A functional approach to characterising the microbial communities underpinning low temperature anaerobic digestion
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Low-temperature anaerobic digestion (LTAD) presents a sustainable, cost-efficient technology for the treatment of a vast array of wastewater streams. However, this microbially-mediated process requires further understanding and experimental characterisation if large-scale application of LTAD is to be realised. The objective of this thesis was to employ a functional-based approach to characterise the microbial communities underpinning low-temperature anaerobic digestion. Firstly, the key microbial functional groups present in end-point samples taken from three, laboratory-scale, expanded granular sludge bed (EGSB) bioreactors; R1 (37°C), R2 (15°C) and R3 (7°C) were characterised. Metaproteomics, in conjunction with 16S rRNA gene phylogenetic approaches (clone libraries, qPCR), was applied to record microbial community composition and metaproteomic profiles as a function of bioreactor operating temperature. Clone libraries indicated a predominance of the Chloroflexi (21%) and ¿-Proteobacteria (61%) bacterial groups in R1, with Firmicutes (24%) and Bacteroidetes (46%) prominent in both R2 and R3. The Methanosaeta genus was strongly represented in archaeal clone libraries (29% [R1], 76% [R2] and 91% [R3]). This was reflected in the metaproteomic results with 26 (65%) differentially expressed proteins assigned to this methanogenic group. Also evident from the metaproteomic data were proteins assigned to the bacterial phyla Proteobacteria, Firmicutes and Actinobacteria, while the archaeal orders Methanobacteriales, and Methanomicrobiales were also represented. Interestingly, the identification of a protein assigned to Methanosarcina sp. was not consistent with DNA-based community profiling data, where this methanogenic group was not detected, confirming the importance of employing a functional-based approach in this study. A pure culture proteomic (iTRAQ, 2-DGE) approach was then employed to uncover the sub-mesophilic functional characteristics of a Methanosarcina strain (optimum growth temperature, 37¿C), with proteins assigned to this group detected in previous LTAD bioreactor trials. New insights into the low-temperature adaptation capacity of this mesophilic methanogen, including differentially expressed proteins during low temperature growth, e.g. elongation factor protein expression during sub-mesophilic adaptation, with high levels of viable cells recorded through this 'adaptation' stage (84% [±9.65% SE; n = 10] of cells were viable after 17 days of growth at 15degrees C). Therefore, through this polyphasic approach, the psychrotolerant capacity of this organism was characterised. Finally, two EGSB bioreactors (R1 & R2) were operated, initially at 37°C with a subsequent temperature drop to 15°C, with biomass samples being taken throughout the trial. PCR-based (clone libraries, qPCR, DGGE [RNA- DNA- derived]) and PCR-independent (specific methanogenic activity [SMA] profiling, microautoradiography fluorescent in situ hybridisation [MAR-FISH], and metaproteomics) approaches were employed to investigate the microbial community structure and key functional groups throughout the trial, with particular emphasis on the methanogenic archaea. Once again Methanosaeata were prominenet in archaeal community and functional analysis with consistent proteomic profiles recorded between the two LTAD bioreator trials investigated in this study. For example, a bifunctional protein (Mcon_1383) with possible function in riboulose monophosphate (RuMP) pathway was significantly expressed in low temperature biomass for both studies. Overall, community profiling techniques (clone libraries, qPCR) linked with functional-based (RNA-based DGGE, metaproteomics) approaches employed in this research illustrated the importance and metabolic complexity of Methanosaeta in a well functioning LTAD system. Also, the incorporation of functional analysis was justified through the uncovering of discrete community information missed through traditional DNA-based community profiling methods e.g. Methanospirillum importance in low temperature biomass during LTAD trial confirmed through metaproteomic and RNA-based DGGE profiles.
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