The life-cycle of methanogenic granular biofilms

Abstract

Anaerobic granules, sometimes referred to as methanogenic granules, underpin the sustainable treatment of wastewater in high-rate anaerobic bioreactors. They are spontaneously-forming self-immobilised biofilm aggregates that contain the entire microbial community necessary to completely mineralise complex organics into a methane-based biogas. Their discovery in the 1970s completely revolutionised wastewater treatment engineering and prompted the design of numerous new, sustainable, and efficient systems based around anaerobic digestion (AD). Moreover, the success of the anaerobic granule has driven the engineered design of various type of granules, each with unique functions. Indeed, the aerobic granule has played a significant role in upgrading conventional aerobic treatment processes, and the anaerobic ammonium oxidation (anammox) granule has challenged the well-established practices in nutrient removal – providing a sustainable path for nitrogen transformations. The unique properties of granules including their settleability, and their densely-packed, diverse microbial communities have been consistently cited as the primary characteristics influencing their success. However, while granules have had a significant impact on environmental engineering, they are recently catching the interest of microbial ecologists. In no other form in Nature do biofilms exist with such defined boundaries, wide applications, and complex communities, creating whole-ecosystems in a 1-4 mm diameter. The potential of such microbial aggregates for high-throughput investigations into ecological theory is endless. They can be used to pursue the nature of microbial stress responses, community assembly, community expansion and succession, mechanisms around biofilm disintegration, cellular communication and signaling, and so on.

The objective of this thesis was to use anaerobic granules as a means to study biofilm growth, life-cycle, function, and microbial diversity. Each experimental chapter herein operates on the hypothesis that size can be used as an indicator of growth-stage, where small granules are ‘young’ and large granules are ‘old’. Each experimental setup involved the separation of anaerobic granules into discrete size fractions, and the comparison of these sizes in terms of structure, function, and microbial diversity.

The first experimental chapter (Chapter 2) describes a characterisation-based study in which granules were separated into 10 discrete size fractions. Each size was compared based on several different physico-chemical, and molecular parameters. It was determined that size matters for the structure and function of anaerobic granular biofilms as strong gradients were observed across the granular ultrastructure using scanning electron microscopy (SEM); in the makeup of the loosely-bound extracellular polymeric substances (EPS); in granular density and settleability; and in the rates of methane generation. Additionally, as granule size increased – representing granule growth – the rarefied richness and Shannon entropy of the microbial community decreased linearly, becoming increasingly dominated by a core group of methanogenic archaea. The diversity of these Euryarchaeota, however, was not increasing with increasing granule size. It is therefore likely that a functional group theory explains the pronounced diversity shifts in these communities. Furthermore, it is proposed that these granular biofilms may undergo a predictable life-cycle and growth model, in which it is possible that medium-sized granules may be the most optimal for efficient bioreactor function.

In the second experimental chapter (Chapter 3) the growth hypothesis was tested by operating a series of lab-scale bioreactors each inoculated with granules from within a defined size range (small, medium, or large). Following a 51-day trial, the size distribution of the sludge was reexamined and was found to have diversified beyond the size fraction used for inoculation. In particular, a full range of sizes was found in the bioreactors initially containing only large granules indicating the emergence of both ‘de novo’ and bigger granules. Sequencing of 16S rRNA gene amplicons from granules from each of these emerging sizes revealed that Methanobacterium, Aminobacterium, Propionibacteriaceae and Desulfovibrio made up the majority of the microbial community. Based on the relative abundances of microbial taxa, a clear shift to the hydrogenotrophic pathway for methanogenesis was apparent, and the responsive emergence of several syntrophic bacteria was observed. This chapter describes how, over a short trial, granules do ‘grow’ in a progressive manner – from small into medium and, eventually, large. It remains unclear, however, whether or not, or by which mechanisms, this process is cyclical.

To help elucidate the nature of the life-cycle, in Chapter 4 a cDNA-based approach was used to analyse the active microbiome of the size fractions. Each fraction was separately supplied with a series of methanogenic substrates: acetate, propionate, butyrate and H2/CO2. The biomass was sampled from incubations during the exponential phase of biogas production, and the cDNA was used for 16S rRNA amplicon sequence analysis. The makeup of the active microbiome, and specifically the beta-diversity analyses, indicated that a granule biofilm life-cycle is likely in such systems; the active community structure of the largest and smallest sizes was more similar than previously observed with DNA-based analyses. Moreover, the active microbiome provided insights into key organisms involved in the degradation of specific substrates. However, size remained a more significant driver than substrate for overall active community structure.

In summary, this thesis provides evidence in support of a growth, and life-cycle model, for methanogenic granular biofilms.