Molecular biological characterisation of the functional microbial communities in anaerobic digestors
Saimon Malhotra1, Alan J McCarthy1 & Jason Snape2
1School of Biological Sciences, Life Sciences Building, University of Liverpool, Liverpool L69 7ZB. UK, 2AstraZeneca
plc, Brixham Environmental Laboratory, Devon. TQ5 8BA, UK

The microbial population in sludge taken from a domestic anaerobic digestor was investigated by methods not
requiring direct cultivation. Molecular techniques based on direct nucleic acid recovery were used to target key
bacterial functional groups extracted from both raw and digesting sludge. In comparison to digesting sludge, lower
levels of archaebacterial DNA could be amplified from raw sludge. Hybridisation using group-specific probes indicated
the presence of large populations belonging to the orders Methanobacteriales, Methanococcales,
Methanomicrobiales and Methanosarcinaceae. In the latter group, members of the genus Methanosaeta (which
produces methane from acetate only) were found to predominate. The majority of the sulphate-reducing bacteria
(SRB) are divided into six phylogenetic subgroups based on 16S rRNA sequence information. PCR
amplification primers and confirmatory oligonucleotide probes were applied to detect the six genus-level subgroups,
Desulfotomaculum; Desulfobulbus; Desulfobacterium; Desulfobacter; Desulfococcus-Desulfonema-Desulfosarcina;
Desulfovibrio-Desulfomicrobium. Direct PCR amplification enabled detection of Desulfobacter; Desulfococcus-
Desulfonema-Desulfosarcina and Desulfovibrio-Desulfomicobium as predominant in raw sludge but only one
dominant subgroup, the Desulfococcus group, was found in digesting sludge. All other groups were detected in both
sludge types via a more sensitive nested PCR approach, implying their presence in lower numbers than the dominant
subgroups detected by direct PCR. This implies that the digestion process imposes a selection on the incoming SRB
population. Primer sets specific for four clostridial groups containing cellulolytic, proteolytic and mesophilic
representatives were used for amplification of digestor DNA. The results demonstrated the presence of clostridial
clusters I, III and XIV, but members of cluster IV could not be detected in preliminary analysis.
The Society for General Microbiology, Irish Branch Spring Meeting, Recent Advances in Molecular Microbial
Ecology, Venue: Ardilaun House Hotel, Galway, 7th–8th April 2000