A quantitative and qualitative analysis of microbial community development during low-temperature anaerobic digestion of dairy wastewater

The objective of this thesis was to link microbial community structure, population dynamics and microbial activity with performance of two different bioreactor configurations during low-temperature anaerobic digestion of dairy wastewater. Firstly, the efficiency of the process was assessed in two different bioreactor configurations: an Expanded Granular Sludge Bed (EGSB) and an Inverted Fluidized Bed (IFB) bioreactor operated at 37oC (Chapter 2). Distinct methanogenic communities developed in the IFB and EGSB reactors reflecting step-wise reductions in the applied hydraulic retention time from 72 to 12 h. Variations in reactor performance and in-reactor VFA concentrations during the 200-day trial influenced the microbial community structure of each reactor. Nonmetric multidimensional scaling (NMS) and moving window analyses, based on absolute and relative abundance quantification of the 16S rRNA gene concentration obtained by qPCR, were demonstrated to be useful tools to link methanogenic population shifts and reactor performance. The aceticlastic family Methanosarcinaceae was only detected in the IFB and the order Methanomicrobiales was also much more abundant in this reactor, while the aceticlastic family Methanosaetaceae was more abundant in the EGSB. The hydrogenotrophic order, Methanobacteriales, predominated in both reactors under all applied operational conditions. Subsequently, IFB and EGSB reactors were employed to investigate performance efficiency and methanogenic community structure and population dynamics, during operating temperature transitions from 37oC to 25oC, and from 25oC to 15oC, over a 430-day trial (Chapter 3). A comparable level of performance was recorded for both systems at 37 and 25oC, but a more dynamic and diverse microbial community in the IFB reactors supported better stability and adaptive capacity towards low-temperature operation. The emergence and maintenance of particular bacterial genotypes (phyla Firmicutes and Bacteroidetes) was possibly correlated with efficient protein hydrolysis in the IFB, while protein hydrolysis was inefficient in the EGSB. A significant community shift towards hydrogenotrophic methanogens of the order Methanomicrobiales, and specifically, Methanocorpusculum-like organisms, was demonstrated during operation at 15oC in both reactor configurations. Finally the reactors were deployed to explore the feasibility of low-temperature anaerobic biotreatment at 10oC (Chapters 4 & 5). Stable and efficient biotreatment of complex dairy based wastewaters at 10oC was found to be feasible in the EGSB bioreactor at applied organic loading rates (OLR) of 0.5-2 kg COD m-3 d-1 with mean chemical oxygen demand removal efficiency (COD RE) >85% (Chapter 4). The process was dependent on the OLR applied and values >2 kg COD m-3 d-1 resulted in process deterioration. OLR also influenced microbial community dynamics. An increased abundance of hydrogenotrophic methanogenic groups (Methanomicrobiales and Methanobacteriales) was recorded, using quantitative polymerase chain reaction (qPCR) analysis, by the end of the 335 day trial. Despite the perturbations in OLR applied during the 10oC bioreactor trial, the aceticlastic Methanosaeta spp. were maintained at stable levels. On the other hand, the IFB reactor displayed poor performance throughout the whole 10oC trial with mean COD RE of 54±17% at applied OLRs of 0.5-5 kg COD m-3 d-1 (Chapter 5). The applied OLR above 2 kg COD m-3 d-1 influenced the microbial composition and dynamics. Hydrogenotrophic methanogens: Methanomicrobiales and Methanobacteriales were monitored via qPCR and demonstrated 16478-fold and 85-fold decrease in their, abundance, respectively. This suggests that those organisms were inhibited or washed out from the system after the OLR stress was applied and did not regrow even when the conditions were changed and stress removed. The bacterial community in the bioreactor was monitored via denaturing gradient gel electrophoresis (DGGE), and the results of this analysis also suggested an influence of OLR stress on bacterial community structure and population dynamics. Possible shortcomings in the bioreactor operation are indicated, which could be helpful in future design and optimization of fluidized reactors intended for digestion of complex industrial wastewaters during LTAD. This thesis provides new information on the feasibility of anaerobic digestion of dairy wastewater of two different reactor configurations, under variable operating conditions. The influence of operating temperature as well as other operating conditions (i.e. organic loading rate and hydraulic retention time) on the process performance and community structure and population dynamics was investigated in the course of this study. The overall results and findings of this work provide a comparative insight into LTAD. Identification of significant knowledge gaps can be potentially helpful in better future reactor design and process control