Biosolids are residues from settleable solids in the raw wastewater (primary sludge), and the biomass sludge from the activated sludge process (waste activated sludge). Anaerobic microbes with different functional processes (hydrolysis, acidogenesis, acetogenesis and methanogenesis) work together in anaerobic digestion to degrade organics in biosolids and produce economically beneficial methane gas. Temperature phased anaerobic digestion (TPAD) became a potentially superior method of treating waste as it allows enrichment of different groups of microbes by spatially separating them into two reactors. In the first phase (called pre-treatment) that has a short hydraulic retention time (a few days), hydrolytic and acidogenic/fermentative bacteria degrade polymers to monomers and produce organic acids. In the second stage (with a much longer retention time), these products are then converted into methane gas. However, detailed knowledge of the pre-treatment microbial community structure and their function is lacking. Thermophilic pre-treatment (TP) often offers improved solubilisation. Comparison of TP and mesophilic pre-treatment (MP) microbial communities with rigorous phylogenetic interpretations and functional analysis is absent. As pre-treatment communities supply vital intermediates for methane production, detailed knowledge of the community structure, composition and function is critical for process maintenance and improvement. Therefore, the core objective of this research was to determine the microbial community structure and function in a TPAD system and to examine microbial agents responsible for the improved process of thermal pre-treatment.
Microbes in functionally different laboratory scale pre-treatment reactors, TP (>50°C) and MP (35 °C) of TPAD systems were characterized and monitored over time while treating two different types of biosolids i.e., primary and activated sludge. Microbial community structures were monitored by 16S rRNA gene terminal restriction fragment length polymorphism (TRFLP) and identified by 16S rRNA gene cloning, probe design, fluorescent in situ hybridisation (FISH) and 16S rRNA gene pyrosequencing. Community profiles were correlated to reactor parameters and performance by relevant statistical analysis. Microbial function in TP and MP reactors were analysed by a metaproteomic investigation to gain insights of the key microbial pathways in thermophilic and mesophilic anaerobic digestion.
This was the first study to examine the diversity and dynamics of bacteria involved in primary sludge anaerobic digestion. Microbial communities in TP and MP reactors were different. TP communities were always predominated by a few abundant populations while MP communities were more even. Superior waste solubilisation and methane production was achieved in TP in comparison to MP, suggesting the different microbial communities of those were responsible for the improved function. Abundant populations detected in TP were affiliated with bacteria characterised by elevated hydrolysis and organic acid production capabilities. While Proteobacteria was an important phylum in all pre-treatment reactors, members of Thermotogae and Firmicutes were abundant in TP treating activated sludge. Interestingly, unclassified and uncultured bacteria dominated the pre-treatment microbial communities. Over 90% of the sequences were previously undetected in TP treating primary sludge. Similarly, approximately 50% of the sequences from both TP and MP reactor treating activated sludge were previously undescribed.
Community structure in TP was dynamic in response to alterated reactor conditions of temperature and pH for both primary and activated sludge. Statistical analysis of TRFLP profiles showed that increasing temperature in TP treating activated sludge had a significant effect on the community composition, increasing the abundance of Thermotogae sp. and Lutispora thermophila of the Firmicutes. However, community structure was relatively steady in the TP reactor treating activated sludge while the reactor temperature was constant at 55°C. Notably higher solubilisation of sludge and higher production of organic acids occurred in TP in comparison to MP. A Fervidobacterium species, a member of the Clostridiales and a Methanosarcina species were the abundant organisms in this TP reactor as determined by pyrosequencing. In comparison the microbial communities in MP were more influenced by those of the incoming feed sludge (Feed).
Metaproteomic analysis identified 225 proteins from TP, MP and Feed samples. The majority of the proteins identified in TP were affiliated to Betaproteobacteria, followed by Firmicutes and Thermotogae on a phylum basis while the proteins in MP were affiliated mainly to Betaproteobacteria and Nitrospirae. Proteins from members of Fervidobacterium, Clostridium, and Methanosarcina were exclusively present in TP and these were the abundant microorganisms in this reactor. Proteins detected in the TP extracellular fraction suggested elevated activites of nutrient transfer after polymer breakdown. Additionally, proteins affiliated with acidogenic, acetogenic and methanogenic activities were detected, indicating both the aceticlastic and the CO2 reduction methanogenesis pathways were active in TP. The comparison of proteins in TP and MP thus showed the presence and abundance of enzymes for different steps of anaerobic digestion in TP indicating the mechanism of enhanced hydrolysis-fermentation and methanogenesis performance in TP.
This study provides new insight of anaerobic digestion as it unveils previously unknown detail of the microbial community important for the enhanced solubilisation during a pre-treatment stage. Remarkably, considerable methane production was achieved during this TP operation, and insight of the community composition and function was obtained.