Sponsor Day: Seminar on Animal Feeding 15th-16th May 2014 IRTA Mas Bover

1. Evaluation of nutritional strategies that
affect manure characteristics and
gaseous emissions
Francesc Prenafeta (francesc.prenafeta@irta.cat)
Belén Fernández, Marc Viñas
GIRO – IRTA
Rosil Lizardo, Joaquim Brufau
Mas de Bover – IRTA

2. Global emissions from pig supply chains
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Sponsor Day – Seminar on animal feeding
55% N (ammonium, urea, organic N)55% N (ammonium, urea, organic N)
22% C (fibres, VFA)22% C (fibres, VFA)
44% P (phosphate, organic P)44% P (phosphate, organic P)
45% N45% N
56% P56% P
10% C10% C
Methane (CHMethane (CH44))
Ammonia (NHAmmonia (NH33))
Hydrogen sulphide (SHHydrogen sulphide (SH22))
Jørgensen et al. (2013). J Anim Sci Biotechnol 4:42
Additives!Additives!

3. Global emissions from pig supply chains
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Sponsor Day – Seminar on animal feeding
Sliggers & Bull (2007). Ammonia emissions in agriculture. pp. 31-37
Regulatory conventions and protocols

4. Global emissions from pig supply chains
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Sponsor Day – Seminar on animal feeding
Greenhouse Gases (GHG)

5. Nutritional strategies for reduced emissions: Feed additivation
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Sponsor Day – Seminar on animal feeding
Non-starch polysaccharides enzymes: Improve pig performance by removing the anti-
nutritional effects of fermentable fibre in cereals.
Acidifying salts: Dietary electrolyte balance impacts renal regulation to maintain constant
blood pH, having an important effect on the pH of urine and slurry, thus lowering NH3
volatilization.
Urease inhibitors: Herbal extracts associated with saponins and synthetic compounds such
CHPT and PPDA.
Ammonium binding: Microporous aluminosilicate minerals (zeolites) characterized by large
internal surface area and high cation exchange capacity.
Probiotics: Micro-organisms which, when administered in adequate amounts, confer a health
benefit on the host. The use of probiotics in livestock is prompted from a demand for
alternatives to the need for antibiotics.
Philippe et al. (2011). Agric Ecosyst Environ 141:245– 260
…how to measure the effects of these additives on emissions?

6. How to measure the effects of additives on emissions?
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Sponsor Day – Seminar on animal feeding
To characterize physicochemical parameters of pig slurries collected during growth
performance assays
…we still don’t have a standard method for the direct and long-term
measurements of emissions!
Dispersibility (related to viscosity)
Dry matter, volatile compounds

7. Global emissions from pig supply chains: GHG
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Sponsor Day – Seminar on animal feeding
Biochemical Methane Potential (BMP) according to the IPCC (2006)
Incubation of samples of organic wastes in anoxic vials and monitoring of headspace gases.
Vials are previously inoculated with methanogenic biomass

8. Anaerobic Digestion Process
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Sponsor Day – Seminar on animal feeding
Desired process
Waste-to-energy conversion (biogas)
and nutrient recovery.
Undesired process
Uncontrolled emissions during manure management.

9. The anaerobic digestion process
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Sponsor Day – Seminar on animal feeding
Methanogenesis: Microbial conversion of the organic matter in the absence of oxygen into
CH4 and CO2

10. Nitrogen emissions
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Sponsor Day – Seminar on animal feeding
Ammonification: Microbial conversion of the organic nitrogen from manure into ammonium (NH4
+
)

11. Proposed method
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Sponsor Day – Seminar on animal feeding
Slurry collection
Treatment 1
Treatment 2
Control (-)
Growth Performance Trial
…
Treatment 1
Treatment 2
Control (-)
Biochemical Methane Potential
…

12. Objectives
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Sponsor Day – Seminar on animal feeding
To implement and validate the BMP methodology for testing the effect of direct-fed
microbials (probiotics) on grow/finish pigs fed with high-fiber diets.
The study focused on the effect of two tested probiotics (P-α and P-β) on:
1.Physicochemical characteristics of the slurry, regarding the composition of organic mater and
nitrogen compounds.
2.Potential emission of harmful gases (CH4, H2S, and NH3) when the pig slurry is kept under
anaerobic conditions.
3.The microbial community structure and functional genes of the resulting fresh and digested
pig slurry, concerning both Eubacteria and Archaeobacteria domains.

