Analysis of Clostridium botulinum serotype E strains by using multilocus sequence typing, amplified fragment length polymorphism, variable-number tandem-repeat analysis, and botulinum neurotoxin gene sequencing

A total of 41 Clostridium botulinum serotype E strains from different geographic regions, including Canada, Denmark, Finland, France, Greenland, Japan, and the United States, were compared by multilocus sequence typing (MLST), amplified fragment length polymorphism (AFLP) analysis, variable-number tandem-repeat (VNTR) analysis, and botulinum neurotoxin (bont) E gene sequencing. The strains, representing environmental, food-borne, and infant botulism samples collected from 1932 to 2007, were analyzed to compare serotype E strains from different geographic regions and types of botulism and to determine whether each of the strains contained the transposon-associated recombinase rarA, involved with bont/E insertion. MLST examination using 15 genes clustered the strains into several clades, with most members within a cluster sharing the same BoNT/E subtype (BoNT/E1, E2, E3, or E6). Sequencing of the bont/E gene identified two new variants (E7, E8) that showed regions of recombination with other E subtypes. The AFLP dendrogram clustered the 41 strains similarly to the MLST dendrogram. Strains that could not be differentiated by AFLP, MLST, or bont gene sequencing were further examined using three VNTR regions. Both intact and split rarA genes were amplified by PCR in each of the strains, and their identities were confirmed in 11 strains by amplicon sequencing. The findings suggest that (i) the C. botulinum serotype E strains result from the targeted insertion of the bont/E gene into genetically conserved bacteria and (ii) recombination events (not random mutations) within bont/E result in toxin variants or subtypes within strains.     

Genetic analysis of type E botulinum toxin-producing Clostridium butyricum strains

Type E botulinum toxin (BoNT/E)-producing Clostridium butyricum strains isolated from botulism cases or soil specimens in Italy and China were analyzed by using nucleotide sequencing of the bont/E gene, random amplified polymorphic DNA (RAPD) assay, pulsed-field gel electrophoresis (PFGE), and Southern blot hybridization for the bont/E gene. Nucleotide sequences of the bont/E genes of 11 Chinese isolates and of the Italian strain BL 6340 were determined. The nucleotide sequences of the bont/E genes of 11 C. butyricum isolates from China were identical. The deduced amino acid sequence of BoNT/E from the Chinese isolates showed 95.0 and 96.9% identity with those of BoNT/E from C. butyricum BL 6340 and Clostridium botulinum type E, respectively. The BoNT/E-producing C. butyricum strains were divided into the following three clusters based on the results of RAPD assay, PFGE profiles of genomic DNA digested with SmaI or XhoI, and Southern blot hybridization: strains associated with infant botulism in Italy, strains associated with food-borne botulism in China, and isolates from soil specimens of the Weishan lake area in China. A DNA probe for the bont/E gene hybridized with the nondigested chromosomal DNA of all toxigenic strains tested, indicating chromosomal localization of the bont/E gene in C. butyricum. The present results suggest that BoNT/E-producing C. butyricum is clonally distributed over a vast area.     

Multiplex real-time PCR for exhaustive detection of diarrhoeagenic Escherichia coli

Aims:  The source and routes of diarrhoeagenic Escherichia coli (DEC) have not been clarified because it is difficult to detect these organisms in samples with numerous coliform bacteria. We have developed multiplex real-time PCR assays for exhaustive detection of DEC.
Methods and Results:  Primers and TaqMan probes were designed to amplify and quantify one gene ( eae , stx1 , stx2 , elt , est , virB , aggR , astA, and afaB ) from each of seven pathotypes of DEC, in duplex or triplex reactions under the same PCR cycling conditions. Specificity was confirmed using 860 strains including 88 DEC strains. The fluorescence threshold cycle and DNA concentrations correlated with decision coefficients of more than 0·99. Subsequently, meat samples and enrichment broths were spiked with DEC and the assays used to detect the genes. The detection limits varied from 7·1 × 10² to 1·1 × 10⁴ CFU ml⁻¹, depending on the target genes. All meat samples spiked with a variety of DEC (more than 10 CFU 10 g⁻¹) were found to be positive by the method.
Conclusions:  The present system allows for the efficient and simultaneous determination of various DEC pathotypes.
Significance and Impact of the Study:  This system makes epidemiological investigations for DEC sensitive and quick, and is a useful tool to clarify the source and routes of DEC.     

