Microbiology of Ensiling - Luonnonvarakeskus...Cons Multiple species having the same length (LHsame...
Transcript of Microbiology of Ensiling - Luonnonvarakeskus...Cons Multiple species having the same length (LHsame...
Microbiology of Ensiling
R. E. Muck
U.S. Dairy Forage R h C tResearch Center Madison, WI
Silage Microbiology g gy– The Way It Was Selective media for each group of
i imicroorganisms
Counts of culturable microorganisms Counts of culturable microorganisms
Very laborious procedures to identify theVery laborious procedures to identify the species of the colonies on the agar
And still struggled to know cause and effect in the siloeffect in the silo
Outline Recent microbial techniques
N il i di d New silage species discovered Factors affecting silage populationsg g p p Aerobic deterioration How inoculants affect silage, livestock Strides to find new inoculants Strides to find new inoculants Conclusions and future directions
Recent Microbial TechniquesPolymerase Chain Reaction (PCR)Reaction (PCR)
Key to most new yanalyses
Many copies of a Many copies of a specific portion of the DNADNA
Cycle 1 Cycle 2 Cycle 3
TaxonomyBacteria Yeasts and Moulds
16S ribosomal RNA gene (16S rRNA)
18S ribosomal RNA gene (18S rRNA)g ( ) g ( )
These genes are highly variable in g g ynucleotide sequence from one species to another, permitting classification but..Th hi hl d i f th There are highly conserved regions of the genes across species for PCR primers, allowing amplification of the gene or geneallowing amplification of the gene or gene regions
Use of PCR for Individual Species/StrainsIdentify an unknown species
Pick a colony from a plate
Amplify the 16S rRNA gene using PCR
S th Sequence the gene
Use a program such as BLAST to identify Use a program such as BLAST to identify the most likely species
Use of PCR for Individual Species/StrainsQuantify a known species (real-time or quantitative real-time PCR)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 pcopies, you have a measure of the quantity of that species
Use of PCR for Community yAnalysis Snapshots of what species are most
prevalent at a given timeprevalent at a given time
Four different community techniques have y qbeen used in silage studies: LH-PCR LH PCR T-RFLP ARISA ARISA DGGE
Use of PCR for Community yAnalysis 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 i f th 16S RNA 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 Amplify 16S rRNA gene, cut DNA with endonuclease (McEniry et
al., 2008)
ARISA: automated ribosomal intergenic spacer analysis A lif th i b t th 16S RNA d 23S RNA Amplify the region between the 16S rRNA and 23S rRNA genes (Brusetti et al. 2008)
Community Analysis by Length-y y y gBased Techniques
Pros Consistent results Easy to port into statistical
programs
Cons Multiple species having the
same length (LH-PCR, T-same length (LH PCR, TRFLP, particularly)
Multiple peaks for one species (ARISA)species (ARISA)
Considerable work to identify specific species
(Brusetti et al. 2006)
DGGE: Denaturing Gradient gGel Electrophoresis Amplify 16S rRNA
gene or region of thatgene or region of that gene
Separated on a gel
Distance travelled Distance travelled affected by nucleotide sequencenucleotide sequence as well as length
(Li and Nishino 2011)
DGGE: Denaturing Gradient gGel Electrophoresis Pros Cons
Bands can be excised and cloned
More qualitative results
for species identification Variability from one
gel to the nextgel to the next
New/Unusual Species Isolated pFrom Silages Lactic acid bacteria
Enterococcus Leuconostoc lactis Paralactobacillus Enterococcus
flavescens Entercoccus mundtii
selangorensis Pediococus dextrinicus
P di l lii ( l Lactobacillus acetotolerans
Lactobacillus panis
Pediococcus lolii (newly described)
Pediococcus parvulus Lactobacillus panis Lactobacillus reuteri Lactobacillus
p Weissella cibaria Weissella kimchii
taiwanensis (newly described)
Weissellaparamesenteriodes
New/Unusual Species Isolated pFrom Silages Anaerobic Spore Formers
Clostridium baratiiP ib ill
Yeasts Candida apicola
C did i di Paenibacillus macerans
Bacillus Bacillus megaterium
Candida intermedia Candida glabrata Candida magnolia
Bacillus megaterium
Enterobacteria Erwinia persicina
Candida mesenterica Candida quercitrusa Saccharomyces martiniae
Pantoea agglomerans Rahnella aquatilis
Acetic Acid Bacteria
Pichia deserticola Pichia kudriavzevii
Acetic Acid Bacteria Acetobacter pasteurianus
New/Unusual Species Isolated pFrom SilagesWhat does it mean to find all these new species in silage?species in silage?
