Tratamiento MBR

3.2.1.1. Overview

16S rRNA PCR primers, reported to be anammox specific (see table), were investigated as to their suitability for investigating anammox ecology. Primarily two candidate PCR assays were investigated: An7F & An1388R and Amx368F & Amx820R/BS820R.

3.2.1.2. Environmental Samples and PCR Controls

Primers were initially tested for specificity using a number of controls including environmental samples (Medway Estuary site M6, collected 23/03/2010), three environmental clones positively identified as Ca. Jettenia sp. Ca. Brocadia sp. and

Ca. Scalindua sp. and the entire synthesised 16S rRNA gene of Ca. Kuenenia stuttgartiensis (GenScript, USA) and genomic DNA extracted from a pure culture of

Planctomyces maris DSM-8797 (DSMZ, Germany) along with other DNA samples from the laboratory including archaeal DNA and DNA extracted from isolates of non-anammox bacteria. Site M6 was chosen as it had previously demonstrated high anammox rates and the presence of anammox bacteria (Rooks, et al., 2012). DNA was extracted from environmental samples and P. maris cultures using the method outlined in Purdy (2005). PCRs were performed as outlined in section 2.3.2.1.

3.2.1.3. 454 Pyrosequencing

Following initial investigations as to the specificity of these primers in the laboratory, their suitability for measuring anammox ecology using high-throughput sequencing technologies (i.e. 454 pyrosequencing) was tested. PCR product was obtained in triplicate from environmental samples. Triplicate PCR products were pooled, purified (PCR Purification Kit, Qiagen, UK) and submitted for pyrosequencing (as outlined in section 2.3.5).

However, the amplicon produced by primers An7F & An1388R (~1380 bp) was too large to be used with 454 pyrosequencing (at time of sequencing maximum amplicon

46 size was around 800 bp)*. Hence this primer set was unsuitable for use with this technology. Therefore, in order to obtain a usable amplicon for pyrosequencing, amplification was attempted with the anammox-specific An7F primer and a universal bacterial reverse 16S rRNA primer. Potential reverse primers were initially checked for suitability by aligning them against anammox 16S rRNA sequences from NCBI and SILVA (Pruesse, et al., 2007) and calculating primer sensitivity and specificity using ThermoPhyl (Oakley, et al., 2011), BLAST (Altschul, et al., 1990; Zhang, et al., 2000) and ARB (Ludwig, et al., 2004). Primer 518R (see Table 2.1) was highlighted as a suitable reverse primer as it targeted all known anammox 16S rRNA sequences and theoretical calculations showed that the primer could work thermodynamically with primer An7F. Primers An7F & 518R were used as a second round nested PCR on purified product from An7F & An1388R (see section 2.3 for full methods) as, although the addition of a universal reverse primer (i.e. 518R) would likely target organisms outside of the anammox clade, it was hoped that the specificity of the first round PCR would ensure that primarily anammox related sequences were obtained from the 2nd round reaction. This methodology produced a clear band of the correct size. Non-specific bands were also observed in this PCR but these were distinct from the band of correct size and the correct band was easily purified by gel extraction.

3.2.1.4. PCR Validation

Primers Amx368F & Amx820R were further validated as to their ability to be able to target non-Scalindua anammox organisms from environmental DNA. Unfortunately, due to the difficulties associated with investigating anammox organisms, such samples were not readily available. Nevertheless, a collection of samples were generously donated by several other research groups (see Table 3.1) which had been positively identified to contain a range of anammox genera. These samples were amplified using the suite of primers discussed above to test their specificity and their ability to amplify target DNA assessed on a simple presence/absence basis. All samples underwent a nested PCR approach with primers Pla46F & 1390R as a first round PCR. This PCR product was gel extracted and purified prior to the second round of amplification (see chapter 2.3.2.4). Samples which exhibited negative

* _{N.B. The amplicon of primers Amx368F & Amx820R/BS820R was already of a suitable size for use} with pyrosequencing technologies.

47 results after this two-step PCR were then further optimised in order to ascertain that the lack of amplifiable product was indeed due to the inability of this primer set to amplify DNA from that sample and not due to other complications associated with PCR (e.g. impurities, low DNA yields etc). All samples which failed to be amplified after the nested PCR could not be amplified following this further optimisation.

Table 3.1: Table of collaborations with other research groups who generously donated environmental DNA

and positive controls which were used in the optimisation and validation of anammox specific PCR primers during this study.

Table 3.2: Identity of individual samples donated by the Zopfi and Amano Laboratories (see Table 3.1)

including sample names, used throughout this study.

To further investigate the ability of primers Amx368F & Amx820R to amplify anammox DNA from non-Scalindua genera, a small clone library was constructed using the samples mentioned above (Table 3.2). PCR product was purified using a commercial PCR Purification Kit (Qiagen, UK) and then cloned into the pGEMT- Easy vector (Promega, UK). Cloned plasmids were transformed into JM109 competent E. coli cells (Promega, UK) and single colonies isolated. Colonies were

Lead Researcher Institution Sample Location Sample Description Reference

Jakob Zopfi

University of Lausanne, Switzerland

Lake Neuchâtel,

Switzerland DNA from bulk soil (Humbert et al., 2010)

Teruki Amano Kyoto University, Japan Haipong, Vietnam

DNA from sediment collected from Mangrove forest and

shrimp pond sites

(Amano et al., 2011) Josh Neufeld University of Waterloo, Canada Ontario, Canada Cloned 16S rRNA genes extractd from

groundwater samples

(Moore et al., 2011)

Sample Quantity

(ng/ul) A260/A280 Description

Reported anammox genera Z o p fi S a m p le

s Z1 132 1.68 DNA ext. from anammox enriched _soil Unknown Z2 82 1.68 DNA ext. from soil LnA Ca. Brocadia spp. Z3 201 1.85 Plasmid DNA from enrichment _culture Ca. Jettenia sp.

A m a n o S a m p le

s _{MF1 109 1.8 Mangrove Forest 1} All anammox (dominated

by Ca. Scalindua spp.)

MF2 157 1.7 Mangrove Forest 2 All anammox (dominated

by Ca. Scalindua spp.) SP1 134 1.58 Shrimp Pond 1 All anammox (dominated

by Ca. Kuenenia spp.)

CH 67 1.63 Channel All anammox (dominated

48 screened for containing the insert and then sequenced via Sanger sequencing (Natural History Museum, UK) using both the M13F and M13R primers. Five colonies were sequenced for each of the seven samples. Forward and reverse reads were checked and trimmed manually in SeqMan II (DNASTAR, USA) and assembled into contigs. Contigs were aligned against reference sequences and phylogenetic relationships calculated in MEGA4 and MEGA5 (Tamura, et al., 2007; Tamura, et al., 2011).