on 16S rRNA gene has revealed divergence into two evolutionary lineages (Collins et al., 1994; Stackebrandt and Rainey, 1997; Stackebrandt et al., 1999). In phylogenetic analysis it was also demonstrated that the genus Clostridium is a very heterogeneous group with 19 distinct clusters. Most of the clostridial species were shown to belong to cluster I. Pathogenic clostridia of concern for animal health (C. botulinum, C. chauvoei, C. novy, C. perfringens, C. septicum and C. tetani), were found to be affiliated to cluster I. Some other pathogenic clostridia of concern for animal health were included in cluster XI, for instance C. difficile and C. sordellii. Clostridium botulinum have been shown to represent a phylogenetically heterogeneous group, present in many subclusters of cluster I and more than one toxin type can be affiliated to one single subcluster. Furthermore, species have been described, that are phylogenetically very close to C. botulinum, but they lack the toxin gene, e.g. Clostridium sporogenes.
Many bacterial species have multiple 16S rRNA operons, and the homologous constituting genes are not necessarily identical within one strain (Stackebrandt and Rainey, 1997). These sequence differences are known as polymorphism. Members of the genera Bacillus and Clostridium have unusually large numbers of rRNA operons, which sometimes makes interpreting of the sequence data difficult, particularly if there are also sequences length polymorphisms.
Non pathogenic spore-forming bacteria can be used as indicators and reflect the effect on the more pathogenic ones. Spore-forming bacteria from biowaste survive after pasteurisation and digestion. Therefore, these spore-forming bacteria may pose a hygienic problem, when spreading digested residues as fertiliser.
The aim of this screening study was to identify and quantify species of Bacillus spp. and Clostridium spp. in manure, slaughterhouse waste and in substrates from different stages in biogas plants process.
faecal samples were collected at one occasion between January and April 2003. All cattle were healthy at the sampling occasion. The samples were taken by local veterinarians during regular visits. Approximately 100 g of each sample were collected and stored in clean pots.
At the sampling occasion a questionnaire was filled in concerning feeding, health, manure spreading practice, size of the farm and grazing routines. The biggest farm housed between 100-120 cows and around 200 heifers. The smallest farm housed 12 cows and 5 heifers (Table 1).
2.1.2 Slaughterhouses:
Two slaughterhouses, U and K, took samples from waste intended for biogas production once a day during ten days. The slaughterhouse U was located close to the farms included in this study. The samples from this slaughterhouse were fetched daily by employees at the National Veterinary Institute (SVA). Samples from slaughterhouse K were daily sent by mail to SVA. The samples were taken by the employees at the slaughterhouse.
Approximately 100 g of each sample were collected and stored in clean pots.
2.1.3 Biogas plants:
Samples were collected from two biogas plants, K and L. From both biogas plants, samples were taken from the homogenisation tank (before pasteurisation, BP), after pasteurisation (AP) and after digestion (AD), once a week during ten weeks. The employees at the biogas plants carried out the sampling. The samples were without delay sent by mail to SVA.
Approximately 100 g of each sample were collected and stored in clean pots.
The slaughterhouse K was located close to the biogas plant K. This slaughterhouse sent the animal by-products to biogas plants K. Hydrochloric acid (HCl) was added to the digester of biogas plant L to increase the gas production, but the amount was not available.
2.2 Culture
2.2.1 Quantitative methods
The samples were analysed immediate after arrival at the laboratory. Ten gram of substrate (faeces, slaughterhouse waste or biogas plant material) were placed in a bottle and heated at 65°C for 10 min. Quantitative analyses were made after performing 10-fold dilution series in peptone saline solution (0.9%).
Sulphite Cycloserine agar plates (TSC, perfringens agar base, Oxoid, Basingstoke, England) the level was reported to be 10 cfu/mL. The detection levels for Clostridium spp. on Fastidious Anaerobic Agar plates (FAA, LabM, Bury, Lancashire, England), with 5% defibrinated horse blood, and for Bacillus spp. on horse blood agar plates, were reported to be 100 cfu/mL.
2.2.2 Clostridium spp.
For analysis of Clostridium spp. the samples were cultured on TSC and FAA.
One millilitre from different steps in the 10-fold dilution series was mixed with melted TSC and 0.1 mL was streaked onto FAA plates. All agar plates were incubated at 37°C for 24 h in anaerobic jars (ANAEROgen®, Oxoid, Basingstoke, Hampshire, England). Following incubation different species of Clostridium spp. were quantified and subcultured.
Suspected colonies of C. perfringens were counted and from each sample, 10 colonies were subcultured on egg yolk agar and horse blood agar and incubated in anaerobic jars at 37°C for 24 h. From each sample, two C.
perfringens isolates (lecithinase positive and with haemolysis), were stored at -20°C.
Suspected colonies of Clostridium spp. other than C. perfringens were counted and subcultured onto horse blood agar. Following anaerobic incubation at 37°C for 24 h, the isolated bacteria were Gram-stained.
Clostridium spp. were identified as Gram-positive rods. The identification of clostridia was based on fermentation of glucose, maltose, lactose, sucrose, starch, mannitol and fructose, production of lecithinase, tryptophanase, urease and hydrolysis of aesculin. Other strains suspected to represent Clostridium spp. were identified by 16S rRNA sequencing. All isolates in pure culture were stored at -20°C.
