Microorganisms and their Function

Microorganisms are vital in mediating the earth‟s biogeochemical cycles (Graham et

al., 2016). However, the relationship between microbial community structure and

particular ecosystem processes might not be reflected in total community diversity but rather in the diversity of narrow functional guilds (Graham et al., 2016). For example, although the total bacteria investigated comprised denitrifiers, we found no significant relationship between diversity aspects of the total bacterial community and the estimated denitrification rates in our spatio-temporal study on the BPNS (Chapter

123 particularly between diversity aspects of amoA gene carrying communities of active nitrifiers (AOA and β-AOB) and nitrification rates in the late summer. However, among different sediment types in September, β-AOB richness and diversity followed the same pattern as nitrification rates, with the highest values noted in fine sediments while AOA diversity indices did not show such spatial differences among sediment types (Chapter 3). Apart from the smaller cell volumes (10 to 100 times) in most AOA (Hatzenpichler, 2012), which is thought to be responsible for lower rates of ammonia oxidation in AOA compared to AOB (Hatzenpichler, 2012; Veuger et al., 2013), biochemical adaptations of AOA are thought to contribute to their wide distribution compared to AOB (Hatzenpichler, 2012). Our results in the BPNS revealed that the bacterial (total and β-AOB) community generally showed more spatio-temporal variation than the archaeal (total and AOA) community as sedimentation of organic matter and the subsequent changes in the environment had a stronger impact on bacteria (total and β-AOB) community composition and diversity indices. In addition, fine sandy sediments in our study display a high macrofaunal abundance, biomass and functional diversity (high BPc) (Chapter 2), which transfer water and solutes into the sediment. There are reports indicating the ability of AOA to live in low ammonia and oxygen concentration due to high affinities for the substrate (Abell et al., 2010; Park et al., 2010; Hatzenpichler, 2012). Furthermore, AOA exhibit a variety of metabolic pathways compared to AOB. They are able to obtain energy through different autotrophic carbon-fixing pathways (Hatzenpichler, 2012) as well as by heterotrophic metabolism through oxidizing organic matter (Mosier, 2011; Qin et al., 2014). In our study area, both AOA and β-AOB seem to play roles in sedimentary nitrification in fine sediments in September (Chapter 3, Table 2) but differentiation of their individual contributions was not investigated.

Correlations between nitrifying and denitrifying communities and ecosystem processes have been observed within numerous studies (e.g. Smith et al., 2007; Wankel et al., 2011). This information is mainly based on diversity and/or abundance data of a particular microbial community (e.g. Wankel et al., 2011). However, the uncoupled relation has also been reported (Zheng et al., 2014; Hou et al., 2013). For example, in a study in the intertidal sediments of the Yangtze Estuary, where bacterial amoA genes were more abundant than archaeal amoA genes, potential nitrification rates were not correlated with amoA gene abundance of AOB, but with AOA‟s (Zheng et al., 2014). Such lack of correlation between functional gene

124 abundance and the associated activity was also reported in other studies (Wuchter et

al., 2006; Caffrey et al., 2007; Bernhard et al., 2010; Hou et al., 2013). Considering

the fact that biogeochemical processes are mediated by microorganisms, this lack of correlation between abundance of microorganisms and the rate of their functions can be due to methodological artefacts (Bernhard et al., 2010) in microbial techniques, targeting DNA rather than RNA as a template in cultivation-independent studies of microbial population (Spring et al., 2000; Philippot and Hallin, 2005), the effect of other controlling factors on the ultimate expression of a gene and activities or limitations in accuracy of the method applied in measuring the rate of microbial processes. Still other factors might be also involved.

Here, we explain briefly these different possible causes for the lack of correlation between microorganisms and their functionality, and how our research strategy avoided some of these issues. In general, methodological considerations are discussed below.

5.2.1. Methodological Considerations

Methodological artefacts (Bernhard et al., 2010) in microbial techniques may include PCR bias (Acinas et al., 2005; Aird et al., 2011) and underestimation of microbial diversity due to the limitation of the primer pair (Yu et al., 2008; Liu et al., 2013b). For example, it seems the currently widely used primers for denitrification pathway often fail to detect Gram-positive denitrifiers (Verbaendert et al., 2014). In our study in

Chapter 4, we also aimed to investigate bacterial and archaeal amoA genes involved

in nitrification but the bacterial amoA gene failed to amplify. The optimal primer melting temperature for custom MiSeq primers is 65°C (Personal communication with the Genomics Core). However, it was less for the custom primers for this library which may have contributed to the failed amplification (β-AOB primer sets: amoA-1F (forward primer), 5'-GGGGHTTYTACTGGTGGT-3' and amoAr-new (reverse primer), 5'-CCCCTCBGSAAAVCCTTCTTC-3').

A weak point of many cultivation-independent population studies is the use of DNA as a template. It has been argued that genomic DNA of dead cells (extracellular DNA) can be very stable, surviving for long periods of time in the environment (Spring

et al., 2000). Extracellular DNA concentrations in aquatic sediments are 3 to 4 orders

of magnitude greater than those in the water column and account for a large fraction of the total DNA in the sediment, compared with intracellular DNA concentrations

125 (Corinaldesi et al., 2005). Therefore, we removed extracellular DNA from sediment samples in our research on total bacterial and archaeal communities (Chapter 3), which allowed for studying living cells (dormant and active cells) in the sediment. Considering dormant cells account for a large fraction of living cells in marine sediments (Luna et al., 2002), the use of extracted RNA as PCR template (see