Detection of Clostridium botulinum types A, B, E and F in foods by PCR and DNA probe

A PCR procedure was developed for the detection of Clostridium botulinum in foods. PCR products were detected in agarose gels and by Southern hybridization. The sensitivity of PCR was tested in broth cultures and in canned asparagus, dry cured ham and honey. The sensitivity of the method in broth was high (2·1–8·1 cfu ml⁻¹) for types A and B, but rather low (10⁴ cfu ml⁻¹) for types E and F. However, after enrichment at 37°C for 18 h, it was possible to detect Cl. botulinum types A, B, E and F in food samples at initial levels of about 1 cfu 10 g⁻¹ of food. This PCR detection protocol provides a sensitive and relatively rapid technique for the routine detection of Cl. botulinum in foods.     

A Comparison of Toxin Production by Clostridium botulinum Type E in Irradiated and Unirradiated Vacuum Packed Fish

S ummary . The rates of toxin production at 10° by inocula of 10⁵, 10⁴, 10³ and 10² spores of Clostridium botulinum type E in vacuum packed herring, cod and haddock were compared with that of equivalent numbers of spores surviving γ-irradiation at 0.3 Mrad. There was little difference between the rates of toxin production in unirradiated and irradiated fish. More toxin was produced in irradiated herring than in unirradiated, but in haddock the reverse was obtained. In cod about equal amounts of toxin were found without trypsinization; after trypsinization toxin levels were generally higher in irradiated samples.     

Polymerase chain reaction for the rapid identification of Clostridium botulinum type A strains and detection in food samples

A polymerase chain reaction (PCR) was developed for the detection of Clostridium botulinum type A, a cause of human botulism. A two primer set and an oligonucleotide detection probe were used to specifically detect Cl. botulinum type A neurotoxin gene (BoNT/A). After 40 cycles of amplification, detection of a 798 bp amplified DNA fragment was carried out by agarose gel electrophoresis and Southern blot hybridization. This assay was able to detect 12.5 fg of purified target DNA or five bacteria per reaction. The sensitivity in artificially contaminated food samples after an 18 h enrichment step ranges from 10 to 10³ bacteria per g according to the type of food samples. No cross-reactions were observed with the other Cl. botulinum toxinotypes and other bacteria found routinely in food. This PCR method may provide a suitable and rapid alternative to standard techniques for detection of Cl. botulinum type A in food samples.     

Neurotoxin gene profiling of clostridium botulinum types C and D native to different countries within Europe

Clostridium botulinum types C and D, as well as their mosaic variants C-D and D-C, are associated with avian and mammalian botulism. This study reports on the development of low-density macroarrays based on the GeneDisc cycler platform (Pall-GeneDisc Technologies) applied to the simultaneous detection of the C. botulinum subtypes C, C-D, D, and D-C. The limit of detection of the PCR assays was 38 fg of total DNA, corresponding to 15 genome copies. Artificially contaminated samples of cecum showed a limit of detection below 50 spores/g. The tests were performed with a large variety of bacterial strains, including C. botulinum types C (n = 12), C-D (n = 29), D (n = 5), and D-C (n = 10), other botulinum neurotoxin (BoNT)-producing Clostridium strains (n = 20), non-BoNT-producing clostridia (n = 20), and other bacterial species (n = 23), and showed a high specificity. These PCR assays were compared to previously published real-time PCRs for the detection of C. botulinum in 292 samples collected from cases of botulism events in four European regions. The majority of the samples originated from wild birds (n = 108), poultry (n = 60), and bovines (n = 56). Among the 292 samples, 144 were positive for either the bont/C-D or the bont/D-C gene by using the GeneDisc arrays. The reliability of the results tallied to 97.94%. Interestingly, only BoNT mosaics, types C-D and D-C, were found in naturally contaminated samples whatever their animal origin and their geographical location. Further investigations should now be performed in order to check that mosaic types dominate in Europe and that acquisition of mosaic types helps in survival or adaptation to particular niche.     