Not sure yet because we still cannot determine cause and effect yet
In spite of identifying new species the In spite of identifying new species, the familiar species are often the most dominant: L plantarum L brevis P pentosaceus PL. plantarum, L. brevis, P. pentosaceus, P. acidilactici, Lactococcus lactis, etc.
Factors Affecting Silage g gMicrobial Populations Silo type:
Baled vs. precision chop perennial ryegrass (McEniry et al. 2010)
Wrapped bale vs. vacuum-packed bag (Naoki and Yuji 2008)( j )
Both studies – little difference in dominant populationspopulations
Factors Affecting Silage g gMicrobial Populations Compaction:
Perennial ryegrass (McEniry et al. 2010) Little effect on most of the prevalent species p p
observed, but increased density had a:• Negative effect on some LAB, enterobacteria g ,
species• Positive effect on clostridia
Factors Affecting Silage g gMicrobial Populations Dry Matter Concentration:
Perennial ryegrass (McEniry et al 2010) Perennial ryegrass (McEniry et al. 2010)• Greater prevalence of enterobacteria in drier silage
(185 vs. 406 g DM/kg)G i (P i d Ni hi 2009) Guinea grass (Parvin and Nishino 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 Silage g gMicrobial Populations Climate?
( 2006) Maize, Italy (Brusetti et al. 2006)• P. pentosaceus and W. confusa most prevalent at
ensiling and present throughout 30 densiling and present throughout 30 d Maize, Colombia (Villa et al. 2010)
V i t d l li t f t ti• Variety grown under cool climate, fermentation was dominated by Lactobacillus and Pediococcus species
• Variety grown under warm climate fermentationVariety grown under warm climate, fermentation appeared to have contributions from Leuconostocspecies in addition to Lactobacillus and Pediococcus.
Factors Affecting Silage g gMicrobial Populations Cultivar
Sugarcane (Ávila et al 2010) 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…g p• By cultivar:
• 1 cultivar – only 2 species of yeast• 3 cultivars – 5 species of yeast• 3 cultivars – 5 species of yeast• 1 cultivar – 7 species of yeast
Aerobic StabilityEffect of plastic film (polyethylene vs. oxygen barrier film) on maize silage stability from bag silos (D l i t l 2011)(Dolci et al. 2011) Both bags inoculated with L. buchneri, L. plantarum
and E faeciumand 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)
Aerobic StabilityEstimating 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
( )
, gtemperature at 200 mm
Measured temperature at 400 mm at center of facemm at center of face
Mould counts, pH were strongly correlated to the difference in temperaturesdifference 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 27 R2=0 84+ 2.27, R =0.84
Aerobic StabilityStability 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) L:A ratio (0.437) DM concentration (-0.373)
Acetic acid (-0 331) Acetic acid (-0.331)
Aerobic StabilityClostridial growth during aerobic deterioration of maize silage (Tabacco et al. 2009)
Silage Inoculants Inoculants have been available for
d ddecades
Three principal types Three principal types Facultative heterofermenters like L.
l t ( l h f t )plantarum (commonly homofermenters) Obligate heterofermenters like L. buchneri Combination products
How Do Inoculants Dominate Silage Fermentation? With homofermenters, we assume the
i l t t i f t th thinoculant strains are faster than the competition.
With L. buchneri, we assume it is a good i b th t i lsurvivor because these strains are slow.