2.2.3 Bacillus spp.
For analysis of Bacillus spp., 0.1 mL from different steps in the 10-fold dilution series was cultured on horse blood agar incubated at 30°C for 24 h.
Following incubation, different species of Bacillus spp. were counted and subcultured.
Suspected colonies of Bacillus spp. were counted and subcultured on horse blood agar. Bacillus cereus was confirmed by culture on Mossel Cereus Selective agar (MCS, Merck, Darmstadt, Germany) at 37°C for 24 h. Other strains of Bacillus spp. were analysed by Gram staining, catalase test, Voges-Proskauer (VP) test, and production of lecithinase and tryptophanase, fermentation of glucose, arabinose, mannitol and citrate and reduction of
nitrate. Bacillus spp. were identified as catalase positive, Gram-positive rods.
Other strains suspected to represent Bacillus spp. were identifying by 16S rRNA sequencing. All isolates in pure culture were stored at -20°C.
2.3 16S rRNA sequencing
2.3.1 DNA preparation
One loopful (approximately 5 µL of bacteria collected with a 10 µL loop) of colony material was suspended in 500 µL of phosphate-buffered saline (PBS, Merck, Darmstadt, Germany) and centrifuged for 10 min at 7,200 x g. The supernatant was discarded and the pellet was washed again by the same procedure. The pellet was re-suspended in 250 µL of double distilled H2O.
The bacterial cells were lysed by boiling the suspension for 10 min before quickly cooling on ice and then stored at -20°C until further analysis.
2.3.2 Amplification
A segment corresponding to about 90-95% of the 16S rRNA genes of Clostridium spp. and Bacillus spp. was amplified with universal primers (RIT593 and kag-001) designed for members of the phylum Firmicutes (Johansson et al., 1998; Johansson et al., 2006). The PCR mixture (50 µL total volume) comprised 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.2 mM of each of the four dNTPs (Applied Biosystems, Foster City, CA, USA), 10 pmol of each oligonucleotide primer, 1 U AmpliTaq DNA polymerase (Applied Biosystems) and 1 µL of template DNA. The template DNA was used at two concentrations, undiluted and diluted 10-1. The amplification program for Bacillus spp. consisted of an initial denaturation at 96°C for 20 s followed by 30 cycles of denaturation at 96°C for 20 s, primer annealing at 55°C for 35 s, extension at 72°C for 45 s and a last extension at 72°C for 2 min. The amplification program for Clostridium spp. consisted of 30 cycles of denaturation at 96°C for 32 s, primer annealing and extension at 60°C for 2.15 min, and a last extension at 72°C for 4 min.
The PCR experiments were performed in 200 µL tubes with individual lids in a MJ research Peltier Thermal Cycler (PTC 200) (MJ Research Inc, Watertown, Massachusetts, USA). The PCR products were separated by electrophoresis in 1.5% (w/v) agarose gel (Agarose NA, GE Healthcare, Uppsala, Sweden), stained with ethidium bromide and visualized by ultraviolet transillumination. Mycoplasma capricolum subsp. capricolum (Calif.
T
2.3.3 Sequencing of the 16S rRNA gene
Amplicons of 50 strains of Clostridium spp. and 51 strains of Bacillus spp. that could not be typed by standard methods were analysed by 16S rRNA gene sequencing. The amplicons were purified prior to sequencing by using the GFX PCR DNA and Gel Band Purification Kit (Amersham Bioscience Europe, GmbH, Freiburg, Germany).
The purified amplicons were sequenced with previously described forward (RIT583, RIT584, RIT538, 006) and reverse (RIT631, kag-011, kag-002) primers (Johansson et al., 1998; Johansson et al., 2006). The amplicons were diluted and cycle sequencing reactions were carried out with BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems Foster City, CA, USA) in combination with ethanol/EDTA/sodium acetate precipitation, according to the protocol of the manufacturer. Thermocycling was performed in a GeneAmp 2700 Thermocycler (Applied Biosystems), which resulted in seven overlapping DNA fragments. These fragments were subjected to electrophoretic separation and on-line detection on an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems). The resulting sequences were assembled into a contig corresponding to about 90-95% of the 16S rRNA gene by using the program ContigExpress, Vector NTI suite ver. 9.0. Sequence similarity searches were done in GenBank (Benson et al., 2007). The 16S rRNA gene sequence of Clostridium spp. and Bacillus spp.
found in manure, slaughterhouse waste and biogas plant substrates were deposited in GenBank under the accession numbers listed in Table 2.
2.3.4 Phylogenetic analyses
For construction of the 16S rRNA based phylogenetic tree, sequences obtained from this work were aligned manually with sequences retrieved from GenBank and prealigned sequences retrieved from the Ribosomal Database Project II (RDP-II, Cole et al., 2005). The sequences retrieved from GenBank and RDP-II were selected from the high score list obtained from the sequence similarity searches. The phylogenetic relationships among the isolates and the sequences retrieved from GenBank and RDP-II were calculated by Neighbour-Joining.
The GenBank accession numbers of the 16S rRNA gene sequences of the type and reference strains from Clostridium spp. and Bacillus spp. used for construction of phylogenetic trees are presented in Table 3 and Figure 1.
Escherichia coli with accession number J01695 and Corynebacterium ulcerans NCTC 7910T with accession number X84256 were used as outgroups for constructing phylogenetic trees for all Clostridium spp. and Bacillus spp isolates used in this study.