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PCR of 16S rDNA
FEMS Microbiology Ecology 38 (2001) 169^177
www.fems-microbiology.org
Bacterial community dynamics in liquid swine manure during storage: molecular analysis using DGGE/PCR of 16S rDNA Kam Leung a , Edward Topp b
b;
*
a Department of Biology, Lakehead University, 955 Oliver Road, Thunder Bay, ON, Canada P7B 5E1 Agriculture and Agri-Food Canada, Research Branch, 1391 Sandford Street, London, ON, Canada N5V 4T3
Received 12 July 2001; received in revised form 17 September 2001; accepted 18 September 2001 First published online 9 November 2001
Abstract Denaturant gradient gel electrophoresis (DGGE) analysis of polymerase chain reaction-amplified DNA fragments prepared from extracted DNA using universal 16S rDNA primers was used to compare the composition of bacterial communities in liquid swine manure (LSM) during incubation in aerated and in non-aerated laboratory reactors. The LSM was initially dominated by 13 phylotypes whose identities were established by excising and cloning DGGE bands, and comparing the 16S rDNA sequences with those available in GenBank. With varying degrees of similarity, Clostridium butyricum, Clostridium disporicum, a Pedobacter sp., two Rhodanobacter sp., a spirochete, and seven uncultured eubacterial sequences were identified. The chemical composition, total microbial populations determined by direct microscopic count, and DGGE profiles of the LSM were stable during anaerobic storage for 7 weeks. However, the community composition of the LSM changed substantially with aeration, the DGGE bands in the original sample receding in intensity as the manure community became dominated by phylotypes most closely related to the aerobes Bacillus thuringiensis, Sphingobacterium mizutae, a `Sphingobacteriumlike' bacterium, and a Paenibacillus sp. When continuously aerated, the pH rose from 8 to 9.5, the ammonium-N content decreased, populations of culturable aerobes increased 150-fold, and total bacterial populations remained stable. DGGE analysis of manure fractionated into planktonic and biofilm (flocs and aggregates) communities suggested that Clostridium populations were stable in biofilms during aeration, whereas the aerobes that ultimately dominated the LSM community were primarily in the planktonic phase. ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
1. Introduction Animal manures represent an important and valuable source of nutrients for agricultural crop production. At the same time, public health and environmental concerns regarding objectionable odours, chemical contaminants and pathogens pose a challenge for the storage and safe handling of animal wastes [1^4]. Storage conditions which promote the destruction of these contaminants have been the subject of numerous investigations, such as, controlling the range of redox conditions from aeration to anaerobic digestion, increasing storage temperature, drying and ozonation [2,5^13]. It has long been recognised that sub-populations of the global microbial population in liquid swine manure (LSM) may be responsible for breaking down various organic compounds and/or involved in sup-
* Corresponding author. Tel. : +1 (519) 457 1470 ext. 235; Fax: +1 (519) 457 3997. E-mail address : [email protected] (E. Topp).
pressing pathogens during storage of the manure. However, only a few studies have examined the impact of storage conditions on the global microbial composition of LSM. These studies have been generally limited to the use of culture-dependent methods [5,9,14,15]. Recently, a study of the bacterial 16S rDNA sequences extracted and cloned from a human fecal sample found only 24% of the phylotypes corresponded to known organisms [16]. Furthermore, a similar study of bacteria in the colonic and cecal lumens of a pig showed that 59% of the 16S rDNA cloned sequences have less than 95% similarity to sequences from cultivated organisms [17]. Clearly the present information on the swine manure bacterial populations based on cultivation approaches may misrepresent the true diversity of microbial populations in LSM. As part of a study on the impact of aeration on the composition of animal wastes, we undertook a cultureindependent examination of the major bacterial constituents of LSM and how the microbial community in the manure changes during storage in aerated and unaerated reactors.
0168-6496 / 01 / $22.00 ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 6 4 9 6 ( 0 1 ) 0 0 1 8 1 - 7
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Table 1 Chemical analysis of the manure at the start of the incubation, and following 7 weeks of incubation under aerated or unaerated conditions Manurea
Dry matter (%) Organic matterb (%) Mineral Nc (%) NH4 -N (%)
initial
aerated
unaerated
0.6 36 0.24 0.23
0.6 28 0.03 0.02
0.6 33 0.24 0.23
a
Means of two replicates. Organic matter is reported on a dry matter basis. c Mineral constituents are reported on a weight per volume basis. b
2. Materials and methods 2.1. Aerobic and anaerobic manure reactors LSM was collected from a 600-animal farrowing farm located in South Western Ontario, Canada. The LSM consists of urine and faecal material from sows, and was obtained from the barn's manure collecting tank (pit-type storage), which holds the LSM for periods of up to several months until it can be applied to adjacent ¢elds. The tank is buried beneath the barn and thus the LSM would be sheltered from temperature and moisture-content £uctuations typical of an open outdoor lagoon. The composition of the LSM is reported in Table 1. 750-ml portions of fresh manure were dispensed into 1-l mason jars which were then capped with a lid ¢tted with several ports. The reactors were incubated for 7 weeks at room temperature (22 þ 1³C), with continuous agitation by means of a magnetic stirrer. Humidi¢ed compressed air was bubbled into the aerated manure at a £ow rate of 120 ml min31 , whereas anaerobic reactors were left unsparged and hermetically sealed. The headspace pressure in the unsparged reactors was released periodically by inserting a syringe needle through a rubber septum in the lid. All treatments were conducted in triplicate. 2.2. Chemical analyses Manure samples were analysed for chemical composition using standard methods (Table 1; ApL Canada Lab-
oratories East, London, ON, Canada). The pH and redox potential of the manure were measured periodically during the incubations with an Accumet pH/voltmeter (Fisher Scienti¢c, Nepean, ON, Canada) equipped with a combination pH electrode (Orion Research, Beverly, MA, USA) or a platinum redox electrode (Orion Research) (Table 2). Reported redox values are relative to the normal hydrogen electrode. 2.3. Microbiological enumerations Ten-fold dilution serial dilutions of the manure samples were prepared in 0.1% sodium pyrophosphate (w/v of water) and 0.1-ml portions of the sample solutions were spread-plated on Iso-sensitest agar medium (Oxoid, Basingstoke, Hampshire, UK). Colony-forming units (cfu) on the Iso-sensitest agar were determined following 4 days of incubation at 30³C. Direct bacterial counts were performed on 5-[4,6-dichlorotriazin-2-yl]amino£uorescein (DTAF)-stained (Sigma-Aldrich Canada, Oakville, ON, Canada) manure samples [18]. Samples were preserved in formaldehyde (3.7% ¢nal concentration, v/v), and diluted 50-fold with sterile 0.85% NaCl solution. Ten Wl diluted suspension was smeared in a pre-etched area (1.77 cm2 ) on a RITE-ON microscope slide (Gold Seal Products, Highpark, IL, USA). The smears were air-dried, heat-¢xed and stained with 0.02% DTAF. Bacterial counts were determined by epi£uorescent microscope equipped with a £uorescein ¢lter set. 2.4. DNA extraction Nucleic acid extraction was performed using UltraClean soil DNA isolation kits (MoBio Laboratories, Solana Beach, CA, USA) according to the manufacturer's instructions with the following modi¢cations. One ml of manure sample was collected from each manure reactor and pelleted in a 1.5-ml micro-centrifuge tube at 16 000Ug for 10 min. The supernatant was discarded and the cell pellet was resuspended in 50 Wl sterile deionised water and transferred to the 2-ml bead solution tube of the UltraClean soil DNA isolation kit. The sample was mixed with an
Table 2 Redox potentials and pH of aerated and anaerobic manure samplesa Days
0 2 7 14 21 35 49 a
Redox potential (mV)
pH
aerated
anaerobic
aerated
anaerobic
3191 ( þ 2) +170 ( þ 6) +209 ( þ 9) +246 ( þ 5) +256 ( þ 6) +271 ( þ 15) +287 ( þ 5)
3187 3162 3153 3153 3128 3123 3177
8.01 9.25 9.48 9.59 9.44 9.32 9.53
8.01 8.10 8.06 8.11 8.11 8.17 8.11
( þ 1) ( þ 11) ( þ 8) ( þ 14) ( þ 19) ( þ 10) ( þ 7)
Data are means þ S.D. ; n = 3.
