiguchi et al. 2001); hence, we performed cloning to secure pure bacterial DNA for species identification. In TMR silage L. acetotolerans (band 24) was seen even without a detectable corresponding band in ingredient wheat silage. Likewise, although L. buchneri (band 25) was distinctively seen in the October product, only one of five ingredient wheat silages had L. buchneri in their bacterial community. Therefore, TMR silage can be a good habitat for L. acetotolerans and L. buchneri; their growth may have been favored even if the initial population was small. Use of crop silage as a TMR ingredient might have supported inhabitation of L. acetotolerans, because the November product, in which no crop silages were mixed, did not have L. acetotolerans. Likewise, although various commercial products were surveyed before, we did not find L. acetotolerans in the TMR silage with no crop silages (Wang & Nishino 2010). L. buchneri is known to anaerobically metabolize lactic acid into acetic acid and 1,2-propanediol, which can help inhibit aerobic deterioration after silo opening (Oude Elferink et al. 2001). Since corn and sorghum silages had substantial amounts of acetic acid and 1,2-propanediol, detection of the distinctive bands of L. buchneri was reasonable. Also, since L. acetotolerans can tolerate high acetic acid levels such as 30 g/L (Entani et al. 1986), the presence of the bacterium in corn and sorghum silages was plausible. L. acetotolerans has become a normal inhabitant by taking advantage of the culture-independent procedure (Li & Nishino 2011; Wang & Nishino 2013), although isolation of the LAB species from silage has not yet been reported. This study revealed that LAB species can be selected during the ensiling process of TMR silage rather than during transfer and survival of LAB and non-LAB bacteria present in ingredient crop silages. Therefore, it is concluded that silages of various fermentation types can be formulated without interfering with the lactatetype fermentation in TMR silage.
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Animal Science Journal (2015) 86, 45–50
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