Effect of Iysozyme concentration, heating at 90°C, and then incubation at chilled temperatures on growth from spores of non-proteolytic Clostridium botulinum

The heat treatment necessary to inactivate spores of non-proteolytic Clostridium botulinum in refrigerated, processed foods may be influenced by the occurrence of lysozyme in these foods. Spores of six strains of non-proteolytic Cl. botulinum were inoculated into tubes of an anaerobic meat medium, to give 10⁶ spores per tube. Hen egg white lysozyme (0–50 μg ml^-1) was added, and the tubes were given a heat treatment equivalent to 19·8 min at 90°C, cooled, and incubated at 8°, 12°, 16° and 25°C for up to 93 d. In the absence of added lysozyme, neither growth nor toxin formation were observed. A 6–D inactivation was therefore achieved. In tubes to which lysozyme (5–50 μg ml^-1) had been added prior to heating, growth and toxin formation were observed. With lysozyme added at 50 μg ml^-1, growth was first observed after 68 d at 8°C, 31 d at 12°C, 24 d at 16°C, and 9 d at 25°C. Thus, in these circumstances, a heat treatment equivalent to 19·8 min at 90°C was not sufficient, on its own, to give a 6–D inactivation. A combination of the heat treatment, maintenance at less than 12°C, and a shelf-life not more than 4 weeks reduced the risk of growth of non-proteolytic Cl. botulinum by a factor of 10⁶.     

Persistence of Escherichia coli O157:H7 on the rhizosphere and phyllosphere of lettuce

Aims:  The major objective of this study was to determine the effects of low levels of Escherichia coli O157:H7 contamination on plant by monitoring the survival of the pathogen on the rhizosphere and leaf surfaces of lettuce during the growth process.
Methods and Results:  Real-time PCR and plate counts were used to quantify the survival of E. coli O157:H7 in the rhizosphere and leaf surfaces after planting. Real-time PCR assays were designed to amplify the stx 1, stx 2 and the eae genes of E. coli O157:H7. The detection limit for E. coli O157:H7 quantification by real-time PCR was 2·4 × 10³ CFU g⁻¹ of starting DNA in rhizosphere and phyllosphere samples and about 10² CFU g⁻¹ by plate count. The time for pathogens to reach detection limits on the leaf surface by plate counts was 7 days after planting in comparison with 21 days in the rhizosphere. However, real-time PCR continued to detect stx 1, stx 2 and the eae genes throughout the experimental period.
Conclusion:  Escherichia coli O157:H7 survived throughout the growth period as was determined by real-time PCR and by subsequent enrichment and immunomagnetic separation of edible part of plants.
Significance and impact of the Study:  The potential presence of human pathogens in vegetables grown in soils contaminated with E. coli O157:H7 is a serious problem to our national food supply as the pathogen may survive on the leaf surface as they come in contact with contaminated soil during germination.     

Rapid detection of pathogenic Vibrio parahaemolyticus by a sensitive and specific duplex PCR-hybridization probes assay using LightCycler

Aims:  To develop a real-time polymerase chain reaction (PCR) hybridization probe assay for rapid and specific detection of thermostable direct haemolysin-producing Vibrio parahaemolyticus.
Methods and Results:  Primers and hybridization probes were designed to target the toxR and tdh2 genes. Mismatches were introduced in the tdh2 primers for specific amplification of the target. The 3' ends of donor probes for both genes were labelled with fluorescein. The 5' ends of recipient probes for tdh2 and toxR were labelled with LC Red 640 and LC Red 705, respectively. The real-time assay was evaluated against conventional biochemical tests and the KAP-RPLA kit (Kanagawa phenomenon detection kit by reverse passive latex agglutination). toxR and tdh2 were detected in 100% and 91% of clinical V. parahaemolyticus isolates ( n  = 118), respectively. Specificity and sensitivity of the real-time assay for toxR and tdh2 were 100%, respectively. Dynamic range of detection for toxR was 10⁷–10¹ CFU ml⁻¹ and that for tdh2 was 10⁷–10⁴ CFU ml⁻¹.
Conclusions:  The LightCycler assay described is sensitive and highly specific for detection of pathogenic V. parahaemolyticus in a single reaction tube within 80 min.
Significance and Impact of the Study:  The assay developed allows accurate detection of pathogenic V. parahaemolyticus , which is valuable for rapid tracing of infection source during outbreaks.     

