Microbiology of Ensiling. R. E. Muck U.S. Dairy Forage Research Center Madison, WI

Transcription

1 Microbiology of Ensiling R. E. Muck U.S. Dairy Forage Research Center Madison, WI

2 Silage Microbiology The Way It Was Selective media for each group of microorganisms i Counts of culturable microorganisms Very laborious procedures to identify the species of the colonies on the agar And still struggled to know cause and effect in the silo

3 Outline Recent microbial techniques New silage species discoveredd Factors affecting silage populationsp Aerobic deterioration How inoculants affect silage, livestock Strides to find new inoculants Conclusions and future directions

4 Recent Microbial Techniques Polymerase Chain Reaction (PCR) Key to most new analyses Many copies of a specific portion of the DNA Cycle 1 Cycle 2 Cycle 3

5 Taxonomy Bacteria Yeasts and Moulds 16S ribosomal RNA 18S ribosomal RNA gene (16S rrna) gene (18S rrna) These genes are highly variable in nucleotide sequence from one species to another, permitting classification but.. There are highly hl conserved regions of the genes across species for PCR primers, allowing amplification of the gene or gene regions

6 Use of PCR for Individual Species/Strains Identify an unknown species Pick a colony from a plate Amplify the 16S rrna gene using PCR Sequence the gene Use a program such as BLAST to identify Use a program such as BLAST to identify the most likely species

7 Use of PCR for Individual Species/Strains Quantify a known species (real-time or quantitative real-time PCR) Develop primers specific for a species Amplify that section of DNA from a silage extract Based on the number of cycles of amplification to reach a set number of copies, you have a measure of the quantity of that species

8 Use of PCR for Community Analysis Snapshots of what species are most prevalent at a given time Four different community techniques have been used in silage studies: LH-PCR T-RFLP ARISA DGGE

9 Use of PCR for Community Analysis 3 of 4 techniques use differences in the lengths of amplified sequences for identifying species Typically separated by capillary electrophoresis LH-PCR: length-heterogeneity PCR Amplify a region of the 16S rrna gene (Brusetti et al. 2006) T-RFLP: terminal restriction fragment length polymorphism Amplify 16S rrna gene, cut DNA with endonuclease (McEniry et al., 2008) ARISA: automated ribosomal intergenic spacer analysis Amplify the region between the 16S rrna and 23S rrna genes (Brusetti et al. 2008)

10 Community Analysis by Length- Based Techniques (Brusetti et al. 2006) Pros Consistent results Easy to port into statistical programs Cons Multiple species having the same length (LH-PCR, T- RFLP, particularly) Multiple peaks for one species (ARISA) Considerable work to identify specific species

11 DGGE: Denaturing Gradient Gel Electrophoresis Amplify 16S rrna gene or region of that gene Separated on a gel Distance travelled affected by nucleotide sequence as well as length (Li and Nishino 2011)

12 DGGE: Denaturing Gradient Gel Electrophoresis Pros Bands can be excised and cloned for species identification Cons More qualitative results Variability from one gel to the next

13 New/Unusual Species Isolated From Silages Lactic acid bacteria Enterococcus flavescens Entercoccus mundtii Lactobacillus acetotolerans Lactobacillus panis Lactobacillus reuteri Lactobacillus taiwanensis (newly described) Leuconostoc lactis Paralactobacillus selangorensis Pediococus dextrinicus Pediococcus lolii lii (newly described) Pediococcus parvulus Weissella cibaria Weissella kimchii Weissella paramesenteriodes

14 New/Unusual Species Isolated From Silages Anaerobic Spore Formers Clostridium baratii Paenibacillus ill macerans Bacillus Bacillus megaterium Enterobacteria Erwinia persicina Pantoea agglomerans Rahnella aquatilis Acetic Acid Bacteria Acetobacter pasteurianus Yeasts Candida apicola Candida intermedia Candida glabrata Candida magnolia Candida mesenterica Candida quercitrusa Saccharomyces martiniae Pichia deserticola Pichia kudriavzevii

15 New/Unusual Species Isolated From Silages What does it mean to find all these new species in silage? Not sure yet because we still cannot determine cause and effect yet In spite of identifying new species, the familiar species are often the most dominant: L. plantarum, L. brevis, P. pentosaceus, P. acidilactici, Lactococcus lactis, etc.

