Chapter 3 - An investigation into microbial dynamics and
3.5 Conclusions
From this study, it has been demonstrated that pure culture work can add valuable knowledge and insights into how key players in the AD pathway specifically interact on a fundamental scale. Unquestionably, community-based studies are important to get a full picture of how the whole system works, but it is only from pure culture and synthetic co-culture studies that a clear picture can be attained as to how species adapt and interact with each other in an undisturbed way in particular circumstances, such as at a specific temperature or with a key substrate. By peeling away the layers of trophic groups, experimental insights can help to optimise the way in which microorganisms handle specific environmental stresses.
For the M. barkeri temperature study, a decrease in temperature resulted in decreased growth, as expected. CH4 production decreased by 38.5% when temperature was reduced from 35°C to 30°C, but it only decreased by 27.3% when the temperature was reduced from 30°C to 25°C. This implied that there are more substantial growth and substrate changes at differences of higher temperatures.
Similar findings were demonstrated in the acetate consumption profile analysis (sections 3.3.1 and 3.4.1). Additional statistically robust proof for this preliminary conclusion will be obtained if similar studies are conducted at further temperature levels.
For the M. barkeri-M. maripaludis experiment, both species were able to compete for H2-CO2. This co-culture impacted on the respective pure cultures’ data by causing the growth and substrate profiles of M. barkeri to decrease slightly compared to the increase of M. maripaludis profiles. M. maripaludis marginally out-competed M. barkeri for growth. As already demonstrated, Methanococcaceae is a very important species within anaerobic bioreactors, and with its capability of fast growth this data suggests that lower numbers are capable of surviving and thriving in hydrogen-rich anaerobic environments. This experiment represented the first time this co-culture has been analysed according to the literature.
For the M. barkeri-A. woodii H2-CO2 study, a direct competition was observed for the substrate as evident from the CH4 co-culture profile (Figures 3.6 (F) and 3.7 (F)), but overall, they were both able to grow similarly to their individual pure cultures. qPCR results reflect this observation where the cell abundances per ng of
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DNA in co-culture were half of those in respective pure cultures at each time point and the co-cultures contained 50% of each starting inoculum when they were in pure culture. Previous studies have demonstrated a biphasic type of substrate utilisation in the co-culture, with acetate initially accumulating due to A. woodii growth with acetate subsequently decreasing due to acetate-adapted M. barkeri feeding on it after H2-CO2 had been depleted (Winter and Wolfe, 1979, Winter and Wolfe, 1980).
When acetate was the substrate, acetate-adapted M. barkeri consumed it and it was hypothesised that A. woodii would not grow. From the qPCR and metabolic data, it appeared that there was some marginal amount of A. woodii growth where there were increased acetate concentrations and qPCR cell abundances per ng DNA based on the16S rRNA gene copy numbers. Error bars indicate that there may not have been any growth because they extend wider than the basal level starting points for the qPCR data (Figure 3.9 (B)) and the acetic acid data (Figure 3.9 (E)).
Both cultures are capable of growth at low temperatures under H2-CO2-feeding and acetate-adapted M. barkeri could grow under acetate-feeding. A deeper understanding of pure cultures at low temperatures will help to advance the wider LtAD field and to understand how microorganisms adapt and survive at low temperatures within bioreactors. A study which investigates these cultures at a gradient of low temperatures could give accurate information as to the differences in metabolic changes over those temperatures, which would aid and link in with the current understanding of bioreactor performance.
Overall, temperature did influence each of the competitive and/or synergistic relationships within the three studies. In all cases the higher temperature resulted in more growth and methane production.
Advancing from these experiments, an important subsequent step in understanding the pure cultures and co-cultures examined in this study would be to implement a suite of ‘omics techniques to provide additional data, for example, investigating A.
woodii growth under the sole acetate substrate. Under the various combinations of temperature, substrate and pure culture applied here, samples can be used further from each growth phase to look at specific genes that are responsible as the drivers under specific environmental changes using genomics, for example. Pure culture
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studies will help to allow more detailed metabolic modelling of synthetic communities within an AD bioreactor environment.
An additional future direction of this pure culture work would be to build on these synthetic co-cultures with a tri-culture community. Synthetic biology serves as a powerful tool in order to create predictive models of microbial communities but pure culture work significantly add to these databases detailing how microorganisms interact with each other.
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