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( þ 0.07) ( þ 0.04) ( þ 0.03) ( þ 0.04) ( þ 0.05) ( þ 0.06) ( þ 0.02)
( þ 0.10) ( þ 0.02) ( þ 0.02) ( þ 0.01) ( þ 0.06) ( þ 0.19) ( þ 0.23)
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inhibitor removal solution provided by the kit, and agitated twice in a Bio 101 Savant Fast Prep FP120 bead beater at a setting of 5.5 for 30 s. The crude DNA extract was puri¢ed by passing through a spin-column containing a DNA-adsorbing silica matrix as described by the manufacturer. Puri¢ed DNA was eluted from the matrix into 35 Wl sterile bu¡er solution provided by the DNA extraction kit. 2.5. Polymerase chain reaction (PCR)^denaturant gradient gel electrophoresis (DGGE) PCR primers targeting the V3^V5 region of 16S rDNA gene of all bacteria at the nucleotide positions 341^357 and 907^928 (based on the 16S rDNA of Escherichia coli, accession no. J01859) were used to amplify the 16S rDNA fragments of bacterial populations in the manure samples. The sequences of the forward and reverse primers were 5P-CGC CCG CCG CGC CCC GCG CCC GTC CCG CCG CCC CCG CCCG CCT ACG GGA GGC AGC AG-3P (GC clamp underlined) and 5P-CCG TCA ATT CCT TTG AGT TT-3P, respectively [19]. A touchdown PCR program was performed on a 50-Wl PCR mixture containing 1.5 U of Taq DNA polymerase (Promega, Madison, WI, USA), 30 pmol of each primer, 1UPCR bu¡er, 1.5 mM MgCl2 , 200 WM dNTP and 2 Wl of the 10-fold diluted puri¢ed manure DNA extract [20]. DGGE was performed with a DGene 16U16-cm 10% polyacrylamide gel (Bio-Rad, Hercules, CA, USA) maintained at 60³C in 7 l of Tris^acetate^EDTA (TAE) bu¡er (20 mM Tris^acetate, 0.5 M EDTA, pH 8.0). Gradient gels were prepared with 35 and 65% denaturant (100% denaturant de¢ned as 7 M urea plus 40% v/v formamide). Between 0.5 and 1 Wg of the ampli¢ed DNA was loaded per well and run at 100 V for 16 h. Gels were stained in 150 ml TAE bu¡er containing 15 Wl 10 000U concentrated SYBR Green I (Molecular Probes, Eugene, OR, USA) for 1 h and destained in 350 ml TAE for 1 h. Images were captured using a Bio-Rad gel documentation system. Two approaches were used to ensure the correct designation of the DGGE bands in di¡erent samples. First, since the DGGE band pattern (such as bands A, H, J, K, B, C, D and E) of the initial LSM sample was highly consistent, it was used as a standard marker in di¡erent runs to facilitate gel-to-gel (or lane-to-lane) comparison. Second, some major DGGE bands (e.g. bands A, B, F, G and J) were sequenced twice under various occasions, such as under aerated and aggregate conditions, to con¢rm their phylotypes. 2.6. Cloning of 16S rDNA fragments resolved by DGGE Bands in the polyacrylamide gel were excised and rinsed in 1 ml of sterile deionised water. The gel was crushed in 50 Wl sterile deionised water and allowed to equilibrate overnight at 4³C. Five Wl of the DNA extract was used
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as the template in a PCR ampli¢cation as described earlier, except that the forward primer lacked the GC clamp. The PCR products were puri¢ed by electrophoresis through 1.0% low melting agarose and a centrifugation procedure. DNA in the low-melting agarose was extracted by centrifugation through a glass-wool-stu¡ed 0.5-ml micro-centrifuge tube which had a tiny hole punched through its bottom. The DNA samples collected in a 1.5-ml micro-centrifuge tube were puri¢ed either by a phenol^chloroform extraction protocol [21] or the QIAquick PCR Puri¢cation Kit (Qiagen Canada, Mississauga, ON, Canada). The PCR-ampli¢ed products were cloned into the pGEM-T cloning vector (Promega). 2.7. Sequence analysis Three clones from each DGGE band were randomly chosen for sequencing analysis. If the sequence of the three clones from the DGGE band were not identical, three more clones from the same band were sequenced to estimate the number of 16S rDNA sequences co-migrating on the DGGE band. The cloned PCR fragments were sequenced by an ABI-Prism model 377 automatic sequencer (Perkin-Elmer, Forest City, CA, USA) using a T7 primer (5P-TAA TAC GAC TCA CTA TAG GG) targeting the T7 transcription initiation site of the pGEM-T vector. Sequence alignments were performed using the software program DNAMAN (version 4.0; Lynnon BioSoft, Vaudreuil, QC, Canada). Sequences were compared with the GenBank database by using the BLASTN facility of the National Center for Biotechnology (http:// www2.ncbi.nlm.nih.gov/BLAST/) and were also tested for possible chimera formation with the CHECK_CHIMERA program (http://www.cme.msu.edu/rdp/cgis/chimera.cgi?su = SSU). 2.8. Separation of bio¢lm and planktonic cells Bio¢lm bacterial cells attached on the aggregate surfaces and clustered in large £ocs were separated from planktonic bacterial cells by centrifugation. To optimise the separation of the bio¢lm and planktonic cells in the manure samples, the manure samples were centrifuged in a micro-centrifuge at 400Ug for 30, 60 and 150 s. Microbial cells in the supernatant were stained with the LIVE/ DEAD BacLight stain (Molecular Probes) and examined with an epi£uorescent microscope for bacterial £ocs and aggregates. Centrifugation at 400Ug for 60 s was found to provide the best separation. The bio¢lm population in the samples were pelleted by centrifuging a 1-ml sample at 400Ug for 60 s. The supernatant containing the planktonic microbial cells was centrifuged at 16 000Ug for 10 min to recover the planktonic cells from the liquid phase. Samples containing the planktonic, bio¢lm and total microbial populations were stored at 320³C for DNA extraction.
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2.9. Isolation of Bacillus and potential Sphingobacterium spp. Because sequencing analysis of the two major DGGE bands (i.e. bands F and G) showed that the aerated LSM samples were dominated by the genera Bacillus and Sphingobacterium, the traditional cultivation approach was used to isolate potential Bacillus and Sphingobacterium at the highest dilution of the aerated LSM samples. The rDNAs of the isolates were sequenced and compared to the three dominant phylotypes in the aerated LSM identi¢ed by DGGE. A dilution series of the week 7 aerated manure samples were spread-plated on the Iso-Sensitest agar and Bacillus-selective agar (Oxoid). Ten potential Sphingobacterium (yellow-pigmented) colonies were isolated from the Iso-Sensitest agar and re-streaked on tryptic soy agar (TSA) plates. Ten presumptive Bacillus colonies were also isolated from the Bacillus-selective agar and restreaked on TSA plates. Genomic DNAs were extracted from four randomly chosen presumptive Bacillus and Spingobacterium spp. by the InstaGene Matrix DNA puri¢cation kit (Bio-Rad Laboratories) as described by the manufacturer. The DNA extract was used as the template in a PCR ampli¢cation as described earlier, except that the forward primer lacked the GC clamp. The PCR products were gel-puri¢ed as described earlier and the fragments were sequenced by an ABI-Prism model 377 automatic sequencer (Perkin-Elmer) using the DGGE forward primer (without GC clamp). 3. Results 3.1. Physico-chemical composition Initially, the manure contained 0.6% dry matter, about a third of which was organic matter (i.e. lost upon ashing; Table 1). Almost all of the inorganic nitrogen was in the form of ammonium-N. The redox potential was about 3190 mV, and the pH value about 8.0 (Table 2). These parameters remained unchanged throughout the experiment in the unaerated treatment (Tables 1 and 2). In contrast, aeration promoted rapid and signi¢cant changes in the manure. The redox potential increased to +170 mV within 2 days, and up to +287 mV by the end of the incubation. The pH increased to a value of pH 9.25 within 2 days, and remained at about pH 9.5 thereafter. By the end of the experiment the organic matter content had decreased somewhat, and almost all of the inorganic nitrogen and ammonium-nitrogen was lost. The aerated manure rapidly became dark brown, in contrast to the grey^ black of the unaerated material. This colour di¡erence was maintained throughout the incubation. In addition, after 2 days of aeration, the unpleasant odour of the manure disappeared in the aerated samples. The ammonia smell was detected transiently from the aerated sample in the
Fig. 1. Persistence of total bacteria (determined by direct microscopic count) and selected aerobes (determined by plate count) in LSM incubated with or without aeration.
¢rst few days and disappeared afterward. On the other hand, the undesirable odours persisted in the anaerobic manure samples. 3.2. Dynamics of bacterial populations Total bacterial populations enumerated by microscopic direct counts were stable at about 1U1010 cells/ml in the manure regardless of the aeration treatment (Fig. 1). Culturable aerobic bacterial populations decreased somewhat from 1.2U106 to 3.0U105 cfu ml31 by the end of the experiment in the unaerated treatment. However, in the aerated reactors, culturable aerobic bacterial population increased about 150-fold from 7.3U105 to 1.1U108 cfu ml31 in 7 weeks, with most of the growth occurring in the ¢rst 7 days. 3.3. DGGE analysis Background smearing in the DGGE pro¢les indicated the presence of a diverse community in the LSM (Fig. 2). However, the relative prominence of nine distinct bands indicated that the community was dominated by a relatively small number of phylotypes. In the unaerated reactors no changes were observed in the DGGE pro¢les over the course of the experiment (Fig. 2). In contrast, three novel bands (F, G and I) emerged in the DGGE pro¢les of the aerated manure samples. Band F appeared at week 3 and increased in intensity to become one of the major bands by week 7. Band G appeared at week 5 and remained visible thereafter. Band I was a weak band visible at week 7. The appearance of these bands was accompanied by a decrease in the relative intensities of the nine DGGE bands dominating the community at the start of the incubation. The relative composition of the planktonic and bio¢lm bacterial communities were di¡erent (Fig. 3). For example,
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3.5. 16S rDNA of Bacillus and potential Sphingobacterium isolates
Fig. 2. DGGE pro¢les of DNA obtained from aerated (A) and unaerated (B) LSM. Lane 1, start of the incubation ; lane 2, after 1 week; lane 3, after 3 weeks ; lane 4, after 5 weeks ; lane 5, after 7 weeks of incubation.