Identification of type A, B, E, and F botulinum neurotoxin genes and of botulinum neurotoxigenic clostridia by denaturing high-performance liquid chromatography

Denaturing high-performance liquid chromatography (DHPLC) is a recently developed technique for rapid screening of nucleotide polymorphisms in PCR products. We used this technique for the identification of type A, B, E, and F botulinum neurotoxin genes. PCR products amplified from a conserved region of the type A, B, E, and F botulinum toxin genes from Clostridium botulinum, neurotoxigenic C. butyricum type E, and C. baratii type F strains were subjected to both DHPLC analysis and sequencing. Unique DHPLC peak profiles were obtained with each different type of botulinum toxin gene fragment, consistent with nucleotide differences observed in the related sequences. We then evaluated the ability of this technique to identify botulinal neurotoxigenic organisms at the genus and species level. A specific short region of the 16S rRNA gene which contains genus-specific and in some cases species-specific heterogeneity was amplified from botulinum neurotoxigenic clostridia and from different food-borne pathogens and subjected to DHPLC analysis. Different peak profiles were obtained for each genus and species, demonstrating that the technique could be a reliable alternative to sequencing for the rapid identification of food-borne pathogens, specifically of botulinal neurotoxigenic clostridia most frequently implicated in human botulism.     

Clostridia spore formation during aerobic deterioration of maize and sorghum silages as influenced by Lactobacillus buchneri and Lactobacillus plantarum inoculants

Aims:  The effect of the inoculation of maize and sorghum silages with Lactobacillus plantarum (LP) and Lactobacillus buchneri (LB) on the clostridia spore formation during aerobic deterioration has been studied.
Methods and results:  The crops were ensiled in 30 l jars, without a lactic acid bacteria inoculant (C), and with an LP or LB inocula (theoretical rate of 1 × 10⁶). After 90 days of conservation, the silages were analysed for the chemical and microbiological characteristics and subjected to an aerobic stability test, during which pH, temperature, nitrate, yeast, mould and clostridia spores were measured. Compared to the C and LP silages, yeasts were reduced in the LB silages, resulting in an increased aerobic stability. Clostridia spores, determined by most probable number (MPN) procedure, increased to 6 log₁₀ MPN g⁻¹ in the C and LP maize silages, whereas they reached 3 log₁₀ MPN g⁻¹ in C and LP sorghum silages.
Conclusions:  Clostridia spore count only slightly increased in the LB maize silages after 342 h (2·59 log₁₀ MPN g⁻¹), whereas it did not show any increase in the LB sorghum silages for the whole period of air exposure.
Significance and impact of the study:  The data indicated that clostridia spore outgrowth can take place during silo feedout in aerobic-deteriorated silages and that LB inoculation reduces the risk of clostridia outgrowth after silage opening by increasing the aerobic stability.     

Genetic Diversity among Clostridium botulinum Strains Harboring bont/A2 and bont/A3 Genes

Clostridium botulinum type A strains are known to be genetically diverse and widespread throughout the world. Genetic diversity studies have focused mainly on strains harboring one type A botulinum toxin gene, bont/A1, although all reported bont/A gene variants have been associated with botulism cases. Our study provides insight into the genetic diversity of C. botulinum type A strains, which contain bont/A2 (n = 42) and bont/A3 (n = 4) genes, isolated from diverse samples and geographic origins. Genetic diversity was assessed by using bont nucleotide sequencing, content analysis of the bont gene clusters, multilocus sequence typing (MLST), and pulsed-field gel electrophoresis (PFGE). Sequences of bont genes obtained in this study showed 99.9 to 100% identity with other bont/A2 or bont/A3 gene sequences available in public databases. The neurotoxin gene clusters of the subtype A2 and A3 strains analyzed in this study were similar in gene content. C. botulinum strains harboring bont/A2 and bont/A3 genes were divided into six and two MLST profiles, respectively. Four groups of strains shared a similarity of at least 95% by PFGE; the largest group included 21 out of 46 strains. The strains analyzed in this study showed relatively limited genetic diversity using either MLST or PFGE.     

Rapid and simple detection of Clostridium botulinum types A and B by loop-mediated isothermal amplification

Aims:  To develop a convenient and rapid detection method for toxigenic Clostridium botulinum types A and B using a loop-mediated isothermal amplification (LAMP) method.
Methods and results:  The LAMP primer sets for the type A or B botulinum neurotoxin gene, BoNT / A or BoNT / B , were designed. To determine the specificity of the LAMP assay, a total of 14 C. botulinum strains and 17 other Clostridium strains were tested. The assays for the BoNT/A or BoNT/B gene detected only type A or B C. botulinum strains, respectively, but not other types of C. botulinum or strains of other Clostridium species. Using purified chromosomal DNA, the sensitivity of LAMP for the BoNT/A or BoNT/B gene was 1 pg or 10 pg of DNA per assay, respectively. The assay times needed to detect 1 ng of DNA were only 23 and 22 min for types A and B, respectively. In food samples, the detection limit per reaction was one cell for type A and 10 cells for type B.
Conclusions:  The LAMP is a sensitive, specific and rapid detection method for C. botulinum types A and B.
Significance and Impact of the Study:  The LAMP assay would be useful for detection of C. botulinum in environmental samples.     

