Instrumentos

From the results of these investigations it appears as if the diversity of hzo does not match the phylogenetic diversity of the 16S rRNA gene. This contradicts the findings of Schmid, et al. (2008) who reported that the phylogeny of “hzo Cluster 1” was in good agreement with that of the 16S rRNA gene. Conversely, it has been argued that 16S rRNA does not accurately report phylogenetic diversity. Li, et al.

(2010b) concluded that hzo primers were more specific and efficient than those of 16S rRNA targeting primers whereas Hirsch, et al. (2011) argued that the 16S rRNA gene underestimated anammox diversity based on a greater diversity obtained from

hzo sequences which they concluded to be a closer representation of the true diversity. Wang and Gu (2013) further agreed with these statements. However, these claims are unreasonable as firstly, determination of the “true” microbial diversity, especially in complex environments, is impossible to attain due to the inability to accurately and comprehensively enumerate the number of individuals within the environment. Furthermore, it is also impossible to accurately determine bacterial “species” richness as the definition of what constitutes a “species” is neither clear nor universally implemented in bacteria and nevertheless, if a bacterial species could be accurately defined, each individual would have to be isolated from the community to positively determine which species it belonged to beyond doubt. As such measurements of the “true” diversity of a bacterial population cannot be obtained and so any investigations into bacterial ecology can only report a relative diversity. Secondly, with the present data available, it cannot be positively determined that diversity within the hzo gene represents phylogenetic diversity. Therefore it cannot be assumed that the higher measurement of diversity is more correct simply because it is the greater of the two as the assumption that it is overestimating diversity is equally as valid. In addition, regardless of whether the 16S rRNA gene is an accurate measurement of “true” anammox diversity, it remains the method by which the current interpretation of anammox taxonomy was established and as such, must be

78 used as the benchmark until anammox diversity is validly re-classified. Hirsch, et al.

(2011) stated that “16S rRNA sequences provide taxonomic identification of bacteria but no evidence for functional capability. Thus the detection of hzo genes can be more promising to examine functional anammox bacteria in the environment”. This statement is true providing that we can be confident of the role and identity of hzo

and the quality of sequences obtained using primers to specifically target this gene. However, this may be confounded by multiple analogues of the hzo gene in the genomes of anammox bacteria, which may be functionally redundant.

It could be argued that ‘Cluster A’ and ‘Cluster B’ represent diversity within the genus Scalindua whereas the other three clusters represent that of other anammox organisms, however this would be in disagreement with the literature where Ca.

Scalindua spp. hzo/HZO clearly cluster into one distinct clade within “hzo Cluster 1”

(Schmid, et al., 2008; Hirsch, et al., 2011). In fact, these data do not support this claim as no sequences obtained from this investigation were positively identified as belonging to non-Scalindua anammox organisms. Only one sequence (Urea-1-8 clone 9) could be putatively assigned to an unknown, non-Scalindua anammox organism based solely on its association with ‘Cluster E’, which has been inferred to represent a sub-section of non-Scalindua anammox bacteria (Li, et al., 2010b). As such, the ability of these primers to differentiate between hzo genes belonging to Ca.

Scalindua spp. and non-Scalindua organisms cannot be asserted with confidence. Therefore, in the light of these data, it is difficult to confidently infer anammox diversity with the use of these primers.

Another issue with the use of hzo as a molecular marker for anammox is its similarity to hao genes found in aerobic ammonium oxidising bacteria (AOB). hzo

and hao share a close functional and sequence similarity and it is difficult to definitively distinguish between HZO and HAO protein sequences (Klotz, et al., 2008). In fact Klotz, et al. (2008) further speculated that HZO and HAO may be the same protein which oxidises different molecules depending on the upstream metabolic products of that particular organism (i.e. N2H4 in anammox and NH2OH in

AOB). However Shimamura, et al. (2007) managed to separate HZO and HAO like complexes from KSU-1 and demonstrated that the resulting HZO protein could

79 oxidise N2H4 but not NH2OH thus suggesting a different function in at least some of

the proteins translated from hzo/hao like genes.

4.5. Summary

 Three functional genes have been suggested in the literature as suitable for investigating the ecology of anammox organisms: hzo, hzs and nirS.

 nirS does not appear suitable as a phylogenetic marker due to its close relationship to nirK and discrepancies reported in the literature.

 hzs shows promise as a functional and phylogenetic marker for anammox organisms however requires further research to validate these claims.

 Despite contrary findings published in the literature, hzo does not appear to be a suitable molecular marker for measuring anammox diversity based on the data gathered during this investigation. However it may be suitable as a molecular marker for anammox functionality within an environment. Further research is required in order to elucidate the diversity of the hzo gene and its implications for anammox ecology.

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