But are there other factors to their 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 9 positive strains had antibacterial activityp y
How Do Inoculants Dominate Silage Fermentation? Vazquez et al. (2005):
Studied 6 LAB strains that produce bacteriocins Studied 6 LAB strains that produce bacteriocins Bacteriocin from a particular strain increased
both the growth and bacteriocin production ofboth the growth and bacteriocin production of that strain
Bacteriocin from one strain added to another Bacteriocin from one strain added to another most often reduced growth, bacteriocinproduction in the second strain
However, in some cases growth and bacteriocinproduction were increased in the second strain
How Do Inoculants Dominate Silage Fermentation? Antifungal activity (Broberg et al. 2007;
P t l 2010)Prema et al. 2010): L. plantarum strains, 2 of 3 from grass silageg g 3-phenyllactic acid identified in one study
with broad activity against silage mouldswith broad activity against silage moulds 3-phenyllactic acid, 3-hydroxydecanoic acid
in inoculated silages in the other studyin inoculated silages in the other study
How Do Inoculants Affect Animal Performance? Kung and Muck (2007): Approximately
50% f t di i d t d iti50% of studies reviewed reported positive effects of inoculated silage on milk production or gain, averaging 3 to 5% increaseincrease
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 Fermentation Improved Digestibility Improved
fermentation and animal performance across studies
PerformanceImproved
p g y pNo Yes No Yes
No 1 1 1 2No 1 1 1 2Yes 2 6 1 3
Review of Weinberg and Muck (1996)
How Do Inoculants Affect Animal Performance?Adding inoculant bacteria to strained
fl id (W i b t l 2003)rumen fluid (Weinberg et al. 2003)
LAB levels remained relatively constant 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 fermentationfermentation Approximately 2/3 of inoculated lucerne silages
(14 strains/products) produced less gas than(14 strains/products) produced less gas than untreated control (Muck et al. 2007)
A L l t t i TMR il (C t l An L. plantarum strain on TMR silage (Cao et al. 2010)
Reduced methane vs control (9 6 10 5 L/kg DDM) Reduced methane vs. control (9.6, 10.5 L/kg DDM) Increased propionate, reduced butyrate
How Do Inoculants Affect Animal Performance?Effects on microbial biomass production f i it i l f t tifrom in vitro ruminal fermentation
Inoculant Treatment
VFA (mM)
Gas (mL/g DM)
MBY (mg/gTDDM)
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
d ti i i ?production in vivo?Response Control LP PDM Intake, kg/d 25.4 25.8 0.072Milk, kg/d 39.6 40.4 0.027Milk/DMI 1 56 1 57 0 379Milk/DMI 1.56 1.57 0.379Fat, % 3.80 3.79 0.984Protein % 2 81 2 78 0 048Protein, % 2.81 2.78 0.048Lactose, % 4.82 4.89 <0.001Milk Urea N, mg/dL 12.7 11.6 <0.001
Muck et al. (2011)
Strides To Find New InoculantsScreening 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
Strides To Find New InoculantsScreening for new inoculants (Saarisalo et al. 2007) Goal: broad spectrum antimicrobial activity both Goal: broad spectrum antimicrobial activity, both
bacterial and fungal Selected 9 strains with those characteristics Selected 9 strains with those characteristics Grew 9 strains on grass extract (measuring products,
pH, growth rate, NH3-N) and on API 50 CHL test kitsp , g , 3 ) Selected 4 strains: rapid growth, high lactic, grow on
many sugars, low pH, low NH3-Ny g 3
Tested 4 strains in making grass silage, selecting the best
Strides To Find New InoculantsGoals for new inoculants Most common: rapid growth, producing high lactic acid andMost common: rapid growth, producing high lactic acid and low NH3 (e.g., Kim et al. 2008, 2009)Antimicrobial activity (Saarisalo et al. 2007; Marcinakova et al. 2008)2008)Low ethanol and yeast counts (Ávila et al. 2009, 2010)
Hi h d t i l fib (P b i t l 2011)High crude protein, low fibre (Pasebani et al. 2011)Increased methane yield from anaerobic digestion of silage (Banemann et al. 2010)( )Increased animal performance, efficiency?
Conclusions and Future Directions The new PCR-based techniques are making
it easier to know the microbial ecology ofit easier to know the microbial ecology of ensiling
But we have more to learn about cause and But we have more to learn about cause and effect in the silo W d h t f th i bi l We need more snapshots of the microbial community, particularly early in fermentation and over a wide range of conditions andand over a wide range of conditions and locations
Conclusions and Future Directions We need to pair these snapshots with
l f th j t j ilanalyses of more than just major silage fermentation products Metabolomics to identify more minor
productsp Various antimicrobial compounds
Thank you for your attention