Sponsor Day on animal feeding: Evaluation of nutritional strategies that affect manure characteristics and gaseous emissions

Evaluation of nutritional strategies that affect manure characteristics and

gaseous emissions

Francesc Prenafeta ([email protected])

Belén Fernández, Marc Viñas

GIRO – IRTA

Rosil Lizardo, Joaquim Brufau

Mas de Bover – IRTA

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% CMethane (CHMethane (CH44))

Ammonia (NHAmmonia (NH33))

Hydrogen sulphide (SHHydrogen sulphide (SH22))

Jørgensen et al. (2013). J Anim Sci Biotechnol 4:42

Additives!Additives!


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Sliggers & Bull (2007). Ammonia emissions in agriculture. pp. 31-37

Regulatory conventions and protocols


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Greenhouse Gases (GHG)

Nutritional strategies for reduced emissions: Feed additivation

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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?

How to measure the effects of additives on emissions?

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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

Global emissions from pig supply chains: GHG

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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

Anaerobic Digestion Process

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Desired processWaste-to-energy conversion (biogas) and nutrient recovery.

Undesired processUncontrolled emissions during manure management.

The anaerobic digestion process

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Methanogenesis: Microbial conversion of the organic matter in the absence of oxygen into CH4 and CO2

Nitrogen emissions

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Ammonification: Microbial conversion of the organic nitrogen from manure into ammonium (NH4+)

Proposed method

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Slurry collectionTreatment 1

Treatment 2

Control (-)

Growth Performance Trial

…

Treatment 1

Treatment 2

Control (-)

Biochemical Methane Potential

…

Objectives

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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.

Methodological validation

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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

Methodology: Physicochemical characterization

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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

Methodology: Determination of potential emissions

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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.

Results: BMP assay

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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

CH

4 N

ET

O_

25º

C (N

mL

)

days

T1 T2 T3 T4

Results: Emission indexes

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Index

Mean SD Mean SD Mean SD Mean SD

B0 Methane yield NLCH4 kgVSslurry

-1 346 103 314 52 324 54 172 102 Nm3 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

Nm3 Biogas tslurry-1 101 24 69 15 65 27 27 36

Cumulative H2S emissionsNL H2S kgVSslurry

-1 6.1 1.4 9.5 2.4 9.5 3.2 5.4 4.0 Nm3 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 8.2kgN-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 s lurry) 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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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)

http://www.google.es/url?sa=i&rct=j&q=&esrc=s&frm=1&source=images&cd=&cad=rja&docid=xbN3hqGVSZhbOM&tbnid=MOWUYToIryAwrM:&ved=0CAUQjRw&url=http%3A%2F%2Fvet.osu.edu%2Fidmel%2Ffacilities&ei=-kZoUvqPOsur0AWOjICwBg&bvm=bv.55123115,d.Yms&psig=AFQjCNEHFA4HSmDokq7wsywCz6W4u9Prmg&ust=1382651983071034

Results: Quantitative microbial characterization

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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

Gene

copy

num

bers

· m

L-1

16SrRNA gene Eubacteria mcrA gene Metanogenic 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.)

Results: Microbial community structure

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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

Epsilonproteonacteria

Deltaproteobacteria

Betaproteobacteria

Alphaproteobactera


Erysipelotrichia

Clostridia

Bacilli


Sphingobacteria

Flavobacteria

Bacteroidetes


0

10

20

30

40

50

60

70

80

90

100

Others

Gammaproteobacteria

Epsilonbacteria

Deltaproteobacteria

Betaproteobacteria

Alphaptoteobacteria


Bacilli

Clostridia

Negativicutes

Erysipelotrichia


Bacteroidetes incertae sedis

Sphingobacteria

Flavobacteria

Bacteroidia


0

10

20

30

40

50

60

70

80

90

100

Others

Gammaproteobacteria

Epsilonbacteria

Deltaproteobacteria

Betaproteobacteria

Alphaptoteobacteria


Bacilli

Clostridia

Negativicutes

Erysipelotrichia


Bacteroidetes incertae sedis

Sphingobacteria

Flavobacteria

Bacteroidia


Fresh slurry Digested slurry

Useful for characterizing the whole microbial community and for detecting specific microorganisms (e.g. pathogens, probiotics, etc.)

Conclusions

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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)

Acknowledgments

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Francesc PrenafetaMarc ViñasBelén Fernández

Miriam GuivernauLaura Tey

Technology

Sponsor Day on animal feeding: Evaluation of nutritional strategies that affect manure characteristics and gaseous emissions