bands A and L were more prominent in the planktonic community, whereas bands C and B were more prominent in the bio¢lm community. The response of these communities to aeration was distinct. Following 7 weeks of incubation, the bio¢lm community was dominated by six bands (H, I, J, F, K and P) with the other initial bands receding in intensity. In contrast, the planktonic community at the end of the incubation was dominated by two bands (F and G) with other bands receding. 3.4. Sequence analysis The major DGGE bands were cloned, sequenced and matched with the GenBank database (Table 3). Three to six clones were sequenced from each band. None of the cloned sequences were chimeric according to the CHECK_CHIMERA program. Eight bands (A, E, G, H, L, I, J and P) yielded a single 16S rDNA sequence, and the remaining bands (F, K, D, B and C) were composed of two co-migrating 16S rDNA fragments (Table 3). Bands in the initial LSM samples which corresponded to known organisms in the database included Clostridium butyricum (band H), Clostridium disporicum (J), a Spirochaeta sp. (L), a Pedobacter sp. (K), and two members of the genus Rhodanobacter (D). Percent similarities ranged from good (99%) to poor (89%). The three bands which appeared in the aerated samples, were shown to be composed of three genera. Clone F2 was most closely related to Bacillus thuringiensis (94% similarity), clone F4 to Sphingobacterium mizutae (90% similarity), band G to a Sphingobacterium-like bacterium (93% similarity), band I to a Paenibacillus sp. (95% similarity). The remaining bands were most closely related to either uncultured or unidenti¢ed eubacteria at similarity levels of 86^94%. A dominant band appearing in the aerated bio¢lm (band P) was most closely related to an uncultured bacterium isolated from a deteriorating wall painting (94% similarity).
Four randomly selected potential Bacillus isolates, and four potential Sphingobacterium isolates (on the basis of yellow pigmentation), were tentatively identi¢ed on the basis of 16S rDNA sequence analysis. All four isolates recovered from Bacillus selective media showed 100% similarity to B. thuringiensis, Bacillus mycoides or Bacillus cereus, but were only distantly related (94% similarity based on sequence alignment, DNAMAN software program) to clone F2. Of the yellow-pigmented isolates, Iso6, Iso-12 and Iso-14 showed high 16S rDNA similarity to Bacillus silvestris (100%), Caulobacter sp. (99%) and Aureobacterium kitamiense (99%), respectively. Isolate Iso-3 was 91% similar to the 16S rDNA sequence of Sphingobacterium multivorum, but was only distantly related to clones F4 and G1, at 88 and 91% similarity, respectively. 4. Discussion Previous studies have used various non-selective media to characterise bacteria in swine waste [9,14,15]. However, the disadvantages of cultivation approach are likely to result in an underestimate and/or bias in the diversity captured in these studies [14,15]. A few recent studies have used the culture-independent 16S rDNA-based PCR method to determine the diversity and dynamics of micro-organisms in gastrointestinal tracts of pigs [17,22^24]. However, little is known about the culture-independent global microbial composition of LSM and the in£uence of oxygen status on the evolution of the microbial community during storage of LSM. In this study, 13 major bacterial phylotypes were found in the initial manure samples (Table 3). Three phylotypes, identi¢ed as C. butyricum (clone H2), C. disporicum (clone J1), and a Rhodanobacter sp. (clone D2) showed v95% identity with sequences in the GenBank database. Other
Fig. 3. DGGE pro¢les of DNA obtained from aerated LSM fractionated into bio¢lm (A) or supernatant (B). Lane 1, start of the incubation ; lane 2, after 1 week ; lane 3, after 3 weeks; lane 4, after 5 weeks; lane 5, after 7 weeks of incubation.
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phylogenetic groups identi¢ed with lower levels of identity included Pedobacter sp. (clone K1) and Rhodanobacter lindanobacter (clone D1). The clostridia are important constituents of the swine gut community and were probably excreted by the animals [4]. The other genera have not been reported to be enteric in these animals. Pedobacter is a recently described genus, proposed to be a member of the Sphingobacteriaceae fam. nov [25]. Pedobacter sp. are heparinase-producing obligate aerobes sharing the characteristic ability of growing at the expense of heparin [25]. Since heparin is produced in porcine intestinal mucosa (Sigma Life Sciences catalogue), it is possible to ¢nd heparin-degrading bacteria in the LSM. The Rhodanobacter sp. are Gram-negative aerobic bacteria in the Q-Proteobacteria group, distantly related to Xanthomonas and Stenotrophomonas [27]. Members of the Rhodanobacter sp. are soil bacteria but there is insu¤cient information known of this species to speculate on what its role in LSM might be. The closest matching relatives to clones A3, K3, B1, B2, C1, C2 and E were either unculturable or unidenti¢ed eubacteria (Table 3). Interestingly, all these phylotypes
were found in anaerobic environments, anaerobic vinasses reactors (GenBank accession numbers U81676, U81735 and U81735), anoxic rice paddy soil (AJ229216), human gut (AF132261), bovine rumen (AF001778) or anaerobic marine sediments (4995896). Likewise, an uncultured spirochete most closely related to clone L1 is an organism isolated from a hydrogen sul¢de-rich marine sediment (X93326). These results suggest that most of the phylotypes detected in the LSM are anaerobes. Absent from the sequenced phylotypes are some genera commonly found to be well represented in the pig gastrointestinal tract or faeces, notably Lactobacillus, Streptococcus, Peptococcus, Peptostreptococcus, Propionibacterium, Ruminococcus and Bacteroides [9,14,15,17,26]. It is highly probable that these organisms are in the LSM, but in low enough number that they are not prominent in the DGGE pro¢le. Given that LSM consists of a mixture of urine and faeces and other detritus produced in the barn, that the collecting pit under the barn has a residence time of up to several months, and that it is continuously receiving fresh material, it is not surprising that the microbial composi-
Table 3 Identity of phylotypes in DGGE pro¢les of aerated manure Band designation Enriched upon aeration : F Clone F2 Clone F4 G Clone G1 I Clone 1 P Clone P1 Present in initial manure sample: A Clone A3 L Clone L1 H Clone H2 J Clone J1 K Clone K1 Clone K3 B Clone B1 Clone B2 C Clone C1 Clone C2 D Clone D1 Clone D2 E Clone E1
Closest relative
% Similarity
Accession number
B. thuringiensis S. mizutae
94 90
457643 887639
Sphingobacterium-like bacterium
93
6729646
Paenibacillus sp.
95
Y11583
uncultured bacterium
94
UBA400574
unidenti¢ed eubacterium
93
U81676
uncultured spirochete
90
AF211319
C. butyricum
99
X68178
C. disporicum
98
Y18176
Pedobacter sp. unidenti¢ed eubacterium
91 92
AB033630 AJ229216
uncultured bacterium unidenti¢ed bacterium
86 92
AF132261 U81735
uncultured delta proteobacterium uncultured rumen bacterium
86 88
4995896 AF001778
Rhodanobacter lindanoclasticus Rhodanobacter sp.
92 95
AF039167 AF250415
unidenti¢ed eubacterium
89
U81735
Indicated is the identity, % similarity, and accession number of the closest relative found in the GenBank database. Bands that were enriched during 7 weeks of continuous aeration as described in Section 2 are indicated. Band designations correspond to bands identi¢ed in Figs. 2 and 3.
