Credit seminar on

In relation to methane mitigation in ruminants

Speaker: Arumbaka Sudheer Babu,

Saccharomyces cerevisiae

Introduction

Out of the total global green house effects caused by the GH gases, 61% is accounted for by CO2, 19% by CH4 and 6% by N2O. Contribution of different countries to total GHGs. Country China (%) Total 17

United State
European union Indonesia India Russia Brazil

16
11 6 5 5 4
Kyoto protocol 2007

Livestock population (in millions) of India FAO,2006)

Species
Cattle Buffaloes Sheep Goat Pigs Camels Donkeys Yaks, mithuns, mules etc

Population
226.1 96.9 59 124.9 18.5 0.9 0.9 Approx. <0.9

Above population accounts for 11.95% of the global livestock population. Producing 12.45% of the total enteric CH4 emissions

Kamra et al.,2010 Contribution of Indian livestock for methane production as reported by various workers
Reference Khan (1996) Singh (1998) Garg and Shukla (2003) Singhal et al (2005) Reported value (Tg/Year) 8.9 9.023 7.257 10.08

Harmful effects of high concentration of methane in atmosphere

1.

Global warming

Too much of IR radiation trapped causing green house effect

Average global temperatures will rise by the year 2030, world is likely to be 12 C warmer than today.

2.

Ozone layer depletion

Penetration of UV rays to the earths atmosphere Skin cancer Impaired immune system Retinal degeneration

IPCC 2007

Changes in Concentration of different GHGs for the last 300 years.

Gases CO2 CH4 N2O 1750 292 ppm 729 ppb 275 ppb 1998 366 ppm 1763 ppb 314 ppb 2005 379 ppm 1774 ppb 319 ppb

IPCC 2007

Methane contribution through various sources of agriculture sector

Agriculture sector
Enteric fermentation Rice cultivation Crop soils Manure management

CH4 emission (%)

63 21 13 2.7

INCCA 2010

Schematic presentation of the potential targets of decreasing CH4 emissions

Strategies to reduce Methane Emission

Nutritional strategies
Carbohydrates Frequency of feeding Forage species and its maturity Processing of forage Silage feeding Complete feed block/TMR
1. Ionophores 2. Defaunation 3. Addition of fat & oils
4. DFM

Management strategies
Number and productivity of animals Genetic selection of animals

Manipulation of rumen fermentation

5. Propionate enhancers 6. Plant 20 compounds 7. Immunization 8. Use of chemicals 9. Reductive acetogenesis 10. Prebiotics 11. Electron acceptors 12. Rumen methane oxidation 13. Bacteriocins 14. Bacteriphages

Microorganisms used as DFM for ruminants

Genus Lactobacillus Bifidobacterium Streptococcus Enterococcus Megasphaera Propionibacterium Species Lactic acid producing bacteria

L.acidophilus, L.plantarum, L.casei, L.gallinarium, L.salivarius, L.reuteri, L.bulgaricus S.bovis, S.faecium E.faecium, E.faecalis
Lactic acid utilizing bacteria

B.psuedolongum, B.thermophilium, B.longum, B.lactis

M.elsdenii P.shemanii, P.freudenreichii, P.acidipropionci, P.jensenii

Other bacteria

Prevotella
Bacillus

P.bryantii
B.subtilis, B.lichenifromis, B.coagulans A.oryzae, A.niger

Yeast
Fungi, Aspergillus

Saccharomyces cerevisiae, S.boulardii

Seo et al. (2010)

Unicellular Fungi Resistant- to antibiotics, anti-bacterial agents resistance is natural and genetical, and not susceptible to be modified or transmitted to other micro-organisms. Saccharomyces cerevisiae- Industrially important ability to convert sugars (i.e. glucose, maltose) into ethanol and carbon dioxide (baking, brewing, distillery, liquid fuel industries) It is having GRAS status (Generally Recognised As Safe) from the US Food and Drug Administration.

