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