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tion of LSM is quite di¡erent from that excreted by the animal. Both the microbial and chemical composition of swine faeces is in£uenced by such factors as diet and animal age [24,28]. The composition of the LSM will additionally be in£uenced by farm management practices including feeding and watering regimen, temperature of the collecting pit, and the frequency of pit emptying [9]. In spite of this variability, we found DGGE pro¢les prepared from LSM obtained from the same collection pit at 3month intervals to be remarkably similar, suggesting that our results are at least generally representative for this one farm (data not shown). The chemical properties, redox potential, total number of bacteria determined by direct counting, the number of aerobes determined by plate count, and the relative composition of the LSM bacterial community determined by DGGE were remarkably constant in the anaerobic incubations. Because the manure-collecting tank at the farm holds the LSM for periods of up to several months before use, the liquid manure can be quite stabilised both chemically and biologically under anoxic conditions. Aeration caused a large increase in pH and redox potential, and signi¢cant change in chemical composition of the manure, evidenced by the loss of NH4 -N. Presumably volatile fatty acids were oxidised promoting alkalisation of the medium, and ammonium was either stripped out or ¢xed into growing bacteria. These profound changes in the aerated reactors did not result in a signi¢cant change in total bacterial population determined by direct counts. However, aeration promoted a 150-fold increase in aerobic populations detectable by plate count (representing only about 1% of the total population), and a signi¢cant shift in the community composition detected by DGGE. Most of the phylotypes originally present in the manure declined in relative intensity during the aerated incubation. Four phylotypes (clones F2, F3, G and I) were notably enriched by aeration. They were all related aerobic bacteria, the nearest relatives being B. thuringiensis (F2), S. mizutae (F4), a Sphingobacterium-like bacterium (G) and a Paenibacillus sp. (I) (Table 3). Paenibaccillus sp., facultatively aerobic spore-formers, can grow at the expense of complex carbon sources such as whole rice, oats or wheat, and can produce amylolytic, phospholipolytic, lipolytic and proteolytic enzymes suggesting that they could utilise components of the swine diet [29,30]. Sphingobacterium sp., members of the Cytophaga^Flavobacterium^Bacteroides group, are capable of aerobic growth on a variety of carbohydrates, and are commonly isolated from soil and activated sludge [25]. Bacillus sp. are ubiquitous, and commonly but variably able to grow aerobically on complex substrates and lower molecular mass fatty acids including acetate and propionate [31]. Based on intensity in the DGGE gel, band F (clone F2Bacillus, clone F4-Sphingobacterium) represented the most abundant phylotypes in the aerated manure at the end of the aerated incubation. Bacillus isolates randomly selected
175
from agar medium for 16S rDNA sequencing analysis were 100% similar to B. thuringiensis or Bacillus sylvestris, but were more distantly related to clone F2, suggesting that the Bacillus detected in the DGGE analysis was not well-represented in the population recovered by plate counting. Furthermore, potential Sphingobacterium sp. isolates recovered from media did not correspond to the clone F4 band on the basis of 16S rDNA sequence. Taken together, although the number of clones sequenced was small, the results suggest that the plating methods used did not capture two of the dominant phylotypes in the DGGE analysis. A comparison of the DGGE pro¢les prepared from total manure with pro¢les prepared from fractionated (bio¢lm and planktonic communities) manure indicates that changes due to aeration in the total community are re£ective of changes occurring primarily in the planktonic population. At the end of the aerated incubation the planktonic community pro¢le resembled the total community pro¢le in the aerated manure. It exhibited decreases in almost all the initial phylotypes (bands A, H, L, J, K, B and D) and became signi¢cantly less complex. In contrast, the bio¢lm community responded quite di¡erently to aeration than the planktonic community. Despite the disappearing of some of the initial major phylotypes (bands B, C and D), new phylotypes (band I, Paenibacillus ; band F, Bacillus and Sphingomonas mizutae ; band P, unculturable bacterium) started to establish and some initial bio¢lm phylotypes, such as C. butyricum (band H), C. disporicum (band J) and Pedobacter sp. (band K), remained dominant in the bio¢lm throughout the aerobic incubation. It is well-documented that matrix-embedded bacterial cells are more resistant to environmental insults [32]. Oxygen consumption by organisms at the surface of the bio¢lm, and slow di¡usion of oxygen into the bio¢lm matrix can promote anaerobic microsites in aerated systems. Thus, denitri¢cation and sulfate reduction can occur in sludge aggregates in thoroughly aerated water-treatment systems [19,33,34]. Presumably the higher relative abundance of the aerobes in the planktonic fraction is due to the availability of oxygen in the aqueous phase or at the surface of bio¢lms. Clostridium sp. are active in stored LSM, fermenting lipids, sugars and amino acids to produce malodorous volatile organic acids, hydrogen sul¢de and amines [4]. Clostridium perfringens is commonly used as an indicator of faecal pollution of water [35]. The DGGE pro¢les of total manure suggested that aeration promoted a decline in Clostridium populations. However, DGGE pro¢les prepared on fractionated samples suggest that this decline is illusory ; that the mix of PCR products from the total manure sample was biased by the increase in planktonic template during the aerated incubation. Recognising that DGGE-PCR is not quantitative, the pro¢les do indicate that some organisms will preferentially partition into the bio¢lm. This result is signi¢cant for two reasons. Clearly
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organisms embedded in the bio¢lm matrix will be less vulnerable to chemical or other treatment methods that could be used to reduce bacterial, especially pathogenic, populations in stored LSM. This phenomenon has been well-documented in numerous clinical, industrial and other applications [32]. Secondly, the potential for surface or groundwater contamination by indicator or pathogenic bacteria following manure application is likely to be affected by their distribution in the planktonic and bio¢lm phases. Adherence to £ocs is a variable property, particularly in£uenced by cell surface hydrophobicity [36]. Bacteria which adhere to surfaces are less mobile in aquifer sediments than non-adherent bacteria [37]. It would therefore be of interest to determine the distribution of pathogenic and indicator bacteria in the bio¢lm and planktonic phases, how the spatial distribution may evolve during storage, and what in£uence this distribution may have on the persistence and transport potential of these bacteria following manuring of soils. In summary, this is the ¢rst culture-independent study to examine the change of bacterial constituents in aerated LSM. The major bacterial phylotypes identi¢ed in our LSM samples were di¡erent from microbial species commonly found in the pig gastrointestinal tract or faeces by culture-dependent methods. Other than the two Clostridium spp., 11 out of 13 of the phylotypes identi¢ed in the LSM were not shown in any previous studies. The DGGE analysis also suggested that the aerated LSM was dominated by a Bacillus sp. and two Sphingobacterium spp. Although it was not clear if these phylotypes were involved in the biological and chemical changes of the aerated LSM, the ¢ndings provided valuable information to further study the roles of these micro-organisms in aerated LSM. Finally, we also showed that some bacterial phylotypes, such as the Clostridium spp., could be protected in the aggregates of LSM under constant aeration. This may have important implications on the persistence and transport of potential enteric pathogens in environments exposed to LSM. Acknowledgements
[3]
[4] [5]
[6] [7]
[8]
[9] [10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
This work was funded by Ontario Pork, and Agriculture and Agri-Food Canada's Matching Investment Initiative. We thank Y.-C. Tien and Martha Welsh for excellent technical assistance.
[19]
[20]
References [1] Choudhary, M., Bailey, L.B. and Grant, C.A. (1996) Review of the use of swine manure in crop production. E¡ects on yield and composition and on soil and water quality. Waste Manage. Res. 14, 581^ 595. [2] Mawdsley, J.L., Bardgett, R.D., Merry, R.J., Pain, B.F. and Theodorou, M.K. (1995) Pathogens in livestock waste, their potential for
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movement through soil and environmental pollution. Appl. Soil Ecol. 2, 1^15. Peterson, E.W., Davies, R.K. and Orndor¡, H.A. (2000) 17 L-estradiol as an indicator or animal waste contamination in mantled karst aquifers. J. Environ. Qual. 29, 826^834. Zhu, J. (2000) A review of microbiology in swine manure odor control. Agric. Ecosyst. Environ. 78, 93^106. Chikh, G., Pourquie, J., Kaiser, P. and Davila, A.M. (1997) Characterization of the bacterial £ora isolated from a pilot-scale lagoon processing swine manure. Can. J. Microbiol. 43, 1079^1083. Imbeah, M. (1998) Composting piggery waste: a review. Biores. Technol. 63, 197^203. Kearney, T.E., Larkin, M.J. and Everett, P.N. (1993) The e¡ect of slurry storage and anaerobic digestion on survival of pathogenic bacteria. J. Appl. Bacteriol. 74, 86^93. Munch, B., Larsen, H.E. and Aalbaek, B. (1987) Experimental studies on the survival of pathogenic and indicator bacteria in aerated and non-aerated cattle and pig slurry. Biol. Wastes 22, 49^65. Spoelstra, S.F. (1978) Enumeration and isolation of anaerobic microbiota of piggery wastes. Appl. Environ. Microbiol. 35, 841^846. Westerman, P.W. and Zhang, R.H. (1997) Aeration of livestock manure slurry and lagoon liquid for odor control: a review. Appl. Eng. Agric. 13, 245^249. Himathongkham, S. and Riemann, H. (1999) Destruction of Salmonella typhimurium, Escherichia coli O157:H7 and Listeria monocytogenes in chicken manure by drying and/or gassing with ammonia. FEMS Microbiol. Lett. 171, 179^182. Himathongkham, S., Bahari, S., Riemann, H. and Cliver, D. (1999) Survival of Escherichia coli O157:H7 and Salmonella typhimurium in cow manure and cow manure slurry. FEMS Microbiol. Lett. 178, 251^257. Wu, J.J., Hee, P.S., Hengemuehle, S.M., Yokoyama, M.T., Person, H.L. and Masten, S.J. (1998) The e¡ect of storage and ozonation on the physical, chemical, and biological characteristics of swine manure slurries. Ozone Sci. Eng. 20, 35^50. Iannotti, E.L., Fischer, J.R. and Sievers, D.M. (1982) Characterization of bacteria from a swine manure digester. Appl. Environ. Microbiol. 43, 136^143. Salanitro, J.P., Blake, I.G. and Muirhead, P.A. (1977) Isolation and identi¢cation of fecal bacteria from adult swine. Appl. Environ. Microbiol. 33, 79^84. Suau, A., Bonnet, R., Sutren, M., Godon, J.J., Gibson, G.R., Collins, M.D. and Dore, J. (1999) Direct analysis of genes encoding 16S rRNA from complex communities reveals many novel molecular species within the human gut. Appl. Environ. Microbiol. 65, 4799^ 4807. Pryde, S.E., Richardson, A.J., Stewart, C.S. and Flint, H.J. (1999) Molecular analysis of the microbial diversity present in the colonic wall, colonic lumen, and cecal lumen of a pig. Appl. Environ. Microbiol. 65, 5372^5377. Bloem, J., Bolhuis, R.R., Veninga, M.R. and Wieringa, J. (1995) Microscopic methods for counting bacteria and fungi in soil. In: Methods in Applied Soil Microbiology and Biochemistry (Kassem, A. and Nannipieri, P., Eds), pp. 162^173. Academic Press, London. Santegoeds, C.M., Ferdelman, T.G., Muyzer, G. and de Beer, D. (1998) Structural and functional dynamics of sulfate-reducing populations in bacterial bio¢lms. Appl. Environ. Microbiol. 64, 3731^ 3731. Muyzer, G., de Waal, E.C. and Uitterlinden, A.G. (1993) Pro¢ling of microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction ampli¢ed genes coding for 16S rRNA. Appl. Environ. Microbiol. 59, 695^700. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, Cold Spring Harbor, NY. Leser, T.D., Lindecrona, R.H., Jensen, T.K., Jensen, B.B. and Moller, K. (2000) Changes in bacterial community structure in the colon
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of pigs fed di¡erent experimental diets and after infection with Brachyspira hyodysenteriae. Appl. Environ. Microbiol. 66, 3290^3296. Lin, C., Raskin, L. and Stahl, D.A. (1997) Microbial community structure in gastrointestinal tracts of domestic animals : comparitive analyses using rRNA-targeted oligonucleotide probes. FEMS Microbiol. Ecol. 22, 281^294. Simpson, J.M., McCracken, V.J., White, B.A., Gaskins, H.R. and Mackie, R.I. (1999) Application of denaturant gradient gel electrophoresis for the analysis of the porcine gastrointestinal microbiota. J. Microbiol. Methods 36, 167^179. Steyn, P.L., Segers, P., Vancanneyt, M., Sandra, P., Kersters, K. and Joubert, J.J. (1998) Classi¢cation of heparinolytic bacteria into a new genus, Pedobacter, comprising four species :Pedobacter heparinus comb. nov., Pedobacter piscium comb. nov., Pedobacter africanus sp. nov. and Pedobacter saltans sp. nov. Proposal of the family Sphingobacteriaceae fam. nov.. Int. J. Syst. Bacteriol. 48, 165^177. Ahn, M.Y., Shin, K.H., Kim, D.H., Jung, E.A., Toida, T., Linhardt, R.J. and Kim, Y.S. (1998) Characterization of a Bacteroides sp. from human intestine that degrades glycosaminoglycans. Can. J. Microbiol. 44, 423^429. Nalin, R., Simonet, P., Vogel, T.M. and Normand, P. (1999) Rhodanobacter lindaniclasticus gen. nov., sp. nov., a lindane-degrading bacterium. Int. J. Syst. Bacteriol. 49, 19^23. Clanton, C.J., Nichols, D.A., Mosier, R.L. and Ames, D.R. (1991) Swine manure characterization as a¡ected by environmental temperature, dietary level intake, and dietary fat addition. Trans. Am. Soc. Agric. Eng. 34, 2164^2170. Pirttija«rvi, T.S.M., Grae¡e, T.H. and Salkinoja-Salonen, M.S. (1996) Bacterial contaminants in liquid packaging boards: assessment of potential for food spoilage. J. Appl. Bacteriol. 81, 445^458.
