Anaerobic Spore Formers. â« Clostridium baratii. P ib ill. â« Yeasts. â« Candida apicola. C did i di. â« Paenibacillus macerans. â« Bacillus. â« Bacillus megaterium.
Microbiology of Ensiling R. E. Muck U.S. Dairy Forage R Research hC Center t Madison, WI
Silage g Microbiology gy – The Way It Was
Selective media for each group of microorganisms i 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
Outline
Recent microbial techniques
N New silage il species i di discovered d
Factors affecting g silage g p populations p
Aerobic deterioration
How inoculants affect silage, livestock
Strides to find new inoculants
Conclusions and future directions
Recent Microbial Techniques Polymerase Chain Reaction (PCR)
Keyy to most new analyses
Many copies of a specific portion of the DNA
Cycle 1 Cycle 2 Cycle 3
Taxonomy Bacteria
16S ribosomal RNA gene ((16S rRNA)) g
Yeasts and Moulds
18S ribosomal RNA gene ((18S rRNA)) g
These g genes are highly g y variable in nucleotide sequence from one species to another, permitting classification but.. There Th are hi highly hl conserved d regions i off th the genes across species for PCR primers, allowing amplification of the gene or gene regions
Use of PCR for Individual Species/Strains Identify an unknown species
Pick a colony from a plate
Amplify the 16S rRNA gene using PCR
S Sequence the th gene
Use a program such as BLAST to identify the most likely species
Use of PCR for Individual Species/Strains Quantify a known species (real-time or quantitative real-time 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 p to reach a set number of copies, you have a measure of the quantity of that species
Use of PCR for Community y Analysis
Snapshots of what species are most prevalent at a given time
Four different communityy techniques q have been used in silage studies: LH PCR LH-PCR T-RFLP ARISA DGGE
Use of PCR for Community y 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
A lif a region Amplify i off th the 16S rRNA RNA gene (Brusetti et al. 2006)
T-RFLP: terminal restriction fragment length polymorphism
Amplify 16S rRNA gene, gene cut DNA with endonuclease (McEniry et al., 2008)
ARISA: automated ribosomal intergenic spacer analysis
A lif th Amplify the region i b between t th the 16S rRNA RNA and d 23S rRNA RNA genes (Brusetti et al. 2008)
Community y Analysis y byy Lengthg Based Techniques
Pros
Cons
(Brusetti et al. 2006)
Consistent results Easy to port into statistical programs Multiple species having the same length (LH-PCR, (LH PCR, TT RFLP, particularly) Multiple peaks for one species (ARISA) Considerable work to identify specific species
DGGE: Denaturing g 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)
DGGE: Denaturing g Gradient Gel Electrophoresis Pros
Bands can be excised and cloned for species identification
Cons
More qualitative results
Variability from one gel to the next
New/Unusual Species p 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 P di Pediococcus l lii (newly lolii ( l described) Pediococcus p parvulus Weissella cibaria Weissella kimchii Weissella paramesenteriodes
New/Unusual Species p Isolated From Silages
Anaerobic Spore Formers
Bacillus
Bacillus megaterium
Enterobacteria
Clostridium baratii P Paenibacillus ib ill macerans
Erwinia persicina Pantoea agglomerans Rahnella aquatilis
Acetic Acid Bacteria
Acetobacter pasteurianus
Yeasts
Candida apicola C did intermedia Candida i di Candida glabrata Candida magnolia Candida mesenterica Candida quercitrusa Saccharomyces martiniae Pichia deserticola Pichia kudriavzevii
New/Unusual Species p 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 species, the familiar species are often the most dominant: L plantarum, L. plantarum L. L brevis, brevis P. P pentosaceus, pentosaceus P. P acidilactici, Lactococcus lactis, etc.