10 Auclair, 2001

Physiological state of Saccharomyces cerevisiae through digestive tract

(Auclair, 2001)

Saccharomyces cerevisiae remains alive among the digestive tract, but

cannot colonize. Disappeared rapidly from the gastro-intestinal tract at cessation of dosing.

11

Yeast supplementation

(Jouany, 2006)

O2 uptake

Micronutrient supply (Peptides, vitamins, malate)

Lactic Acid metabolism

pH Stabilization Redox potential Total bacteria, cellulolytic bacteria Risk of acidosis Rate of OM digestion Feed intake Energy and amino acid supply

Health disorder

Improved animal productivity

Mechanisms of action of live yeast probiotics on ruminal fiber-degrading communities.

(Chaucheyras-Durand, 2008)

1. Decrease number of Protozoa

(Newbold et al., 1998)

(Lila et al., 2004)

4. Increase propionate production

Yeast reduce methane by 4 methods

2. Promote acetogenesis
(Chaucheyras et al. 1995)

3. Improve animal productivity

(Bruno et al., 2005)

Newbold et al., 1998

In Vitro H2 utilization, methane & acetate production

Ser-8 Acetogenic strain, MF2 methanogenic strain, live or autoclaved S. cerevisiae strain of CnCm-I-1077 @ 108 cells/ml; incubation time 5 days;

Chaucheyras et al., 1995

Effects of Twin-Strain 8417 and 1026 (YST) Saccharomyces cerevisiae live cells (5 x 109 live organisms /g) on in vitro fermentation (n=15)
Exp:1 In the absence of added substrate
Item 0 pH Ammonia-N, mg/dL Total VFA, mM mol/100 mol Acetate Propionate Acetate: Propionate Protozoa,x104 /mL Total gas , mL H2, mM CH4,mM 6h 24 h 68.9 15.6 4.42 2.3 1.47 ND 0.71 0.28 1.14 68.3 16.2 4.22 2.2 1.46 ND 0.71 0.27 1.15 66.7 17.0 3.91 2.4 1.52 ND 0.71 0.28 1.14 65.3 17.8 3.68 2.5 1.60 ND 0.72 0.26 1.17 64.9 17.9 3.63 2.7 1.77 ND 0.72 0.26 1.17 6.23 1.28 16.2 0.33 6.23 1.21 16.2 0.66 6.24 1.21 16.7 YST g/L 0.99 6.22 1.32 16.8 1.32 6.24 1.27 17.4

Lila et al., 2004

Exp:2 Fermentation of the corn starch by the mixed ruminal microorganisms

Item
0 pH Ammonia-N, mg/dL Total VFA, mM mol/100 mol Acetate Propionate Acetate: Propionate Protozoa,x104 /mL Total gas , mL H2, mM CH4,mM 6h 24 h Total viable bacteria, x 108/mL at 24 hr L-lactate, mM at 24 hr 57.2 26.4 2.18 15.5 28.0 0.18 14.5 15.7 13.3 3.25 0.28 57.1 26.8 2.14 15.3 31.1 0.17 14.6 15.9 13.2 4.11 0.21 57.3 27.0 2.12 15.6 35.0 0.18 14.4 15.4 13.3 4.46 0.18 57.1 27.4 2.08 15.2 37.5 0.18 14.4 15.6 13.2 4.57 0.14 57.1 27.8 2.06 15.4 40.0 0.18 14.6 15.9 13.4 4.84 0.09 6.07 5.2 58.4 0.33 6.07 5.0 61.2 0.66 6.06 5.2 62.6