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[30] Shida, O., Takagi, H., Kadowaki, K., Nakamura, L.K. and Komagata, K. (1997) Transfer of Bacillus alginolyticus, Bacillus chondroitinus, Bacillus curdlanolyticus, Bacillus glucanolyticus, Bacillus kobensis, and Bacillus thiaminolyticus to the genus Paenibacillus and emended description of the genus Paenibacillus. Int. J. Syst. Bacteriol. 47, 289^298. [31] Sneath, P.H.A. (1986) Endospore-forming Gram-positive rods and cocci. In: Bergey's Manual of Systematic Bacteriology (Sneath, P.H.A., Mair, N.S., Sharpe, M.E. and Holt, J.G., Eds.), pp. 1104^ 1139. Williams and Wilkins, Baltimore, MD. [32] Costerton, J.W., Lewandowski, Z., Caldwell, D.E., Corber, D.R. and Lappin-Scott, H.M. (1995) Microbial bio¢lms. Ann. Rev. Microbiol. 49, 711^745. [33] de Beer, D., Schramm, A., Santegoeds, C.M. and Nielsen, H.K. (1998) Anaerobic processes in activated sludge. Water Sci. Technol. 37, 605^608. [34] Schramm, A., Santegoeds, C.M., Nielsen, H.K., Ploug, H., Wagner, M., Pribyl, M., Wanner, J., Amann, R. and de Beer, D. (1999) On the occurrence of anoxic microniches, denitri¢cation, and sulfate reduction in aerated activated sludge. Appl. Environ. Microbiol. 65, 4189^4196. [35] Conboy, M.J. and Goss, M.J. (1999) Contamination of rural drinking water wells by fecal origin bacteria : survey ¢ndings. Water Qual. Res. J. Can. 34, 281^303. [36] Olofsson, A.C., Zita, A. and Hermansson, M. (1998) Floc stability and adhesion of green-£uorescent-protein-marked bacteria to £ocs in activated sludge. Microbiol. 144, 519^528. [37] De£aun, M.F., Oppenheimer, S.R., Streger, S., Condee, C.W. and Fletcher, M. (1999) Alterations in adhesion, transport, and membrane characteristics in an adhesion-de¢cient pseudomonad. Appl. Environ. Microbiol. 65, 759^765.
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Bacterial community dynamics in liquid swine manure during storage: molecular analysis using DGGE/PCR of 16S rDNA Kam Leung a , Edward Topp b
b;
*
a Department of Biology, Lakehead University, 955 Oliver Road, Thunder Bay, ON, Canada P7B 5E1 Agriculture and Agri-Food Canada, Research Branch, 1391 Sandford Street, London, ON, Canada N5V 4T3
Received 12 July 2001; received in revised form 17 September 2001; accepted 18 September 2001 First published online 9 November 2001
Abstract Denaturant gradient gel electrophoresis (DGGE) analysis of polymerase chain reaction-amplified DNA fragments prepared from extracted DNA using universal 16S rDNA primers was used to compare the composition of bacterial communities in liquid swine manure (LSM) during incubation in aerated and in non-aerated laboratory reactors. The LSM was initially dominated by 13 phylotypes whose identities were established by excising and cloning DGGE bands, and comparing the 16S rDNA sequences with those available in GenBank. With varying degrees of similarity, Clostridium butyricum, Clostridium disporicum, a Pedobacter sp., two Rhodanobacter sp., a spirochete, and seven uncultured eubacterial sequences were identified. The chemical composition, total microbial populations determined by direct microscopic count, and DGGE profiles of the LSM were stable during anaerobic storage for 7 weeks. However, the community composition of the LSM changed substantially with aeration, the DGGE bands in the original sample receding in intensity as the manure community became dominated by phylotypes most closely related to the aerobes Bacillus thuringiensis, Sphingobacterium mizutae, a `Sphingobacteriumlike' bacterium, and a Paenibacillus sp. When continuously aerated, the pH rose from 8 to 9.5, the ammonium-N content decreased, populations of culturable aerobes increased 150-fold, and total bacterial populations remained stable. DGGE analysis of manure fractionated into planktonic and biofilm (flocs and aggregates) communities suggested that Clostridium populations were stable in biofilms during aeration, whereas the aerobes that ultimately dominated the LSM community were primarily in the planktonic phase. ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
1. Introduction Animal manures represent an important and valuable source of nutrients for agricultural crop production. At the same time, public health and environmental concerns regarding objectionable odours, chemical contaminants and pathogens pose a challenge for the storage and safe handling of animal wastes [1^4]. Storage conditions which promote the destruction of these contaminants have been the subject of numerous investigations, such as, controlling the range of redox conditions from aeration to anaerobic digestion, increasing storage temperature, drying and ozonation [2,5^13]. It has long been recognised that sub-populations of the global microbial population in liquid swine manure (LSM) may be responsible for breaking down various organic compounds and/or involved in sup-
* Corresponding author. Tel. : +1 (519) 457 1470 ext. 235; Fax: +1 (519) 457 3997. E-mail address : [email protected] (E. Topp).
pressing pathogens during storage of the manure. However, only a few studies have examined the impact of storage conditions on the global microbial composition of LSM. These studies have been generally limited to the use of culture-dependent methods [5,9,14,15]. Recently, a study of the bacterial 16S rDNA sequences extracted and cloned from a human fecal sample found only 24% of the phylotypes corresponded to known organisms [16]. Furthermore, a similar study of bacteria in the colonic and cecal lumens of a pig showed that 59% of the 16S rDNA cloned sequences have less than 95% similarity to sequences from cultivated organisms [17]. Clearly the present information on the swine manure bacterial populations based on cultivation approaches may misrepresent the true diversity of microbial populations in LSM. As part of a study on the impact of aeration on the composition of animal wastes, we undertook a cultureindependent examination of the major bacterial constituents of LSM and how the microbial community in the manure changes during storage in aerated and unaerated reactors.