13. Methodological validation
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Sponsor Day – Seminar on animal feeding
36 Pietrain*(Landrace*Duroc) pigs were housed during 3 weeks in 4 pens containing 8 animals
each, located in corners of a G/F room, and were fed with the assigned experimental diets.
24 selected pigs were then transferred to digestibility room and allocated into 12 metabolic
cages of the digestibility barn. Pigs were assigned to 3 treatments in a randomized complete
block design according to their sex and initial body weight. Each treatment contained 3
replications with 2 pigs per pen.
After a 4 days adaptation to cages, the animals were maintained during 3 additional days for
slurry recovery.
Two dietary additives were tested, one of them at two
doses, against a negative control. Each treatment was
performed in 3 independent replicates (A, B, and C).
-T1: non-inclusion negative control (NC)
-T2: NC + 1/2x dose (250g/MT) Probiotic α
-T3: NC + 1x dose (500g/MT) Probiotic α
-T4: NC + 1x dose (500g/MT) Probiotic β
Growth performance trial

14. Methodology: Physicochemical characterization
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Sponsor Day – Seminar on animal feeding
Environmentally relevant physicochemical parameters were determined in fresh
homogenized pig slurry samples from the pig feeding trial:
• Total (TS) and volatile (VS) solids by drying/ combustion
and gravimetry
• Chemical oxygen demand (COD) by redox titration with
potassium permanganate
• Volatile fatty acids (VFA) by gas chromatography (GC).
• Sulfates by ion chromatography
• Total ammonia nitrogen (TAN) and total nitrogen (TN) by
Kjeldahl method

15. Methodology: Determination of potential emissions
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Sponsor Day – Seminar on animal feeding
Biochemical Methane Potential (IPCC, 2006): Pig slurry samples (5 gCOD L-1
) were
incubated at 25°C of 1.2 L glass vials with 0.7 L of liquid medium. Methanogenic conditions
were prompted by adding an external inoculum (5 gVSS L-1
) from an anaerobic digester treating
pig manure.
Incubation of fresh slurry samples in anoxic vials and monitoring of headspace gases
(CH4 and H2S) by GC. NH3 was estimated from liquid phase NH4
+
measurements.

16. Results: BMP assay
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Sponsor Day – Seminar on animal feeding
Supplementation with P-α at the two tested doses (T2 and T3) resulted in a lower BMP yield
in relation to the control (T1), but no significant dose-effect was observed.
BMP values with the tested P-β were even lower.
0
250
500
750
1000
1250
0 10 20 30 40
CH4NETO_25ºC(NmL)
days
T1 T2 T3 T4

17. Results: Emission indexes
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Sponsor Day – Seminar on animal feeding
Index
Mean SD Mean SD Mean SD Mean SD
B0 Methane yield
NLCH4 kgVSslurry
-1
346 103 314 52 324 54 172 102
Nm
3
CH4 tslurry
-1
66 16 44 9 39 16 16 21
Biogas composition (%v/v)
CH4 65.4% 0% 64.7% 2% 60.8% 1% 63.1% 3%
CO2 33.4% 0% 33.3% 2% 37.5% 2% 35.2% 4%
H2S 1.2% 0% 1.9% 0% 1.7% 0% 1.8% 1%
Biogas yield
NL Biogas kgVSslurry
-1
530 157 485 83 533 86 278 177
Nm
3
Biogas tslurry
-1
101 24 69 15 65 27 27 36
Cumulative H2S emissions
NL H2S kgVSslurry
-1
6.1 1.4 9.5 2.4 9.5 3.2 5.4 4.0
Nm
3
H2S tslurry
-1
1.2 0.3 1.3 0.3 1.1 0.2 0.5 0.6
Cumulative NH3 emissions at 25ºC and pH 8.2
kgN-NH3 kgVSslurry
-1
5.4 1.0 3.4 0.2 4.4 0.5 5.6 1.5
kgN-NH3 tslurry
-1
1.14 0.33 0.56 0.14 0.59 0.18 0.65 0.46
N-NH3 (%initial TN of slurry) 8% 2% 5% 0% 7% 1% 9% 1%
T1 T2 T3 T4
Supplementation with P-α resulted in a lower NH3 emissions (20% – 40%; p ≤ 0.05).
Supplementation with P-β resulted in lower CH4 emissions (50%; p ≤ 0.05).

