Degree Name

Master of Philosophy


School of Civil, Mining and Environmental Engineering


Anaerobic digestion (AD) is arguably the most dominant technology for stabilizing organic waste as well as sewage sludge (SS) in wastewater treatment plants (WWTPs). A new AD approach namely anaerobic co-digestion (AcoD) has been an emerging technology during recent years. AcoD involves the concomitant digestion of multiple substrates which can help to enhance biogas production. Successful implementation of AcoD at existing WWTPs requires careful management of the risk associated with operational and environmental disturbances (e.g. organic overloading, high ammonia concentration and temperature fluctuation). Disturbances can result in alteration of microbial community – the core functional component of the system – to an extent that results in digester failure. Thus, achieving a comprehensive understanding of the microbial community during AcoD operation under steady state as well as during various perturbations (e.g., operational disturbance) are necessary for the improvement of process stability and efficiency.

This study examined the changes in the microbial community during AcoD of SS and a carbon-rich organic waste (beverage waste - BW). Microbial community analysis was done using amplicon sequencing of 16S rRNA and mcrA marker genes on the Illumina Miseq platform. The first component of this study investigated microbial diversity and structure in response to AcoD with increasing organic loading rate (OLR). Biomass samples were collected from a lab-scale system at different BW to SS mixing ratios to cover a large range of OLR. Lab-scale digesters had working volume of 20 L and hydraulic retention time (HRT) of 20 days. The results showed a reduction in the community diversity (i.e. richness and evenness) and a shift in community structure as the OLR increased (by 86 and 171%) due to the addition of BW (at 10 and 20% v/v). Despite the decrease in community diversity (by 29% in richness indices and > 14% in evenness indices), biogas production increased proportionally to the increase in OLR of up to 3.03 kg COD/m3/d (corresponding to an increase of 171% of OLR compared to AD of only SS). Further OLR increase (240%) led to the collapse of biogas production as well as significant reduction in both the microbial diversity and methanogenic population (total abundance of methanogens was < 4%). The methanogenic community was more sensitive to the increase in OLR compared to hydrolytic and fermentative bacteria. These results suggest that there is an OLR threshold at which the function and resilience of the anaerobic ecosystem could be maintained. Beyond this threshold, the enrichment of hydrolytic and fermentative bacteria, as well as inhibition of methanogenic community, can cause AD failure.

The second component of this study evaluated the changes in microbial community diversity and composition in response to operational disturbance and recovery in pilot-scale anaerobic digesters. Pilot-scale digesters had working volume of 600 L and HRT of 20 days. An operational disturbance was imposed on the digesters after a stable operation phase by turning off circulation pumps and mixers while keeping hot water circulation to the water jacket at the bottom of the digester operated as normal. This created uneven temperature distribution inside the digesters. After that, new inoculum was added to facilitate digesters recovery. Biomass samples were collected throughout the experiment to cover both stable and unstable operation periods. Operational disturbance led to the enrichment of hydrolytic and fermentative bacteria (total abundance in the two digesters > 57%) and significant reduction of acetogenic bacteria (total abundance in the two digesters < 9%) and methanogenic populations (total abundance in the two digesters < 3%). The imbalance among core microbial groups caused volatile fatty acids accumulation and subsequent deteriorated performance in terms of biogas production (decrease by > 45% compared to the stable phase) and methane content (decrease from 63% to < 49%). Fresh inoculum addition (four times, each time 17% reactor volume) helped supply active microbes and facilitate digesters recovery. Microbial communities did not return to the original structures after recovery although the same level of performance was retrieved in terms of biogas production and methane content. These results suggest that during AcoD different microbial community structure can sustain a similar level of performance, a phenomenon commonly referred to as ‘functional redundancy in microbial systems’.

This thesis is unavailable until Sunday, August 02, 2020



Unless otherwise indicated, the views expressed in this thesis are those of the author and do not necessarily represent the views of the University of Wollongong.