0168-6496 / 01 / $22.00 ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 6 4 9 6 ( 0 1 ) 0 0 1 8 1 - 7
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Table 1 Chemical analysis of the manure at the start of the incubation, and following 7 weeks of incubation under aerated or unaerated conditions Manurea
Dry matter (%) Organic matterb (%) Mineral Nc (%) NH4 -N (%)
initial
aerated
unaerated
0.6 36 0.24 0.23
0.6 28 0.03 0.02
0.6 33 0.24 0.23
a
Means of two replicates. Organic matter is reported on a dry matter basis. c Mineral constituents are reported on a weight per volume basis. b
2. Materials and methods 2.1. Aerobic and anaerobic manure reactors LSM was collected from a 600-animal farrowing farm located in South Western Ontario, Canada. The LSM consists of urine and faecal material from sows, and was obtained from the barn's manure collecting tank (pit-type storage), which holds the LSM for periods of up to several months until it can be applied to adjacent ¢elds. The tank is buried beneath the barn and thus the LSM would be sheltered from temperature and moisture-content £uctuations typical of an open outdoor lagoon. The composition of the LSM is reported in Table 1. 750-ml portions of fresh manure were dispensed into 1-l mason jars which were then capped with a lid ¢tted with several ports. The reactors were incubated for 7 weeks at room temperature (22 þ 1³C), with continuous agitation by means of a magnetic stirrer. Humidi¢ed compressed air was bubbled into the aerated manure at a £ow rate of 120 ml min31 , whereas anaerobic reactors were left unsparged and hermetically sealed. The headspace pressure in the unsparged reactors was released periodically by inserting a syringe needle through a rubber septum in the lid. All treatments were conducted in triplicate. 2.2. Chemical analyses Manure samples were analysed for chemical composition using standard methods (Table 1; ApL Canada Lab-
oratories East, London, ON, Canada). The pH and redox potential of the manure were measured periodically during the incubations with an Accumet pH/voltmeter (Fisher Scienti¢c, Nepean, ON, Canada) equipped with a combination pH electrode (Orion Research, Beverly, MA, USA) or a platinum redox electrode (Orion Research) (Table 2). Reported redox values are relative to the normal hydrogen electrode. 2.3. Microbiological enumerations Ten-fold dilution serial dilutions of the manure samples were prepared in 0.1% sodium pyrophosphate (w/v of water) and 0.1-ml portions of the sample solutions were spread-plated on Iso-sensitest agar medium (Oxoid, Basingstoke, Hampshire, UK). Colony-forming units (cfu) on the Iso-sensitest agar were determined following 4 days of incubation at 30³C. Direct bacterial counts were performed on 5-[4,6-dichlorotriazin-2-yl]amino£uorescein (DTAF)-stained (Sigma-Aldrich Canada, Oakville, ON, Canada) manure samples [18]. Samples were preserved in formaldehyde (3.7% ¢nal concentration, v/v), and diluted 50-fold with sterile 0.85% NaCl solution. Ten Wl diluted suspension was smeared in a pre-etched area (1.77 cm2 ) on a RITE-ON microscope slide (Gold Seal Products, Highpark, IL, USA). The smears were air-dried, heat-¢xed and stained with 0.02% DTAF. Bacterial counts were determined by epi£uorescent microscope equipped with a £uorescein ¢lter set. 2.4. DNA extraction Nucleic acid extraction was performed using UltraClean soil DNA isolation kits (MoBio Laboratories, Solana Beach, CA, USA) according to the manufacturer's instructions with the following modi¢cations. One ml of manure sample was collected from each manure reactor and pelleted in a 1.5-ml micro-centrifuge tube at 16 000Ug for 10 min. The supernatant was discarded and the cell pellet was resuspended in 50 Wl sterile deionised water and transferred to the 2-ml bead solution tube of the UltraClean soil DNA isolation kit. The sample was mixed with an
Table 2 Redox potentials and pH of aerated and anaerobic manure samplesa Days
0 2 7 14 21 35 49 a
Redox potential (mV)
pH
aerated
anaerobic
aerated
anaerobic
3191 ( þ 2) +170 ( þ 6) +209 ( þ 9) +246 ( þ 5) +256 ( þ 6) +271 ( þ 15) +287 ( þ 5)
3187 3162 3153 3153 3128 3123 3177
8.01 9.25 9.48 9.59 9.44 9.32 9.53
8.01 8.10 8.06 8.11 8.11 8.17 8.11
( þ 1) ( þ 11) ( þ 8) ( þ 14) ( þ 19) ( þ 10) ( þ 7)
Data are means þ S.D. ; n = 3.
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( þ 0.07) ( þ 0.04) ( þ 0.03) ( þ 0.04) ( þ 0.05) ( þ 0.06) ( þ 0.02)
( þ 0.10) ( þ 0.02) ( þ 0.02) ( þ 0.01) ( þ 0.06) ( þ 0.19) ( þ 0.23)
K. Leung, E. Topp / FEMS Microbiology Ecology 38 (2001) 169^177
inhibitor removal solution provided by the kit, and agitated twice in a Bio 101 Savant Fast Prep FP120 bead beater at a setting of 5.5 for 30 s. The crude DNA extract was puri¢ed by passing through a spin-column containing a DNA-adsorbing silica matrix as described by the manufacturer. Puri¢ed DNA was eluted from the matrix into 35 Wl sterile bu¡er solution provided by the DNA extraction kit. 2.5. Polymerase chain reaction (PCR)^denaturant gradient gel electrophoresis (DGGE) PCR primers targeting the V3^V5 region of 16S rDNA gene of all bacteria at the nucleotide positions 341^357 and 907^928 (based on the 16S rDNA of Escherichia coli, accession no. J01859) were used to amplify the 16S rDNA fragments of bacterial populations in the manure samples. The sequences of the forward and reverse primers were 5P-CGC CCG CCG CGC CCC GCG CCC GTC CCG CCG CCC CCG CCCG CCT ACG GGA GGC AGC AG-3P (GC clamp underlined) and 5P-CCG TCA ATT CCT TTG AGT TT-3P, respectively [19]. A touchdown PCR program was performed on a 50-Wl PCR mixture containing 1.5 U of Taq DNA polymerase (Promega, Madison, WI, USA), 30 pmol of each primer, 1UPCR bu¡er, 1.5 mM MgCl2 , 200 WM dNTP and 2 Wl of the 10-fold diluted puri¢ed manure DNA extract [20]. DGGE was performed with a DGene 16U16-cm 10% polyacrylamide gel (Bio-Rad, Hercules, CA, USA) maintained at 60³C in 7 l of Tris^acetate^EDTA (TAE) bu¡er (20 mM Tris^acetate, 0.5 M EDTA, pH 8.0). Gradient gels were prepared with 35 and 65% denaturant (100% denaturant de¢ned as 7 M urea plus 40% v/v formamide). Between 0.5 and 1 Wg of the ampli¢ed DNA was loaded per well and run at 100 V for 16 h. Gels were stained in 150 ml TAE bu¡er containing 15 Wl 10 000U concentrated SYBR Green I (Molecular Probes, Eugene, OR, USA) for 1 h and destained in 350 ml TAE for 1 h. Images were captured using a Bio-Rad gel documentation system. Two approaches were used to ensure the correct designation of the DGGE bands in di¡erent samples. First, since the DGGE band pattern (such as bands A, H, J, K, B, C, D and E) of the initial LSM sample was highly consistent, it was used as a standard marker in di¡erent runs to facilitate gel-to-gel (or lane-to-lane) comparison. Second, some major DGGE bands (e.g. bands A, B, F, G and J) were sequenced twice under various occasions, such as under aerated and aggregate conditions, to con¢rm their phylotypes. 2.6. Cloning of 16S rDNA fragments resolved by DGGE Bands in the polyacrylamide gel were excised and rinsed in 1 ml of sterile deionised water. The gel was crushed in 50 Wl sterile deionised water and allowed to equilibrate overnight at 4³C. Five Wl of the DNA extract was used
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as the template in a PCR ampli¢cation as described earlier, except that the forward primer lacked the GC clamp. The PCR products were puri¢ed by electrophoresis through 1.0% low melting agarose and a centrifugation procedure. DNA in the low-melting agarose was extracted by centrifugation through a glass-wool-stu¡ed 0.5-ml micro-centrifuge tube which had a tiny hole punched through its bottom. The DNA samples collected in a 1.5-ml micro-centrifuge tube were puri¢ed either by a phenol^chloroform extraction protocol [21] or the QIAquick PCR Puri¢cation Kit (Qiagen Canada, Mississauga, ON, Canada). The PCR-ampli¢ed products were cloned into the pGEM-T cloning vector (Promega). 2.7. Sequence analysis Three clones from each DGGE band were randomly chosen for sequencing analysis. If the sequence of the three clones from the DGGE band were not identical, three more clones from the same band were sequenced to estimate the number of 16S rDNA sequences co-migrating on the DGGE band. The cloned PCR fragments were sequenced by an ABI-Prism model 377 automatic sequencer (Perkin-Elmer, Forest City, CA, USA) using a T7 primer (5P-TAA TAC GAC TCA CTA TAG GG) targeting the T7 transcription initiation site of the pGEM-T vector. Sequence alignments were performed using the software program DNAMAN (version 4.0; Lynnon BioSoft, Vaudreuil, QC, Canada). Sequences were compared with the GenBank database by using the BLASTN facility of the National Center for Biotechnology (http:// www2.ncbi.nlm.nih.gov/BLAST/) and were also tested for possible chimera formation with the CHECK_CHIMERA program (http://www.cme.msu.edu/rdp/cgis/chimera.cgi?su = SSU). 2.8. Separation of bio¢lm and planktonic cells Bio¢lm bacterial cells attached on the aggregate surfaces and clustered in large £ocs were separated from planktonic bacterial cells by centrifugation. To optimise the separation of the bio¢lm and planktonic cells in the manure samples, the manure samples were centrifuged in a micro-centrifuge at 400Ug for 30, 60 and 150 s. Microbial cells in the supernatant were stained with the LIVE/ DEAD BacLight stain (Molecular Probes) and examined with an epi£uorescent microscope for bacterial £ocs and aggregates. Centrifugation at 400Ug for 60 s was found to provide the best separation. The bio¢lm population in the samples were pelleted by centrifuging a 1-ml sample at 400Ug for 60 s. The supernatant containing the planktonic microbial cells was centrifuged at 16 000Ug for 10 min to recover the planktonic cells from the liquid phase. Samples containing the planktonic, bio¢lm and total microbial populations were stored at 320³C for DNA extraction.