Investigation of animal botulism outbreaks by PCR and standard methods

Abstract A double PCR procedure is proposed for identification of Clostridium botulinum C and D. This method consists of a first PCR amplification with a degenerate primer pair able to amplify a 340 bp common DNA fragment from botulinum neurotoxin (BoNT) C1 and D genes, followed by two subsequent PCR amplifications with two primer pairs specific for BoNT/C1 and D respectively (198 bp DNA fragment). This method was found to be specific for C. botulinum C and D, amongst 81 strains of C. botulinum and 21 different species of other Clostridium and bacteria tested. The detection limit ranged from 10 to 10³ bacteria in the reaction volume according to the C. botulinum C and D strains. In 160 naturally contaminated animal and food samples submitted to a 48 h enrichment culture, the double PCR showed an 89.4% correlation rate with the standard mouse bioassay. A clear distinction between botulism type C and D was obtained. The double PCR provides a reliable alternative for detection and identification of C. botulinum C and D in clinical and food samples.     

Applicability of the 16S–23S rDNA internal spacer for PCR detection of the phytostimulatory PGPR inoculant Azospirillum lipoferum CRT1 in field soil

Aims:  To assess the applicability of the 16S–23S rDNA internal spacer regions (ISR) as targets for PCR detection of Azospirillum ssp. and the phytostimulatory plant growth-promoting rhizobacteria seed inoculant Azospirillum lipoferum CRT1 in soil.
Methods and Results:  Primer sets were designed after sequence analysis of the ISR of A. lipoferum CRT1 and Azospirillum brasilense Sp245. The primers fAZO/rAZO targeting the Azospirillum genus successfully yielded PCR amplicons (400–550 bp) from Azospirillum strains but also from certain non- Azospirillum strains in vitro , therefore they were not appropriate to monitor indigenous Azospirillum soil populations. The primers fCRT1/rCRT1 targeting A. lipoferum CRT1 generated a single 249-bp PCR product but could also amplify other strains from the same species. However, with DNA extracts from the rhizosphere of field-grown maize, both fAZO/rAZO and fCRT1/rCRT1 primer sets could be used to evidence strain CRT1 in inoculated plants by nested PCR, after a first ISR amplification with universal ribosomal primers. In soil, a 7-log dynamic range of detection (10²–10⁸ CFU g⁻¹ soil) was obtained.
Conclusions:  The PCR primers targeting 16S–23S rDNA ISR sequences enabled detection of the inoculant A. lipoferum CRT1 in field soil.
Significance and Impact of the Study:  Convenient methods to monitor Azospirillum phytostimulators in the soil are lacking. The PCR protocols designed based on ISR sequences will be useful for detection of the crop inoculant A. lipoferum CRT1 under field conditions.     

Genetic homogeneity of Clostridium botulinum type A1 strains with unique toxin gene clusters

A group of five clonally related Clostridium botulinum type A strains isolated from different sources over a period of nearly 40 years harbored several conserved genetic properties. These strains contained a variant bont/A1 with five nucleotide polymorphisms compared to the gene in C. botulinum strain ATCC 3502. The strains also had a common toxin gene cluster composition (ha-/orfX+) similar to that associated with bont/A in type A strains containing an unexpressed bont/B [termed A(B) strains]. However, bont/B was not identified in the strains examined. Comparative genomic hybridization demonstrated identical genomic content among the strains relative to C. botulinum strain ATCC 3502. In addition, microarray data demonstrated the absence of several genes flanking the toxin gene cluster among the ha-/orfX+ A1 strains, suggesting the presence of genomic rearrangements with respect to this region compared to the C. botulinum ATCC 3502 strain. All five strains were shown to have identical flaA variable region nucleotide sequences. The pulsed-field gel electrophoresis patterns of the strains were indistinguishable when digested with SmaI, and a shift in the size of at least one band was observed in a single strain when digested with XhoI. These results demonstrate surprising genomic homogeneity among a cluster of unique C. botulinum type A strains of diverse origin.     