16 Factors Affecting Silage Microbial Populations Silo type: Baled vs. precision chop perennial ryegrass (McEniry et al. 2010) Wrapped bale vs. vacuum-packed bag (Naoki and Yuji 2008) Both studies little difference in dominant populations

17 Factors Affecting Silage Microbial Populations Compaction: Perennial ryegrass (McEniry et al. 2010) Little effect on most of the prevalent species observed, but increased density had a: Negative effect on some LAB, enterobacteria species Positive effect on clostridia

18 Factors Affecting Silage Microbial Populations Dry Matter Concentration: Perennial ryegrass (McEniry et al. 2010) Greater prevalence of enterobacteria in drier silage (185 vs. 406 g DM/kg) Guinea grass (Parvin and Nishino 2009) Lactococcus lactis and L. brevis important at 15 d in both DM s (286, 443 g/kg) g) During storage, L. lactis declined and L. pentosus increased in wetter silage along with a shift to acetic acid L. plantarum - important in drier silage; L. pentosus appeared but little apparent effect on fermentation

19 Factors Affecting Silage Microbial Populations Climate? Maize, Italy (Brusetti et al. 2006) P. pentosaceus and W. confusa most prevalent at ensiling and present throughout 30 d Maize, Colombia (Villa et al. 2010) Variety grown under cool climate, fermentation ti was dominated by Lactobacillus and Pediococcus species Variety grown under warm climate, fermentation appeared to have contributions from Leuconostoc species in addition to Lactobacillus and Pediococcus.

20 Factors Affecting Silage Microbial Populations Cultivar Sugarcane (Ávila et al. 2010) Yeast counts in 5 cultivars at 10, 20, 30 and 40 d Highest counts at 10 d in 3 cultivars; 30 d in other 2 cultivars 4 species at 10 d (Torulaspora delbrueckii, Pichia anomala, Saccharomyces cerevisiae and Candida glabrata) Increasing species with time but By cultivar: 1 cultivar only 2 species of yeast 3cultivars 5 species of yeast 1 cultivar 7 species of yeast

21 Aerobic Stability Effect of plastic film (polyethylene vs. oxygen barrier film) on maize silage stability from bag silos (Dolci et al. 2011) Both bags inoculated with L. buchneri, L. plantarum and E. faecium. L. buchneri dominant band at opening in both silages Heating in polyethylene treatment appeared linked to the rise of Acetobacter pasteurianus Heating in the oxygen barrier treatment was linked to the rise of yeast (Kazachstania exigua)

22 Aerobic Stability Estimating mould counts, ph on the faces of maize bunker silos (Borreani and Tabacco 2010) Took samples (200 mm depth) across the face of 54 farm silos, also measuring temperature at 200 mm Measured temperature at 400 mm at center of face Mould counts, ph were strongly correlated to the difference in temperatures Yeasts, lactic acid were less well correlated (R 2 =0.51) Mould Count = 6.12 M dt 0.13 M dt , R 2 =0.84

23 Aerobic Stability Stability of maize from bunker silos (Tabacco et al. 2011) Yeast counts correlated with: Feed out rate (-0.579) Lactic acid (0.549) ph (-0.456) DM density (-0.451) L:A ratio (0.437) DM concentration (-0.373) Acetic acid (-0.331)

24 Aerobic Stability Clostridial growth during aerobic deterioration of maize silage (Tabacco et al. 2009)

25 Silage Inoculants Inoculants have been available for decadesd Three principal types Facultative heterofermenters like L. plantarum (commonly homofermenters) Obligate heterofermenters like L. buchneri Combination products

26 How Do Inoculants Dominate Silage Fermentation? With homofermenters, we assume the inoculant strains are faster than the competition. With L. buchneri, we assume it is a good survivor because these strains are slow. But are there other factors to their But are there other factors to their success?

