PLANIFICACION HELIOTÉRMICA

PCR-DGGE was used to screen fractions collected from ultracentrifuge tubes (data not shown). For all incubations, one fraction from the ultracentrifuge tubes was then selected as representative of the “light” (unlabeled) DNA, and another fraction was selected as

representative of the “heavy” (labeled) DNA. These fractions generally corresponded to the highest and lowest positions in the tubes, respectively, from which a strong PCR product could be obtained. In samples with unlabeled salicylate, there were no PCR products in the fractions where 13_{C-enriched DNA would be expected to be present.}

Figure 4.4. Negative image of DGGE profiles of PCR-amplified 16S rRNA gene fragments for the bacterial community in reactor slurry before salicylate addition (lane 13) and for DNA fractions from SIP incubations with [13_{C]salicylate. Lanes 1-4 represent 2-day spike}

incubations, lanes 5-8 represent 10-day spike incubations, and lanes 9-12 represent 10-day continuous incubations. Lanes correspond to the following fractions: (1) [13_{C]salicylate 2-day} spike heavy fraction, (2) [13_{C]salicylate 2-day spike light fraction, (3) [}13_{C]salicylate 2-day} spike heavy fraction, (4) [13_{C]salicylate 2-day light fraction, (5) [}13_{C]salicylate 10-day spike} heavy fraction, (6) [13_{C]salicylate 10-day spike light fraction, (7) [}13_{C]salicylate 10-day spike} heavy fraction, (8) [13_{C]salicylate 10-day spike light fraction, (9) [}13_{C]salicylate 10-day} continuous heavy fraction, (10) [13_{C]salicylate 10-day continuous light fraction, (11)}

[13_{C]salicylate 10-day continuous heavy fraction, (12) [}13_{C]salicylate 10-day continuous light} fraction, (13) day 0 reactor slurry, (14) negative PCR control.

Light and heavy fractions from each incubation condition (2-day spike, 10-day spike, 10-day continuous) were amplified with bacterial primers for the 16S rRNA gene, and the resulting PCR products were analyzed on a DGGE gel. Replicate incubations had DGGE profiles with no significant differences (Figure 4.4). DGGE profiles of heavy DNA from 2- day spike and 10-day spike incubations were similar (lanes 1 and 3, lanes 5 and 7). A pair of closely spaced bands dominated heavy DNA from these incubations. In contrast, the 10-day continuous incubations had a third band which migrated below the pair of closely spaced bands (lanes 9 and 11).

4.4.5. 16S rRNA gene clone libraries

Clone libraries of 16S rRNA genes were constructed from the fractions containing “heavy” DNA from 10-day spike and 10-day continuous incubations. Primer 8f was used to partially sequence 43 clones derived from spike incubations and 42 clones derived from the continuous incubations. Five clones were completely sequenced: salsp08, salcon01,

salcon26, salcon39, and salcon44. Clones were grouped at the >99% similarity level to define operational taxonomic units (OTUs) before construction of the phylogenetic tree (Figure 4.5). Only 7 clones were not grouped into OTUs.

In agreement with the DGGE profiles, clone libraries derived from the spike and continuous incubations had some overlap, while the clone library representing the continuous incubation contained many unique sequences (Figure 4.5). Sequences salcon26, salcon39, salsp08, salsp33, and salsp53 shared high similarity with sequences in the Ralstonia genus of the β-Proteobacteria. Ralstonia sequences have been found before in PAH-contaminated soils (Widada et al. 2002; Dionisi et al. 2004). A sequence from the continuous incubation

which appeared only once in the clone library, salcon28, was most closely related to six sequences previously found in a clone library of organisms from the bioreactor (Singleton et al. submitted). Several other sequences from the continuous incubation, salcon44, salcon41, salcon23, and salcon36, were closely related to various uncultured organisms found in freshwater environments (Figure 4.5). The sequences from the continuous incubation were less closely related to cultured organisms than were the sequences from the spike

incubations.

Sequences recovered from the 10-day spike incubations and some sequences from the 10-day continuous incubations were very similar to sequences recovered from a previous 2- day spike addition of salicylate and naphthalene to the same bioreactor slurry (Singleton et al. 2005), illustrating that at least some of the identified salicylate degraders are also naphthalene-degrading organisms. Interestingly, some clones recovered in this study were very similar to sequences found in other SIP studies with various substrates. Salcon08 was most closely related to uncultured clones 18 and 42 from an SIP study of naphthalene- degrading organisms in a PAH-contaminated soil (Yu and Chu 2005). Clone Salsp15 was closely related to uncultured clones 521 and 545, recovered from an SIP investigation with phenol at an agricultural field site (DeRito et al. 2005). Clone Salcon31 was closely related to uncultured bacterium clone LO13.11 from an SIP study examining methanotrophs in peat soil (Morris et al. 2002).

Figure 4.5. Unrooted phylogenetic tree of 16S rRNA gene sequences recovered from heavy (13_{C-enriched) DNA in SIP incubations and their closest relatives. Clones from this study are} shown in bold and follow the naming scheme of: salsp (salicylate spike) or salcon (salicylate continuous) and a number assigned to each clone for identification purposes. The abundance of each clone in clone libraries derived from spike (S) and continuous (C) incubations is indicated in parentheses after the clone name. Reference sequences are shown with the GenBank accession number in parentheses. The tree was based on near-complete sequence of the 16S rRNA gene (~1500 bp) for clones salsp08, salcon01, salcon26, salcon39, and

salcon44, while partial sequences (~800 bp) were used for all other clones. The qPCR primer set used to quantify the abundance of each clone is indicated in parantheses.

LIBSHUFF analysis was used to determine if differences in the microbial

communities resulting from enrichment with salicylate added as a spike or continuously were statistically significant; the null hypothesis was that the communities were different.

incubation (p=0.001). Conversely, the clone library from the continuous incubation was significantly different from the clone library derived from the spike incubations (p=0.892). As shown by the phylogenetic tree (Figure 4.5), the two clone libraries share many

sequences, but the clone library derived from continuous incubations contained many additional sequences not present in the clone library derived from the spike incubations.