YST g/L
0.99 6.05 5.3 64.0 1.32 6.03 5.2 65.3

Lila et al.,2004

Exp:3 Fermentation of the soluble potato starch by the mixed ruminal microorganisms
Item
0 pH Ammonia-N, mg/dL Total VFA, mM mol/100 mol Acetate Propionate Acetate: Propionate Protozoa,x104 /mL Total gas , mL H2, mL/incubation CH4,mL/incubation 6h 24 h Total viable bacteria, x 108/mL at 24 hr L-lactate, mM at 24 hr 58.7 25.5 2.30 13.4 31.0 0.11 12.0 12.4 11.60 2.08 18.70 58.7 26.5 2.22 13.2 34.6 0.11 12.1 12.6 11.54 3.14 16.33 58.5 26.9 2.18 13.6 37.5 0.10 12.0 12.5 11.42 3.56 15.76 58.5 27.6 2.13 13.3 39.0 0.11 11.8 12.3 11.26 3.73 14.34 58.8 28.0 2.10 13.5 41.1 0.10 12.1 12.6 11.63 3.88 13.12 5.83 7.7 51.3 0.33 5.82 7.6 54.3 0.66 5.80 7.8 55.6

YST g/L
0.99 5.79 7.8 57.2 1.32 5.78 7.7 58.4

Lila et al.,2004

Exp:4 Fermentation of the hay plus concentrate by the mixed ruminal microorganisms
Item
0 pH Ammonia-N, mg/dL Total VFA, mM mol/100 mol Acetate Propionate Acetate: Propionate Protozoa,x104 /mL Total gas , mL H2, mL/incubation CH4,mL/incubation 6h 24 h IVDDM, % 24hr Total viable bacteria, x 108/mL at 24 hr Cellulolytic bact.,x106/mL L-lactate, mM at 24 hr 60.2 24.3 2.49 12.5 29.5 0.18 17.1 17.7 16.4 45.0 6.98 4.77 0.19 60.4 24.7 2.45 12.4 36.5 0.17 16.5 17.7 15.4 46.1 7.46 5.34 0.11 60.7 24.9 2.44 12.8 38.5 0.18 16.4 17.7 15.1 47.4 7.73 5.81 0.09 60.9 25.1 2.25 12.4 41.5 0.18 16.3 17.7 14.8 48.8 7.98 5.97 0.06 61.2 25.2 2.19 12.5 43.0 0.18 16.3 17.8 14.8 49.7 8.44 6.49 0.04 6.22 15.6 73.9 0.33 6.20 15.4 76.8 0.66 6.18 15.4 77.9

YST g/L
0.99 6.19 15.4 80.6 1.32 6.21 15.6 82.3

Lila et al.,2004

Effects of 2 active dried yeast strains on ruminal acidosis and methane production in nonlactating Holstein cows
Yeast added via rumen canula @1x1010 CFU/head/day, fed TMR for 35 days Treatment Parameter Control Strain 1 Strain 2 DMI kg/day Intial B.W (kg) Final B.W (kg) 14.1 785 822 13.9 825 852 27
Treatment Control 62.9 Strain 1 61.7 Strain 2 61.2

n=15

15.2 797 832 35

Change in BW(kg) 37
Parameter DM,%

OM,%
CP,% NDF,% ADF,% Digestible DMI, kg/day

63.8
63.6a 40.0 24.6 9.0

62.7
61.2b 38.3 24.1 8.5

62.1
62.4ab 37.7 23.4 9.0

Chung et al., 2011

Parameter Mean Ruminal pH Total Protozoa,x106 cells /mL Total VFA, mM Individual VFA, mol/100 mol Acetate Propionate

Treatment Control 6.24a 1.5 111 63.0ab 19.0b Strain 1 6.34a 1.8 116 63.3a 18.3b Strain 2 5.98b 1.8 120 61.4b 20.4a

Butyrate
A:P ratio Lactate,mM NH3-N,mM

13.3
3.3ab 0.24 8.4

13.5
3.5a 0.26 8.5

13.7
3.0b 0.22 7.5

Chung et al., 2011

Parameter
Methane total emission, g/d

Treatment

Control
268

Strain 1
261 15.1 17.5a 5.8a

Strain 2
263 16.7 15.7b 5.2b

DMI during CH4 collection,kg/d 16.1 Ratio, g of CH4/kg of DMI Percentage of gross energy intake 16.9ab 5.6ab

Methane emission intensity

Chung et al., 2011

Effect of Saccharomyces cerevisiae (S.C), supplementation on the In vitro rumen fermentation