Factors Affecting g Silage g Microbial Populations
Silo type: Baled vs. precision chop perennial ryegrass (McEniry et al. 2010) Wrapped bale vs. vacuum-packed bag ((Naoki and Yujij 2008)) Both studies – little difference in dominant populations
Factors Affecting g Silage g Microbial Populations
Compaction: Perennial ryegrass (McEniry et al. 2010) Little effect on most of the p prevalent species p observed, but increased density had a:
• Negative g effect on some LAB,, enterobacteria species • Positive effect on clostridia
Factors Affecting g Silage g Microbial Populations
Dry Matter Concentration:
Perennial ryegrass (McEniry et al al. 2010) • Greater prevalence of enterobacteria in drier silage (185 vs. 406 g DM/kg)
G i Guinea grass (P (Parvin i and d Nishino Ni hi 2009) • Lactococcus lactis and L. brevis important at 15 d in both DM’s ((286,, 443 g/kg) g 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
Factors Affecting g Silage g 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) • V Variety i t grown under d cooll climate, li t ffermentation t ti was dominated by Lactobacillus and Pediococcus species • Variety grown under warm climate, climate fermentation appeared to have contributions from Leuconostoc species in addition to Lactobacillus and Pediococcus.
Factors Affecting g Silage g Microbial Populations
Cultivar
Sugarcane (Ávila et al 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 g species p with time but… • By cultivar: • 1 cultivar – only 2 species of yeast • 3 cultivars – 5 species of yeast • 1 cultivar – 7 species of yeast
Aerobic Stability Effect of plastic film (polyethylene vs. oxygen barrier film) on maize silage stability from bag silos (D l i ett al. (Dolci l 2011)
Both bags inoculated with L. buchneri, L. plantarum and E. E faecium. 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)
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 g 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 (R2=0.51)
Mould Count = 6.12 M + 0.10 dT – 0.13 M dT +2 2.27, 27 R2=0.84 =0 84
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 (0.437) 437) DM concentration (-0.373) Acetic acid (-0 (-0.331 331)
Aerobic Stability Clostridial growth during aerobic deterioration of maize silage (Tabacco et al. 2009)
Silage Inoculants
Inoculants have been available for d decades d
Three principal types Facultative heterofermenters like L. plantarum l t ( (commonly l h homofermenters) f t ) Obligate heterofermenters like L. buchneri Combination products
How Do Inoculants Dominate Silage Fermentation?
With homofermenters, we assume the i inoculant l t strains t i are ffaster t than th the th competition.
With L. buchneri, we assume it is a good survivor i b because th these strains t i are slow. l
But are there other factors to their success?
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 9p positive strains had antibacterial activity y
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
How Do Inoculants Dominate Silage Fermentation?
Antifungal activity (Broberg et al. 2007; P Prema ett al.l 2010): 2010) L. plantarum strains, 2 of 3 from g grass silage g 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
How Do Inoculants Affect Animal Performance?
Kung and Muck (2007): Approximately 50% off studies t di reviewed i d reported t d positive iti effects of inoculated silage on milk production or gain, averaging 3 to 5% increase
But how can LAB cause such increases?
How Do Inoculants Affect Animal Performance? Comparison of the effects of L. plantarum MTD/1 on silage fermentation and animal performance across studies Animal Performance Improved
Fermentation Improved p Digestibility g y Improved p No
Yes
No
Yes
No
1
1
1
2
Yes
2
6
1
3
Review of Weinberg and Muck (1996)
How Do Inoculants Affect Animal Performance? Adding inoculant bacteria to strained rumen fluid fl id (Weinberg (W i b ett al.l 2003)
LAB levels remained relatively constant over 72 h incubation
Little effect on volatile fatty acids
Most strains raised pH vs. control
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. A L plantarum l t strain t i on TMR silage il (C (Cao ett al. l 2010)
Reduced methane vs. vs control (9.6, (9 6 10.5 10 5 L/kg DDM) Increased propionate, reduced butyrate
How Do Inoculants Affect Animal Performance? Effects on microbial biomass production f from i vitro in it ruminal i l fermentation f t ti Inoculant Treatment
VFA (mM)
Gas (mL/g DM)
MBY (mg/g TDDM)
Control
51.7
158
354b
LP-EF
50.3
158
355b
LP
50.5
157
379a
LPe
52.4
162
390a
LL
53.3
162
380a
Contreras-Govea et al. (2011)
How Do Inoculants Affect Animal Performance? Increased ruminal microbial biomass production d ti in i vivo? i ? Response
Control
LP
P
DM Intake, kg/d
25.4
25.8
0.072
Milk, kg/d
39.6
40.4
0.027
Milk/DMI
1 56 1.56
1 57 1.57
0 379 0.379
Fat, %
3.80
3.79
0.984
Protein % Protein,
2 81 2.81
2 78 2.78
0 048 0.048
Lactose, %
4.82
4.89
