Impact of slurry storage and management on pig manure microbial

community dynamics, cultural and molecular approaches

P. Peu*(1), P. Dabert(2), H Brugère(3), A.-M. Pourcher(4), J.-J Godon(2), J.-P. Delgenes(2)
(1) Cemagref, Livestock and Municipal Waste Management Unit. 17 Avenue de Cucillé, CS 64427, 35 044
Rennes cedex, France. E-mail: pascal.peu@cemagref.fr
(2) INRA, Laboratoire de Biotechnologie de l'Environnement, Av. des Etangs, 11100 Narbonne, France.
(3) UMR INRA/ENVT 1225 Interaction Hôtes-Agents pathogène – Ecole Nationale Vétérinaire de Toulouse, 23,
chemin des Capelles – F 31076 Toulouse cedex, France.
(3) Université d'Angers, UFR Sciences, 2, boulevard Lavoisier, 49045 Angers cedex 01, France.
[Results obtained within the research initiative "Porcherie verte"]

Introduction
Intensive pig production in developed countries generates a large amount of pig slurry.
This biological effluent contains about 1010 microorganisms per ml (Rivière et al., 1974). Its
microbial transformation over the farm management generates air pollution by emission of a
great number of gas compounds (ammonia, green house gas, odours) (Zhu et al., 2000).
Moreover, it may contain pathogenic microorganisms and present a potential risk to the
recipient ecosystem and finally to human populations. The objective of the present work was
to monitor the evolution of a pig slurry microbial community throughout its management
process. Dominant microbial populations were followed with a molecular typing technique
using small-subunit ribosomal DNA analysis: PCR-SSCP (Polymerase Chain Reaction and
Single Strand Conformation Polymorphism electrophoresis) (Delbès et al., 2000) while
faecal indicators were numerated with culturable techniques.
Materials and methods
Sampling was carried out on a commercial pig farm, holding 220 sows plus finishers,
that produces about 4500 m3 of pig slurry per year. 54 samples were collected from
December 2002 to June 2003: faeces, slurries and soils. Analysis of the slurry microbial
community was performed by PCR amplification of the microbial 16S rDNA V3 region
using either general bacterial primers or specific primers targeting the Clostridiacea, the
Bacillus-Lactobacillus-Streptococcus (BSL), and the Cytophaga-Flexibacter-Bacteroides
(CFB) groups according to Dabert et al. (2004). PCR products were separated and visualised
by SSCP capillary electrophoresis on an ABI 310 genetic analyser. The microbial community
appears as a profile of peaks that corresponds to dominant microbial populations of the
ecosystem. Peaks migrating at the same position are assumed to correspond to the same
species. Faecal indicator were monitored using plating on selective media or MPN (Most
Probable Number) technique using normalised culturable media. Number of microorganisms
was expressed as a function of the samples dry matter content determined by drying at
105°C.
Results and discussion
Dynamics of the pig slurry microbial community was followed by aligning the SSCP
electrophoregrams obtained from all slurry samples. The bacterial SSCP profiles appeared
very complex with a high number of peaks reflecting the pig slurry microbial diversity (black
arrows, fig. 1a). The comparison of the SSCP profiles revealed an unexpected stability of the
pig manure slurry microbial community during time within the storage pit. From December
2002 to March 2003, the profiles remained about the same with no major apparent changes in
peak number and composition. As expected, no change in SSCP profiles was observed when
manure went through the auger press. On the opposite, SSCP profiles observed from lagoon
samples showed a slow evolution of the community through time (data not shown). Finally,
none of the peaks present in the profile from the lagoon liquid used for spreading were found
in soil profiles after spreading. This could be explained by the high dilution rate of manure
microorganisms within soil at the time of spreading, and shows that we reached the detection
limits of the PCR-SSCP approach. Globally, the same results were obtained with the analysis
of the specific microbial groups Clostridiacea, BSL, and CFB (data not shown).
Figure 1. a : Evolution of the pig slurry bacterial community SSCP profile in the storage tank
from December 2002 to march 2003. b : Average numbers and MPN* of faecal indicators
numerated at different pig slurry management steps.

Faeces
log cfu /g dry mater. Pig slurry under stalled floor
1.0E+09 Pig Slurry in storage tank
Soil before spreading
1.0E+08 Soil 3 days after spreading
Soil 15days after spreading
1.0E+07
Tank on day 0

1.0E+06

1.0E+05

1.0E+04
Tank on day 21
1.0E+03

1.0E+02

1.0E+01

Tank on day 105

1.0E+00
Enterobacteria Total Escherichia Salmonella* Listeria* Staphylococcus Enterococcus
Coliforms Coli

The dynamics of the faecal indicator microorganisms observed using the cultural approach
revealed also a relative stability of the numeration over time for the different sampling
locations (data not shown). However, in the same way as before, numeration changed from
one step of slurry management to another (figure 1b). Slight variations took place when the
faeces were mixed with urine to form slurry, but important numeration decay happened
principally between the slurry from the storage tank and the separated liquid and solid phases
from the lagoon and compost respectively. Pig slurry spreading did not seem to change
significantly soil numeration of the studied groups.
Conclusion
The microbial community of pig slurry was studied from faeces to soil spreading with
two complementary approaches. The molecular approach allows the detection of uncultured
dominant microorganisms, originating from the pork faecal flora, which persist through the
different manure management steps. The cultural approach allows to monitor faecal
indicators. Both approach conducted to the same conclusions: the manure slurry microbial
community does not evolve rapidly during passive anaerobic storage. Significant changes
occurs primarily when manure move from one step of the management process to another.
Acknowledgements
The authors thank G. Meynaud, veterinary from the ASAMIP (Castanet Tolosan, 31 France)
who realised all sampling.
Reference
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