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Sponsor Day – Seminar on animal feeding
Methodology: Microbial characterization
Quantification of target genes that provide useful phylogenetic/
functional information by qPCR (Stratagene™)
•Total bacteria: ribosomal 16S rRNA genes that are specific of the
Eubacteria domain.
•Methanogenic archaea: Genes of the Methyl-coenzyme M
reductase (mcrA) , which codify for the enzyme involved in the last
step of the methanogenesis.
Characterization of the microbial community structure for
Eubacteria and Archaeobacteria by high throughput sequencing
(NGS) of 16S rRNA genes (Ion Torrent PGM™; Life Technologies)

19. Results: Quantitative microbial characterization
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Sponsor Day – Seminar on animal feeding
0
0.05
0.1
0.15
0.2
0.25
0.3
1.00E+05
1.00E+06
1.00E+07
1.00E+08
1.00E+09
1.00E+10
Genecopynumbers·mL-1
16SrRNA gene Eubacteria mcrA geneMetanogenic Archaea Ratio mcrA/16SrRNA
Bacterial and archaeal gene
copy numbers from fresh and
digested slurry samples
remained within the same
order of magnitude.
Variability was slightly higher
in the fresh pig slurry samples.
A significantly higher ratio
archaea/bacteria ratio was
observed in the inoculated
methanogenic biomass.
Very useful for tracking microbial functional genes of interest (e.g. antibiotic resistance,
hydrolytic- and emissions-related enzymes, etc.)

20. Results: Microbial community structure
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Sponsor Day – Seminar on animal feeding
0
10
20
30
40
50
60
70
80
90
100
Others
Gammaproteobacteria
Epsilonproteonacteria
Deltaproteobacteria
Betaproteobacteria
Alphaproteobactera
Unclassified Proteobacteria
Erysipelotrichia
Clostridia
Bacilli
Unclassified Firmicutes
Sphingobacteria
Flavobacteria
Bacteroidetes
Unclassified Bacteroidetes
0
10
20
30
40
50
60
70
80
90
100
Others
Gammaproteobacteria
Epsilonbacteria
Deltaproteobacteria
Betaproteobacteria
Alphaptoteobacteria
Unclassified Proteobacteria
Bacilli
Clostridia
Negativicutes
Erysipelotrichia
Unclassified Firmicutes
Bacteroidetesincertae sedis
Sphingobacteria
Flavobacteria
Bacteroidia
Unclassified Bacteroidetes
Fresh slurry Digested slurry
Useful for characterizing the whole microbial community and for detecting specific
microorganisms (e.g. pathogens, probiotics, etc.)

21. Conclusions
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Sponsor Day – Seminar on animal feeding
1. The combination of growth performance trials with the Biochemical Methane Potential
test (IPCC 2006) is proposed as a method for estimating the potential emissions (CH4,
NH3, SH2) associated with pig slurries arising from different nutritional strategies.
2. The method was technically viable. Significant differences were observed between the
non-inclusion basal diet (negative control) and treatments supplemented with
probiotics.
3. Specific mechanisms aimed towards reduced NH3 and CH4 emissions were highlighted
in the two tested products.
4. Microbial community structure was also relatively diverse among replicates and
treatments, and no clear pattern could be identified. Yet, specific microorganisms and
functional genes (methanogenesis) were monitored by qPCR and NGS.
Future work
We are currently developing a lab “pig slurry pit” in which we will be able to monitor
space/time gradients (i.e. red-ox depth changes in relation to N2O emissions)

22. Acknowledgments
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Sponsor Day – Seminar on animal feeding
Francesc Prenafeta
Marc Viñas
Belén Fernández
Miriam Guivernau
Laura Tey