12 cultures of SC put in @ 109 CFU/conical flask & Rumen liquor from adult buffalo Probiotic IVDMD (%) 41.99 41.62 42.71 42.51 41.43 42.66 43.77 41.56 42.06 46.20 41.58 46.08 47.16 TVFA (meq/ 100ml) 6.37 6.13 7.07 6.70 6.47 6.53 7.43 6.67 6.57 7.37 6.60 7.77 7.63 Ammonia N (mg/ 100ml) 11.27 10.67 10.63 10.30 10.43 11.47 9.70 10.03 9.63 9.13 9.47 9.53 9.67 Total Gas Production (ml/g ubstrate) 57.50 56.67 56.70 58.57 57.70 57.30 59.63 55.50 58.17 62.37 61.03 61.23 62.80 CH4(ml/100 g OM digested 15.63 15.53 14.60 14.17 14.87 14.17 12.60 13.53 15.23 12.87 14.23 14.00 13.23 Bacterial counts Nos/ml) 5.3108 5.7 108 5.2 108 5.6 108 6.0 108 6.2 108 6.3 108 5.6 108 6.1 108 6.3 108 6.3 108 6.6 108 6.6 108

Control S.C 39 S.C 40 S.C 41 S.C 42 S.C 43 S.C 45 S.C 46 S.C 47 S.C 50 S.C 186 S.C 189 S.C 225

Raman Malik & Rameshwer Singh, 2009

Effect of Dried Beer Yeast on the In vitro roughage degradability and methane production
Degradation characteristics of dried beer yeast Item Treatme nt Dry matter degradability (%) 3h 6h 12 h 24 h a (%) b (%) c (%)

Dried beer

32.6

33.1

56.8

80.3

11.5

88.6

6.2

Effect of dried beer yeast on the degradability of roughages Average No addition Yeast 17.0 16.6 17.0 19.5* 23.6 29.8* 28.0 38.9** 14.8 12.9 85.2 87.1 0.8 1.6**

Effect of dried beer yeast extract, DNA and Albumin on the degradability of roughages Average No addition Yeast 13.3 16.1 17.7 19.3* 25.4 28.8 35.0 40.7 10.6 11.5 89.4 88.5 1.2 1.6

Average

No addition
DNA No addition Albumin

10.8
14.8 10.8 15.6

15.3
21.8** 15.3 20.4**

22.8
29.1** 22.8 29.8

42.6
45.5* 42.6 45.6

3.0
9.1* 3.0 9.6**

97.0
91.0* 97.0 90.4**

2.2
2.2 2.2 2.2

Average

*p<0.05; **P<0.01

Whole crop corn, rice straw and Italian ryegrass

Ando et al., 2003

Difference between no addition and added samples

Item Average Treatment 3h Yeast DNA Albumin -0.4a 4.0b 4.8b Yeast Extract 2.2ab Dry matter degradability (%) 6h 2.4a 2.2a 6.4b 5.1b 12 h 6.2 2.8 6.3 6.4 24 h 10.9a 6.4a 3.0b 3.0b

In vitro methane production

Incubation time 12 h 24 h Treatment Control 14.1 28.4 Beer yeast 44.1** 60.2**

Yeast extract is equivalent to 0.2g of dried beer yeast DNA added was 0.006g which gives nucleic acid content equivalent to 0.2g dried beer yeast Albumin added was 0.0994 g which gives a CP content equivalent to 0.2g dried beer yeast

*p<0.05; **P<0.01

Ando et al., 2003

Ad libitum dry matter intake and ruminal fermentation variables for cattle fed on high forage diets with various yeast
Item Treatments

control
Ad libitum DMI, kg/d pH Total VFA, mM Acetate Propionate A:P Item Control DMI, kg/day CH4 g/ steer CH4 g/kg DMI CH4 % of GE intake CH4 % of DE intake 7.18 179.0 25.05 7.13 11.36 Levucell SC yeast (1 g/d) 6.71 171.9 26.43 7.53 11.81 8.84 6.92 73.6 67.9 18.9 3.63

