Bacterial diversity

There were significant changes in bacterial diversity between sites and months in the River Wensum. The increases in abundance of some OTUs may proportionately reduce the importance of other OTUs, especially affecting rare OTUs. Bacterial diversity was found to decrease at downstream sites of the river (during warm and dry months), whereas decreases in the abundance of OTUs during wet and cold months resulted in an increase in bacterial diversity.

125 Similar results are reported by Sekiguchi et al. (2002) who found that bacterial community diversity decreased downstream in the River Changjiang in China, and attributed this pattern to physical and chemical properties of the river water, such as water temperature, nutrient concentrations and flow rate. Similarly, Lemian et al. (2012) also found that bacterioplankton diversity decreased downstream in the River Xiamen Houxi in China and attributed this decrease to higher values of pH and total nitrogen (TN) at downstream sites, explaining about 48% of the total variation of bacterial community composition. However, other studies have found different trends of bacterial diversity. For example, Bushaw-Newton et al. (2012) found that bacterial diversity in sediments of the River Anacostia in the US increased moving downstream, attributing this to high concentrations of TC and TN at downstream sites. Bacterial diversity was also negatively related to water temperature in the Wensum and this in agreement with Henne et al. (2013) who found that bacterial diversity in different types of freshwater was higher when water temperature is low. Dong et al. (2010) found that bacterial abundance in Tibetan Plateau lakes in China were positively related to water temperature and dry periods, while diversity was positively related to wet periods and water temperature. The evolution of the community as water moves down a river (as mentioned in Chapter 1, section 1.6.1 and Chapter 3, section 3.5.1.1) depends on inputs of exogenous bacteria and on the relative growth rates of the OTUs present. Bacterial diversity increases at upstream sites and during the wet (high river flow) and cold (low water temperature) conditions, and is positively related to TN and TC. Then diversity declines as water moves downstream. Presumably, there are differences between OTUs in terms of their population growth rates in response to water temperature and pH. Then, these OTUs grow faster and come to make up a greater proportion of the bacteria present than other OTUs that are resources limited or are unable to grow in river water. These processes are especially important during periods of high water temperature or when residence time is long. Then, these fast growing OTUs come to dominate the community as water moves downstream, resulting in the decreases of overall bacterial diversity, an effect that is especially marked in periods of higher water temperatures. The data obtained here show that there are significant, but rather weak, differences between sites in terms of bacterial composition and abundance. This is perhaps because water in the river environment is homogeneous, in contrast to environments, such as soil (which are more heterogeneous), and where we can find large differences in the bacterial community at fine scales (Ranjard et al. 2001; Kang and Mills 2006). So, this study suggests that there is not competition between bacterial OTUs. There are significant correlations between bacterial community composition and abundance and environmental parameters, but these correlations are not strong and thus do not clearly distinguish between sites that are more or less impacted by human activities. Consequently, bacteria do not appear to be good indicators of the ecological status of the river water, so will be of limited value for assessing compliance with the European Water Framework Directive. However, bacterial communities may be of use as an indicator for ecological health in more nutrient poor waters and the trophic state needs more research using different molecular techniques (Lear et al. 2009), such as Illumina sequencing and may require the development of new statistical methods to deal with this homogeneous environment.

126 4.6 Summary

In the research reported in this chapter, bacterial community composition was demonstrated to be significantly different between sites and sampling dates. Bacterial diversity decreases as water moves downstream, while bacterial abundance increases as water moves downstream, with only some fragment sizes able to grow and multiply at downstream sites. The dominant OTUs may control the substrates affecting other rarer OTUs and so reducing their abundance. Spatially, the large difference of bacterial community composition was between upstream and downstream sites. Temporarily, the large differences were between December 2011 and 2012 and also between September 2011 and 2012. Bacterial diversity and abundance showed significant relationships with some environmental parameters. However, there may be other chemical, physical and biological parameters beyond the current study that have potential effects on bacterial community composition in the River Wensum. For example, the effects of biological factors such as viruses and flagellates and chemical factors such as chlorophyll a. Also, the effect of heavy metals on bacterial community composition such as chromium, lead, cadmium, nickel and zinc.

127 Chapter Five

454 pyrosequencing technique for the analysis of bacterial communities in the River Wensum

5.1 Introduction

Microbial sequence data have played a fundamental role in classifying organisms. It is more informative than phenotypic information, can be readily interpreted (Woese 1987) and organisms can be taxonomically classified based on sequence differences (Wolska and Szweda 2012).

Metagenomics, the sequencing of genes from mixed communities of microorganisms and directly from environmental samples, reveals extensive microbial diversity overlooked by culture based methods (Hugenholtz et al. 1998; Petrosino et al. 2009). A widely used approach involves the amplification of regions of 16S rRNA, which has been widely used to construct bacterial phylogenies (Petrosino et al. 2009; Dall'Agnol et al. 2012).

Since its introduction in 2005, pyrosequencing has been shown to be an effective tool in assembling genomes of bacteria retrieved from short reads, which can be used to identify bacteria at genus and species level (Margulies et al. 2006). It has been used to study bacterial communities in different environments, including a deep underground mine in the US (Edwards et al. 2006), foods (Humblot and Guyot 2009), the Amazon River (Bai et al. 2010) and sea water (Thompson et al. 2011).

5.2 Aims

As introduced in Section 1.7, the aims of the research presented in this chapter are to use 454 pyrosequencing to characterise bacterial communities in the River Wensum including (i) spatial and temporal variations and associations with environmental factors, (ii) to determine the dominant bacterial phyla, (iii) to determine the commonest bacterial OTUs between sites and in time (December 2012), (iv) to describe the trend of the shift of abundance of the commonest bacterial OTUs when water moves to downstream sites, and (v) to identify the

128 taxonomic affinities of the commonest OTUs, based upon the most similar 16S sequences from cultured strains and the most similar environmental 16S sequences. This is to seek to infer their potential functional significance based on the characteristics of their nearest relatives.

5.3 Methods and materials