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2.9. Isolation of Bacillus and potential Sphingobacterium spp. Because sequencing analysis of the two major DGGE bands (i.e. bands F and G) showed that the aerated LSM samples were dominated by the genera Bacillus and Sphingobacterium, the traditional cultivation approach was used to isolate potential Bacillus and Sphingobacterium at the highest dilution of the aerated LSM samples. The rDNAs of the isolates were sequenced and compared to the three dominant phylotypes in the aerated LSM identi¢ed by DGGE. A dilution series of the week 7 aerated manure samples were spread-plated on the Iso-Sensitest agar and Bacillus-selective agar (Oxoid). Ten potential Sphingobacterium (yellow-pigmented) colonies were isolated from the Iso-Sensitest agar and re-streaked on tryptic soy agar (TSA) plates. Ten presumptive Bacillus colonies were also isolated from the Bacillus-selective agar and restreaked on TSA plates. Genomic DNAs were extracted from four randomly chosen presumptive Bacillus and Spingobacterium spp. by the InstaGene Matrix DNA puri¢cation kit (Bio-Rad Laboratories) as described by the manufacturer. The DNA extract was used as the template in a PCR ampli¢cation as described earlier, except that the forward primer lacked the GC clamp. The PCR products were gel-puri¢ed as described earlier and the fragments were sequenced by an ABI-Prism model 377 automatic sequencer (Perkin-Elmer) using the DGGE forward primer (without GC clamp). 3. Results 3.1. Physico-chemical composition Initially, the manure contained 0.6% dry matter, about a third of which was organic matter (i.e. lost upon ashing; Table 1). Almost all of the inorganic nitrogen was in the form of ammonium-N. The redox potential was about 3190 mV, and the pH value about 8.0 (Table 2). These parameters remained unchanged throughout the experiment in the unaerated treatment (Tables 1 and 2). In contrast, aeration promoted rapid and signi¢cant changes in the manure. The redox potential increased to +170 mV within 2 days, and up to +287 mV by the end of the incubation. The pH increased to a value of pH 9.25 within 2 days, and remained at about pH 9.5 thereafter. By the end of the experiment the organic matter content had decreased somewhat, and almost all of the inorganic nitrogen and ammonium-nitrogen was lost. The aerated manure rapidly became dark brown, in contrast to the grey^ black of the unaerated material. This colour di¡erence was maintained throughout the incubation. In addition, after 2 days of aeration, the unpleasant odour of the manure disappeared in the aerated samples. The ammonia smell was detected transiently from the aerated sample in the
Fig. 1. Persistence of total bacteria (determined by direct microscopic count) and selected aerobes (determined by plate count) in LSM incubated with or without aeration.
¢rst few days and disappeared afterward. On the other hand, the undesirable odours persisted in the anaerobic manure samples. 3.2. Dynamics of bacterial populations Total bacterial populations enumerated by microscopic direct counts were stable at about 1U1010 cells/ml in the manure regardless of the aeration treatment (Fig. 1). Culturable aerobic bacterial populations decreased somewhat from 1.2U106 to 3.0U105 cfu ml31 by the end of the experiment in the unaerated treatment. However, in the aerated reactors, culturable aerobic bacterial population increased about 150-fold from 7.3U105 to 1.1U108 cfu ml31 in 7 weeks, with most of the growth occurring in the ¢rst 7 days. 3.3. DGGE analysis Background smearing in the DGGE pro¢les indicated the presence of a diverse community in the LSM (Fig. 2). However, the relative prominence of nine distinct bands indicated that the community was dominated by a relatively small number of phylotypes. In the unaerated reactors no changes were observed in the DGGE pro¢les over the course of the experiment (Fig. 2). In contrast, three novel bands (F, G and I) emerged in the DGGE pro¢les of the aerated manure samples. Band F appeared at week 3 and increased in intensity to become one of the major bands by week 7. Band G appeared at week 5 and remained visible thereafter. Band I was a weak band visible at week 7. The appearance of these bands was accompanied by a decrease in the relative intensities of the nine DGGE bands dominating the community at the start of the incubation. The relative composition of the planktonic and bio¢lm bacterial communities were di¡erent (Fig. 3). For example,
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3.5. 16S rDNA of Bacillus and potential Sphingobacterium isolates
Fig. 2. DGGE pro¢les of DNA obtained from aerated (A) and unaerated (B) LSM. Lane 1, start of the incubation ; lane 2, after 1 week; lane 3, after 3 weeks ; lane 4, after 5 weeks ; lane 5, after 7 weeks of incubation.
bands A and L were more prominent in the planktonic community, whereas bands C and B were more prominent in the bio¢lm community. The response of these communities to aeration was distinct. Following 7 weeks of incubation, the bio¢lm community was dominated by six bands (H, I, J, F, K and P) with the other initial bands receding in intensity. In contrast, the planktonic community at the end of the incubation was dominated by two bands (F and G) with other bands receding. 3.4. Sequence analysis The major DGGE bands were cloned, sequenced and matched with the GenBank database (Table 3). Three to six clones were sequenced from each band. None of the cloned sequences were chimeric according to the CHECK_CHIMERA program. Eight bands (A, E, G, H, L, I, J and P) yielded a single 16S rDNA sequence, and the remaining bands (F, K, D, B and C) were composed of two co-migrating 16S rDNA fragments (Table 3). Bands in the initial LSM samples which corresponded to known organisms in the database included Clostridium butyricum (band H), Clostridium disporicum (J), a Spirochaeta sp. (L), a Pedobacter sp. (K), and two members of the genus Rhodanobacter (D). Percent similarities ranged from good (99%) to poor (89%). The three bands which appeared in the aerated samples, were shown to be composed of three genera. Clone F2 was most closely related to Bacillus thuringiensis (94% similarity), clone F4 to Sphingobacterium mizutae (90% similarity), band G to a Sphingobacterium-like bacterium (93% similarity), band I to a Paenibacillus sp. (95% similarity). The remaining bands were most closely related to either uncultured or unidenti¢ed eubacteria at similarity levels of 86^94%. A dominant band appearing in the aerated bio¢lm (band P) was most closely related to an uncultured bacterium isolated from a deteriorating wall painting (94% similarity).
Four randomly selected potential Bacillus isolates, and four potential Sphingobacterium isolates (on the basis of yellow pigmentation), were tentatively identi¢ed on the basis of 16S rDNA sequence analysis. All four isolates recovered from Bacillus selective media showed 100% similarity to B. thuringiensis, Bacillus mycoides or Bacillus cereus, but were only distantly related (94% similarity based on sequence alignment, DNAMAN software program) to clone F2. Of the yellow-pigmented isolates, Iso6, Iso-12 and Iso-14 showed high 16S rDNA similarity to Bacillus silvestris (100%), Caulobacter sp. (99%) and Aureobacterium kitamiense (99%), respectively. Isolate Iso-3 was 91% similar to the 16S rDNA sequence of Sphingobacterium multivorum, but was only distantly related to clones F4 and G1, at 88 and 91% similarity, respectively. 4. Discussion Previous studies have used various non-selective media to characterise bacteria in swine waste [9,14,15]. However, the disadvantages of cultivation approach are likely to result in an underestimate and/or bias in the diversity captured in these studies [14,15]. A few recent studies have used the culture-independent 16S rDNA-based PCR method to determine the diversity and dynamics of micro-organisms in gastrointestinal tracts of pigs [17,22^24]. However, little is known about the culture-independent global microbial composition of LSM and the in£uence of oxygen status on the evolution of the microbial community during storage of LSM. In this study, 13 major bacterial phylotypes were found in the initial manure samples (Table 3). Three phylotypes, identi¢ed as C. butyricum (clone H2), C. disporicum (clone J1), and a Rhodanobacter sp. (clone D2) showed v95% identity with sequences in the GenBank database. Other
Fig. 3. DGGE pro¢les of DNA obtained from aerated LSM fractionated into bio¢lm (A) or supernatant (B). Lane 1, start of the incubation ; lane 2, after 1 week ; lane 3, after 3 weeks; lane 4, after 5 weeks; lane 5, after 7 weeks of incubation.
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phylogenetic groups identi¢ed with lower levels of identity included Pedobacter sp. (clone K1) and Rhodanobacter lindanobacter (clone D1). The clostridia are important constituents of the swine gut community and were probably excreted by the animals [4]. The other genera have not been reported to be enteric in these animals. Pedobacter is a recently described genus, proposed to be a member of the Sphingobacteriaceae fam. nov [25]. Pedobacter sp. are heparinase-producing obligate aerobes sharing the characteristic ability of growing at the expense of heparin [25]. Since heparin is produced in porcine intestinal mucosa (Sigma Life Sciences catalogue), it is possible to ¢nd heparin-degrading bacteria in the LSM. The Rhodanobacter sp. are Gram-negative aerobic bacteria in the Q-Proteobacteria group, distantly related to Xanthomonas and Stenotrophomonas [27]. Members of the Rhodanobacter sp. are soil bacteria but there is insu¤cient information known of this species to speculate on what its role in LSM might be. The closest matching relatives to clones A3, K3, B1, B2, C1, C2 and E were either unculturable or unidenti¢ed eubacteria (Table 3). Interestingly, all these phylotypes
were found in anaerobic environments, anaerobic vinasses reactors (GenBank accession numbers U81676, U81735 and U81735), anoxic rice paddy soil (AJ229216), human gut (AF132261), bovine rumen (AF001778) or anaerobic marine sediments (4995896). Likewise, an uncultured spirochete most closely related to clone L1 is an organism isolated from a hydrogen sul¢de-rich marine sediment (X93326). These results suggest that most of the phylotypes detected in the LSM are anaerobes. Absent from the sequenced phylotypes are some genera commonly found to be well represented in the pig gastrointestinal tract or faeces, notably Lactobacillus, Streptococcus, Peptococcus, Peptostreptococcus, Propionibacterium, Ruminococcus and Bacteroides [9,14,15,17,26]. It is highly probable that these organisms are in the LSM, but in low enough number that they are not prominent in the DGGE pro¢le. Given that LSM consists of a mixture of urine and faeces and other detritus produced in the barn, that the collecting pit under the barn has a residence time of up to several months, and that it is continuously receiving fresh material, it is not surprising that the microbial composi-
Table 3 Identity of phylotypes in DGGE pro¢les of aerated manure Band designation Enriched upon aeration : F Clone F2 Clone F4 G Clone G1 I Clone 1 P Clone P1 Present in initial manure sample: A Clone A3 L Clone L1 H Clone H2 J Clone J1 K Clone K1 Clone K3 B Clone B1 Clone B2 C Clone C1 Clone C2 D Clone D1 Clone D2 E Clone E1
Closest relative
% Similarity
Accession number
B. thuringiensis S. mizutae
94 90
457643 887639
Sphingobacterium-like bacterium
93
6729646
Paenibacillus sp.