Development and use of a multiplex PCR system for the rapid screening of heat labile toxin I, heat stable toxin II and shiga-like toxin I and II genes of Escherichia coli in water

Enterotoxigenic Escherichia coli (ETEC) may produce heat-labile toxin (LT) I and LTII and heat-stable toxin (ST) I and STII, while shiga toxin producing E. coli (STEC) strains, including enterohaemorrhagic E. coli (EHEC), may produce shiga-like toxin (SLT) I and/or SLTII. Both ETEC and STEC are pathogenic to humans, pigs and cattle. As contamination of environmental water by any of these pathogenic E. coli cells is possible, a multiplex polymerase chain reaction (PCR) system for the rapid screening of LTI, STII, and SLTI and SLTII genes of E. coli was developed. The PCR primers used were the SLTI and SLTII genes specific primers developed by the present authors and the LTI and STII genes specific primers reported by other laboratories. The detection specificity of this multiplex PCR system was confirmed by PCR assay of ETEC, STEC and other E. coli cells as well as non- E. coli bacteria. Its detection limit was 10²–10³ cfu each of the target cells per assay. When this multiplex PCR system was used for the rapid screening of LTI, STII ETEC and STEC in water samples such as tap, underground and lake waters, it was found that after the enrichment step, as few as 10⁰ cells 100 ml⁻¹ of the water sample could be detected. Therefore, this PCR system could be used for the rapid monitoring of ETEC and/or STEC cells contaminating water samples.     

A new rapid and sensitive detection method for cereulide-producing Bacillus cereus using a cycleave real-time PCR

Aims:  To develop a rapid and sensitive detection method for cereulide-producing Bacillus cereus using a real-time PCR based on the sequence of the cereulide synthesis gene.
Methods and Results:  A total of 56 cereulide-producing B. cereus and 15 cereulide-negative strains were tested. We designed specific primers and probes for the detection of cereulide-producing B. cereus . The new cycleave real-time PCR assay gave positive detections for all of 56 cereulide-producing B. cereus strains, whereas all other strains including 10 systemic infectious disease strains were negative. No cross-reaction was observed and the internal control showed positive for all samples.
Conclusions:  The performance of the assay was highly reproducible and specific for cereulide-producing B. cereus . The positive detection was obtained within only 2 h for cereulide-producing strains. The detection limit of this assay was evaluated as 10⁴ CFU g⁻¹ food sample. The assay also confirmed that strains from systemic infectious cases were cereulide-negative.
Significance and Impact of the Study:  This assay is applicable for contaminated foods as well as specimens from infectious disease cases. We recommend this assay for routine examination of suspected B. cereus food poisonings.     

Stimulation of biomethanation by Clostridium sp. PXYL1 in coculture with a Methanosarcina strain PMET1 at psychrophilic temperatures

Aim:  Bioaugumentation of low temperature biogas production was attempted by addition of cold-adapted Clostridium and a methanogen.
Methods and Results:  A psychrotrophic xylanolytic acetogenic strain Clostridium sp. PXYL1 growing optimally at 20°C and pH 5·3 and a Methanosarcina strain, PMET1, growing optimally on acetate and producing methane at 15°C were isolated from a cattle manure digester. Anaerobic conversion of xylose at 15°C with the coculture of the two strains was performed, and batch culture methane production characteristics indicated that methanogenesis occurred via acetate through 'acetoclastic' pathway. Stimulation studies were also undertaken to evaluate the effect of exogenous addition of the coculture on biogas yields at 15°C. Addition of 3 ml of PXYL1 at the rate of 12 × 10² CFU ml⁻¹ increased the biogas 1·7-fold (33 l per kg cowdung) when compared to control (19·3 l per kg cowdung) as well as increased the volatile fatty acid (VFA) levels to 3210 mg l⁻¹ when compared to 1140 mg l⁻¹ in controls. Exogenous of addition of 10 ml PMET1 inoculum at the rate of 6·8 ± 10² CFU ml⁻¹ in addition to PXYL1 served to further improve the biogas yields to 46 l kg⁻¹ as well as significantly brought down the VFA levels to 1350 mg l⁻¹.
Conclusions:  Our results suggest that the rate-limiting methanogenic step at low temperatures could be overcome and that biogas yields improved by manipulating the population of the acetoclastic methanogens.
Significance and Impact of the Study:  Stimulation of biomethanation at low temperature by coculture.