Final Activity Report Summary - AD_COMPOCHIP (Design and evaluation of an oligonucleotide microarray for the detection of signature microorganisms in composts from anaerobic digestion of biowastes)
Anaerobic digestion is the biotechnological process by which the organic matter contained in organic waste is degraded in the absence of oxygen. The anaerobic microorganisms that drive this process produce biogas, which is mainly composed by methane and carbon dioxide, and can be transformed into heat and electricity. Therefore, anaerobic digestion is both an alternative source of renewable energy from biomass and a means of recycling organic wastes. Unravelling the prokaryotic diversity and dynamics in methane-producing bioreactors is among the challenges to optimise the production of biogas from wastes. To help accomplishing this purpose, we have developed the AnaeroChip, a microarray targeting the 16S rRNA gene of prokaryotes involved in the process of anaerobic digestion.
Microarrays consist of a solid matrix to which up to tens of thousands of DNA probes targeting complementary known sequences are attached in a precise location, allowing the simultaneous hybridisation with DNA from complex samples. This molecular tool offers the possibility to screen for the presence of an entire array of microorganisms from a particular sample in a single assay. The prototype AnaeroChip holds 103 probes, each printed in triplicate: a universal probe for archaea; 98 probes for methanogenic archaea (2 probes at family level, 4 probes targeting multiple genera, 79 probes at genus level and 13 probes at species level); two negative controls (bacterial probes) and two hybridisation controls (a positive control and a blank). The specificity of the probes was tested with pure cultures, and from the total of 1854 individual probe-target hybridisation reactions performed, there were only 43 false positive (2.3 %) and 16 false negative signals (0.86 %). The exclusion of two probes from the array resulted in a reduction of the false positive rate to 1.1 %. The sensitivity of the array was also tested, and it was found that 0.4 pg of DNA from a pure culture subjected to PCR (polymerase chain reaction) amplification gave signals above the detection limit. Also, the application of 25 ng of PCR product from a pure culture to an array resulted in detectable signals.
The information generated by the microarrays (presence/absence) can be used as a basis for conducting quantitative assays for specific targets. We have designed primers and optimised real-time quantitative PCR assays for most lineages of methanogenic archaea involved in anaerobic digestion under mesophilic and thermophilic conditions, which are detectable with the AnaeroChip. Combining both techniques we have investigated the diversity and dynamics of the methanogenic communities during the start-up of laboratory-scale bioreactors, which permitted us optimising the most efficient strategy to start-up a full-scale manure-fed biogas plant in Tyrol (Austria). We have also applied the newly developed molecular strategy to follow the methanogens during a co-digestion assay of cattle manure and two-phase olive mill wastes. This led us to conclude that Methanosarcina, metabolically the most versatile methanogens, were responsible for an increase in biogas production by 337% of reactors co-digesting both residues as compared to reactors digesting only cattle manure.
The future development of the AnaeroChip will broaden the spectrum of target microorganisms, including also bacteria involved in the food chain leading to methane production, as well as other organisms that reduce the efficiency of the process (e.g. sulphate-reducing bacteria).
Microarrays consist of a solid matrix to which up to tens of thousands of DNA probes targeting complementary known sequences are attached in a precise location, allowing the simultaneous hybridisation with DNA from complex samples. This molecular tool offers the possibility to screen for the presence of an entire array of microorganisms from a particular sample in a single assay. The prototype AnaeroChip holds 103 probes, each printed in triplicate: a universal probe for archaea; 98 probes for methanogenic archaea (2 probes at family level, 4 probes targeting multiple genera, 79 probes at genus level and 13 probes at species level); two negative controls (bacterial probes) and two hybridisation controls (a positive control and a blank). The specificity of the probes was tested with pure cultures, and from the total of 1854 individual probe-target hybridisation reactions performed, there were only 43 false positive (2.3 %) and 16 false negative signals (0.86 %). The exclusion of two probes from the array resulted in a reduction of the false positive rate to 1.1 %. The sensitivity of the array was also tested, and it was found that 0.4 pg of DNA from a pure culture subjected to PCR (polymerase chain reaction) amplification gave signals above the detection limit. Also, the application of 25 ng of PCR product from a pure culture to an array resulted in detectable signals.
The information generated by the microarrays (presence/absence) can be used as a basis for conducting quantitative assays for specific targets. We have designed primers and optimised real-time quantitative PCR assays for most lineages of methanogenic archaea involved in anaerobic digestion under mesophilic and thermophilic conditions, which are detectable with the AnaeroChip. Combining both techniques we have investigated the diversity and dynamics of the methanogenic communities during the start-up of laboratory-scale bioreactors, which permitted us optimising the most efficient strategy to start-up a full-scale manure-fed biogas plant in Tyrol (Austria). We have also applied the newly developed molecular strategy to follow the methanogens during a co-digestion assay of cattle manure and two-phase olive mill wastes. This led us to conclude that Methanosarcina, metabolically the most versatile methanogens, were responsible for an increase in biogas production by 337% of reactors co-digesting both residues as compared to reactors digesting only cattle manure.
The future development of the AnaeroChip will broaden the spectrum of target microorganisms, including also bacteria involved in the food chain leading to methane production, as well as other organisms that reduce the efficiency of the process (e.g. sulphate-reducing bacteria).