95
Y11583
uncultured bacterium
94
UBA400574
unidenti¢ed eubacterium
93
U81676
uncultured spirochete
90
AF211319
C. butyricum
99
X68178
C. disporicum
98
Y18176
Pedobacter sp. unidenti¢ed eubacterium
91 92
AB033630 AJ229216
uncultured bacterium unidenti¢ed bacterium
86 92
AF132261 U81735
uncultured delta proteobacterium uncultured rumen bacterium
86 88
4995896 AF001778
Rhodanobacter lindanoclasticus Rhodanobacter sp.
92 95
AF039167 AF250415
unidenti¢ed eubacterium
89
U81735
Indicated is the identity, % similarity, and accession number of the closest relative found in the GenBank database. Bands that were enriched during 7 weeks of continuous aeration as described in Section 2 are indicated. Band designations correspond to bands identi¢ed in Figs. 2 and 3.
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tion of LSM is quite di¡erent from that excreted by the animal. Both the microbial and chemical composition of swine faeces is in£uenced by such factors as diet and animal age [24,28]. The composition of the LSM will additionally be in£uenced by farm management practices including feeding and watering regimen, temperature of the collecting pit, and the frequency of pit emptying [9]. In spite of this variability, we found DGGE pro¢les prepared from LSM obtained from the same collection pit at 3month intervals to be remarkably similar, suggesting that our results are at least generally representative for this one farm (data not shown). The chemical properties, redox potential, total number of bacteria determined by direct counting, the number of aerobes determined by plate count, and the relative composition of the LSM bacterial community determined by DGGE were remarkably constant in the anaerobic incubations. Because the manure-collecting tank at the farm holds the LSM for periods of up to several months before use, the liquid manure can be quite stabilised both chemically and biologically under anoxic conditions. Aeration caused a large increase in pH and redox potential, and signi¢cant change in chemical composition of the manure, evidenced by the loss of NH4 -N. Presumably volatile fatty acids were oxidised promoting alkalisation of the medium, and ammonium was either stripped out or ¢xed into growing bacteria. These profound changes in the aerated reactors did not result in a signi¢cant change in total bacterial population determined by direct counts. However, aeration promoted a 150-fold increase in aerobic populations detectable by plate count (representing only about 1% of the total population), and a signi¢cant shift in the community composition detected by DGGE. Most of the phylotypes originally present in the manure declined in relative intensity during the aerated incubation. Four phylotypes (clones F2, F3, G and I) were notably enriched by aeration. They were all related aerobic bacteria, the nearest relatives being B. thuringiensis (F2), S. mizutae (F4), a Sphingobacterium-like bacterium (G) and a Paenibacillus sp. (I) (Table 3). Paenibaccillus sp., facultatively aerobic spore-formers, can grow at the expense of complex carbon sources such as whole rice, oats or wheat, and can produce amylolytic, phospholipolytic, lipolytic and proteolytic enzymes suggesting that they could utilise components of the swine diet [29,30]. Sphingobacterium sp., members of the Cytophaga^Flavobacterium^Bacteroides group, are capable of aerobic growth on a variety of carbohydrates, and are commonly isolated from soil and activated sludge [25]. Bacillus sp. are ubiquitous, and commonly but variably able to grow aerobically on complex substrates and lower molecular mass fatty acids including acetate and propionate [31]. Based on intensity in the DGGE gel, band F (clone F2Bacillus, clone F4-Sphingobacterium) represented the most abundant phylotypes in the aerated manure at the end of the aerated incubation. Bacillus isolates randomly selected
175
from agar medium for 16S rDNA sequencing analysis were 100% similar to B. thuringiensis or Bacillus sylvestris, but were more distantly related to clone F2, suggesting that the Bacillus detected in the DGGE analysis was not well-represented in the population recovered by plate counting. Furthermore, potential Sphingobacterium sp. isolates recovered from media did not correspond to the clone F4 band on the basis of 16S rDNA sequence. Taken together, although the number of clones sequenced was small, the results suggest that the plating methods used did not capture two of the dominant phylotypes in the DGGE analysis. A comparison of the DGGE pro¢les prepared from total manure with pro¢les prepared from fractionated (bio¢lm and planktonic communities) manure indicates that changes due to aeration in the total community are re£ective of changes occurring primarily in the planktonic population. At the end of the aerated incubation the planktonic community pro¢le resembled the total community pro¢le in the aerated manure. It exhibited decreases in almost all the initial phylotypes (bands A, H, L, J, K, B and D) and became signi¢cantly less complex. In contrast, the bio¢lm community responded quite di¡erently to aeration than the planktonic community. Despite the disappearing of some of the initial major phylotypes (bands B, C and D), new phylotypes (band I, Paenibacillus ; band F, Bacillus and Sphingomonas mizutae ; band P, unculturable bacterium) started to establish and some initial bio¢lm phylotypes, such as C. butyricum (band H), C. disporicum (band J) and Pedobacter sp. (band K), remained dominant in the bio¢lm throughout the aerobic incubation. It is well-documented that matrix-embedded bacterial cells are more resistant to environmental insults [32]. Oxygen consumption by organisms at the surface of the bio¢lm, and slow di¡usion of oxygen into the bio¢lm matrix can promote anaerobic microsites in aerated systems. Thus, denitri¢cation and sulfate reduction can occur in sludge aggregates in thoroughly aerated water-treatment systems [19,33,34]. Presumably the higher relative abundance of the aerobes in the planktonic fraction is due to the availability of oxygen in the aqueous phase or at the surface of bio¢lms. Clostridium sp. are active in stored LSM, fermenting lipids, sugars and amino acids to produce malodorous volatile organic acids, hydrogen sul¢de and amines [4]. Clostridium perfringens is commonly used as an indicator of faecal pollution of water [35]. The DGGE pro¢les of total manure suggested that aeration promoted a decline in Clostridium populations. However, DGGE pro¢les prepared on fractionated samples suggest that this decline is illusory ; that the mix of PCR products from the total manure sample was biased by the increase in planktonic template during the aerated incubation. Recognising that DGGE-PCR is not quantitative, the pro¢les do indicate that some organisms will preferentially partition into the bio¢lm. This result is signi¢cant for two reasons. Clearly
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organisms embedded in the bio¢lm matrix will be less vulnerable to chemical or other treatment methods that could be used to reduce bacterial, especially pathogenic, populations in stored LSM. This phenomenon has been well-documented in numerous clinical, industrial and other applications [32]. Secondly, the potential for surface or groundwater contamination by indicator or pathogenic bacteria following manure application is likely to be affected by their distribution in the planktonic and bio¢lm phases. Adherence to £ocs is a variable property, particularly in£uenced by cell surface hydrophobicity [36]. Bacteria which adhere to surfaces are less mobile in aquifer sediments than non-adherent bacteria [37]. It would therefore be of interest to determine the distribution of pathogenic and indicator bacteria in the bio¢lm and planktonic phases, how the spatial distribution may evolve during storage, and what in£uence this distribution may have on the persistence and transport potential of these bacteria following manuring of soils. In summary, this is the ¢rst culture-independent study to examine the change of bacterial constituents in aerated LSM. The major bacterial phylotypes identi¢ed in our LSM samples were di¡erent from microbial species commonly found in the pig gastrointestinal tract or faeces by culture-dependent methods. Other than the two Clostridium spp., 11 out of 13 of the phylotypes identi¢ed in the LSM were not shown in any previous studies. The DGGE analysis also suggested that the aerated LSM was dominated by a Bacillus sp. and two Sphingobacterium spp. Although it was not clear if these phylotypes were involved in the biological and chemical changes of the aerated LSM, the ¢ndings provided valuable information to further study the roles of these micro-organisms in aerated LSM. Finally, we also showed that some bacterial phylotypes, such as the Clostridium spp., could be protected in the aggregates of LSM under constant aeration. This may have important implications on the persistence and transport of potential enteric pathogens in environments exposed to LSM. Acknowledgements
[3]
[4] [5]
[6] [7]
[8]
[9] [10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
This work was funded by Ontario Pork, and Agriculture and Agri-Food Canada's Matching Investment Initiative. We thank Y.-C. Tien and Martha Welsh for excellent technical assistance.
[19]
[20]
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