27 How Do Inoculants Dominate Silage Fermentation? Gollop et al. (2005): 9 of 10 inoculants/strains produced antibacterial activity when grown in broth Extracts from 15 of 27 silages made with the 9 positive strains had antibacterial activity

28 How Do Inoculants Dominate Silage Fermentation? Vazquez et al. (2005): Studied 6 LAB strains that produce bacteriocins Bacteriocin from a particular strain increased both the growth and bacteriocin production of that strain Bacteriocin from one strain added to another most often reduced growth, bacteriocin production in the second strain However, in some cases growth and bacteriocin production were increased in the second strain

29 How Do Inoculants Dominate Silage Fermentation? Antifungal activity (Broberg et al. 2007; Prema et al. 2010): L. plantarum strains, 2 of 3 from grass silage 3-phenyllactic acid identified in one study with broad activity against silage moulds 3-phenyllactic acid, 3-hydroxydecanoic acid in inoculated silages in the other study

30 How Do Inoculants Affect Animal Performance? Kung and Muck (2007): Approximately 50% of studies reviewed reported positive effects of inoculated silage on milk production or gain, averaging 3 to 5% increase But how can LAB cause such increases?

31 How Do Inoculants Affect Animal Performance? Comparison of the effects of L. plantarum MTD/1 on silage fermentation and animal performance across studies Animal Fermentation Improved Digestibility Improved Performance No Yes No Yes Improved No Yes Review of Weinberg and Muck (1996)

32 How Do Inoculants Affect Animal Performance? Adding inoculant bacteria to strained rumen fluid (Weinberg et al. 2003) LAB levels remained relatively constant over 72 h incubation Little effect on volatile fatty acids Most strains raised ph vs. control

33 How Do Inoculants Affect Animal Performance? Effects on gas production from in vitro ruminal fermentation Approximately 2/3 of inoculated lucerne silages (14 strains/products) produced less gas than untreated control (Muck et al. 2007) An L. plantarum strain on TMR silage (Cao et al. 2010) Reduced methane vs. control (9.6, 10.5 L/kg DDM) Increased propionate, reduced butyrate

34 How Do Inoculants Affect Animal Performance? Effects on microbial biomass production from in vitro ruminal fermentation ti Inoculant Treatment VFA (mm) Gas (ml/g DM) MBY (mg/g TDDM) Control b LP-EF b LP a LPe a LL a Contreras-Govea et al. (2011)

35 How Do Inoculants Affect Animal Performance? Increased ruminal microbial biomass production in vivo?? Response Control LP P DM Intake, kg/d Milk, kg/d Milk/DMI Fat, % Protein, % Lactose, % <0.001 Milk Urea N, mg/dl <0.001 Muck et al. (2011)

36 Strides To Find New Inoculants Screening for new inoculants Start with a goal Find strains that meet that goal Th d t i hi h t i fl i h Then determine which strains can flourish in the silo

37 Strides To Find New Inoculants Screening for new inoculants (Saarisalo et al. 2007) Goal: broad spectrum antimicrobial activity, both bacterial and fungal Selected 9 strains with those characteristics Grew 9 strains on grass extract (measuring products, ph, growth rate, NH 3 -N) and on API 50 CHL test kits Selected 4 strains: rapid growth, high lactic, grow on many sugars, low ph, low NH 3 -N y g 3 Tested 4 strains in making grass silage, selecting the best

38 Strides To Find New Inoculants Goals for new inoculants Most common: rapid growth, producing high lactic acid and low NH 3 (e.g., Kim et al. 2008, 2009) Antimicrobial activity (Saarisalo et al. 2007; Marcinakova et al. 2008) Low ethanol and yeast counts (Ávila et al. 2009, 2010) High h crude protein, low fibre (Pasebani et al. 2011) Increased methane yield from anaerobic digestion of silage (Banemann et al. 2010) Increased animal performance, efficiency?

39 Conclusions and Future Directions The new PCR-based techniques are making it easier to know the microbial ecology of ensiling But we have more to learn about cause and effect in the silo We need more snapshots of the microbial community, particularly early in fermentation and over a wide range of conditions and locations

40 Conclusions and Future Directions We need to pair these snapshots with analyses of more than just major silage fermentation products Metabolomics to identify more minor products Various antimicrobial compounds

41 Thank you for your attention