Microbial ecology of autothermal thermophilic aerobic digestion (ATAD): diversity, dynamics and activity of bacterial communities involved in the treatment of a municipal wastewater

Autothermal thermophilic aerobic digestion (ATAD) is a tertiary sludge processing technology occurring in jacketed, aerated reactors where the temperature increases from the mesophilic (40-50oC) to the thermophilic (55-65oC) range as a result of aeration and biodegradation of the secondary sludge feed. The resultant heat generation gives rise to pasteurisation and sludge stabilization and results in a Class A biosolids, which is suitable for land spread as a fertilizer. We evaluated the ATAD process over a period of one year in a two stage, full scale ATAD plant to determine its ability to eliminate indicator organisms. Although present in the inlet sludge we failed to detect enteric pathogens in the final product using traditional microbial culture or molecular phylogenetic techniques. This evaluation indicated that the ATAD process is in fact fit for purpose and consistently produces a stabilised biosolids product that meets disposal standards. The visual nature and morphology of ATAD sludge has been little studied and the nature of its floc/particle structure unreported. We developed and adapted a number of preservation techniques to allow in situ analysis of ATAD sludge and showed it to possess a unique microscopical appearance with structural characteristics which include: (1) weakly-bonded structural material; (2) material rich in carbohydrates with non-aromatic structures; (3) the presence of a high amount of amorphous cellulose; (4) the exterior of the ATAD sludge-liqour interface is hydrophilic whereas the ATAD sludge liquor is rich in biomass and contains numerous hydrophobic lipid droplets. The nature of biomass present and its location was determined by scanning laser confocal microscopy and offered a unique insight into the ATAD niche and nature of the microbial sludge interaction. Using 13C –NMR to analyse the patterns of sludge biodegradation we found notable variations between the initial sludge at inlet, its treated counterpart, and biosolids after storage. The main spectral differences were observed in the 0-alkyl region (0– 50ppm), in the C-O-alkyl/N-alkyl region (50–110ppm) providing information on the alterations occurring in the different biochemical entities, chiefly lipids and carbohydrates. The original feed organic matter gave distinctive NMR spectra in that it contained a large amount of alkyl carbon. The sub spectra were distinctively alkylrich and close to a microbial biomass spectra which may be due to the large increase in microbial biomass occurring during the ATAD process and which are responsible for the large increase in temperature that one observes in ATAD. Fresh biosolids was rich in alkyl C groups ,where as thermophilic ATAD sludge exhibited a very low aromaticity index compared to any other composted product previously reported and also had a high o- Alkyl/alkyl ratio decomposition value. Prolonged storage of the treated ATAD biosolids under essentially anaerobic mesophilic conditions for 9 days under our experimental conditions also resulted in altered sludge characteristics making it resemble that of a traditional sludge. Our data indicated that cellulose biodegradation was one of the chief driving forces for heat generation. The thermophilic stage provides an extreme niche with specific exo-enzyme activities produced by the mictrobial biomass with unusually a preponderance of esterase activity. many of the activities were thermostable indicating they were produced from the thermophilic population. A key goal of this study was the determination of the microbial community present and active in the ATAD particularly at the thermophilic stage. We demonstrated the inadequacy of culture based techniques for analysis of bacterial communities from ATAD thermophilic wastewater and showed that for such studies at elevated temperatures that direct DNA extraction methods, PCR amplification and 16S rDNA gene sequences, were essential. However rigorous optimisation was necessary to obtain a clear picture of this microbial community due to the presence of PCR inhibitors and a high nuclease activity emanating from lysed cells. Using optimised protocols we monitored the change in microbial community as a function of ATAD stage using denaturing gradient gel electrophoresis (DGGE), and noted a specific population in the thermophilic reactor. Using species specific probes we determined that the Bacterial group was the dominant one at the thermophilic stage. 16S rDNA genes were amplified directly from sludge with universally conserved and Bacteriaspecific rDNA gene primers and a clone library constructed corresponding to the late thermophilic stage (t =23 hours) of the ATAD process. We have deposited a family of fifty unique ATAD sequences in the public genomic database (NCBI and GeneBank), not previously assigned to the ATAD ecological niche. ATAD community members included α– and β- Proteobacteria, Gram-negative bacteria, Actinobacteria with High G+C content and Gram-positive bacteria with a prevalence of phylotypes of the Firmicutes (Low G+C) division (class Clostridia and Bacillus). Most of the ATAD clones showed affiliation with bacterial species previously isolated or detected in other elevated temperature environments, at alkaline pH, or in cellulose rich environments. Several phylotypes associated with Fe(III)- and Mn(IV)-reducing anaerobes were also detected and are of interest as large scale ATAD systems may have sub-optimal aeration and mixing resulting in anaerobic conditions within the reactors. The presence of capnophiles, also suggest the possibility of limited convection and entrapment of CO2 within the ATAD sludge matrix. The abundance of thermophilic, alkalophilic and cellulose-degrading phylotypes suggest that these organisms may be responsible for maintaining the elevated temperature at the later stages of the ATAD process. The ATAD tested had a unique thermophilic community present with unique phylogenetic profiles in comparison to reported profiles of communities in other thermophilic aerobic systems. Many of the observations made during this study have important implications to future ATAD design, potential augmentation strategies, reduced treatment times and offer potential for utilising novel dewatering strategies. The nature of the ATAD microbial community is also of biotechnological potential with unique species, unique microbial activities and enzymes and an untapped biodegradative ability.