Levucell SC yeast
8.89 6.97 72.4 67.1 19.3 3.52 Treatments % greater than control

Procreatin-7 yeast
9.14 6.99 69.1 67.1 19.4 3.50

Procreatin-7 yeast (4 g/d) 7.46 180.9 24.32

% less than control

5.61

6.93 10.77

2.89

Mc Ginn et al., 2004

Incubation for 48hr

In vitro effect of

Diamond V Xp Yeast culture on mixed

ruminal microorganisms

Sullivian & Martin,1999

Combination

Control

Yeast Culture, g/L

Live Cells, g/L

P<0.05
Concentrations of carbon sources component Glucose, mM Malate, mM Succinate, mM Lactate, mM Yeast culture 11.8 17.0 0.72 10.2 Live cells 12.8 10.6 1.0 19.6

0.35
pH CH4, mM Acetate, mM Propionate, mM A:P ratio pH CH4, mM Acetate, mM Propionate, mM A:P ratio pH CH4, mM Lactate, mM Acetate, mM Propionate, mM 6.46a 1.02a 10.6 2.2 4.97 6.49a 1.16a 14.2 2.6 5.65 5.89 11.5 0.10a 24.0a 11.5 6.46a 1.53b 11.0 2.2 5.19 6.46b 2.03b 16.0 3.1 5.27 5.95 11.8 0.14a 22.0ab 10.3

0.73
6.43b 2.36c 9.9 2.0 5.10 6.47b 2.56c 15.8 3.1 5.08 5.85 10.6 0.27b 22.9a 11.4

0.35
6.49c 1.16a 9.4 1.9 5.44 6.52c 2.01b 14.6 2.7 5.49 5.95 11.0 0.10a 19.0bc 9.8

0.73
6.47ac 1.75b 10.7 2.2 5.07 6.51ac 2.54c 16.5 3.1 5.42 5.92 11.8 0.02c 19.2c 9.8

24h without substrate

48h without substrate

With Ground corn 24h

In vitro effect of Diamond V Xp Yeast culture & PMX70SBK live cell yeast on mixed ruminal microorganisms

A:P ratio

2.22a

2.35a

2.12ab

2.06b

2.03b

Lynch & Martin,2002

P<0.05
Combination Control Yeast Culture, g/L 0.35 pH H2, mM CH4, mM Lactate, mM Acetate, mM Propionate, mM A:P ratio pH H2, mM 5.26 0.18a 7.1 20.9 18.2 7.7 2.47a 6.29a 0.03a 5.21 0.26b 6.9 21.4 17.3 7.6 2.40a 6.28b 0.05b 0.73 5.34 0.23ab 7.9 17.2 15.1 7.0 2.20b 6.27b 0.03a 0.35 5.25 0.16a 7.1 19.6 17.4 9.1 1.91c 6.32c 0.03a With Soluble Starch 24h 5.26 0.09c 6.2 19.1 17.2 9.3 1.87c 6.32c 0.03a Live Cells, g/L 0.75

With alfalfa hay 48 h

CH4, mM
Acetate, mM Propionate, mM A:P ratio IVDMD, %

13.8a
43.8a 11.6 3.79 59.4a

13.9a
43.1a 11.3 3.80 56.9b

13.9a
37.4b 10.1 3.72 56.9b

11.0b
35.3b 9.2 3.81 53.8c

13.5a
32.2c 8.5 3.77 54.4c

Lynch & Martin,2002

Saccharomyces cerevisiae (CNCM I-1077 strain) fed @ 0.5 g/animal/day

Srinivas Kumar et al., 2011

Conclusion

Low cost alternative to improve animal production (fed /animal basis) No residual effect on the animal products Possible alternative for methane reduction Strain selection is required which can reduce methane output and ruminal acidosis and also to promote fermentation Additional research required to Identify & elaborate the routes & methods of dosage of yeast or its factors, in vivo

THANK YOU