Identification of ciliate grazers of autotrophic Bacteria in ammonia-oxidizing activated sludge by RNA stable isotope probing

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pdfMoreno Am, Matz C, Kjelleberg S & Manefield M 2010 Identification of ciliate grazers of autotrophic Bacteria in ammonia-oxidizing activated sludge by RNA stable isotope probing. AEM 76:2203-2211

Purpose: To identify the ciliate grazers that control populations of primary producers in ammonia-oxidizing wasterwater sludge.

Although SIP experiments usually target Bacteria or Archaea that growdirectly on specific organic or nitrogenous compounds, in this case the authors are looking one step up the food chain, to the unicellular eukaryotes that eat these Bacteria, and specifically those that eat the primary producers in this environment, i.e. those that incorporate carbon from CO2. The particular environment they're interested in is an ammonia-oxidizing wastewater activated sludge. In this sort of environment, useable nitrogen (NH4+, NO3-, NO2-) is not the primary limiting factor (and probably not phosphorous, either), but rather carbon, and so carbon fixation will predominate. The microbial growth that results will incorporate the ammonia, either directly or after being oxidized to nitrite or nitrate. Protists, mostly ciliates, are present in large numbers in activated (aerobic) sludge, where they graze on the resulting lush bacterial growth. The authors describe conflicting data about whether protist grazing has positive or negative effects on microbial growth or nitrogen cycling. Of particular interest to the authors is whether protists in this environment have feeding preferences, or whether they feed indiscriminately.

The first step in this process was to take a census of the protists in this sludge. They used the same approach we've talked about before; they isolated DNA from this environment, amplified ssu-rRNA (in this case, using protist-specific primers), then cloned and sequenced the products (see Table 1, under "Clones"). This sort of process works just as well with microbial eukaryotes as it does with Bacteria or Archaea.

They only did 29 clones, but calculated that they covered almost 2/3rds of the diversity of the sample, because the sequences were predominated by a single type, an amoeba Arcella. They got several other amoebas (Chaos, Euglypha, Balamuthia, and Stenamoeba), some ciliates (Zoothamnium, Epistylis, and Acineta), and a small number of cercozoans (Cercomonadida) and apicomplexians (Eimeriidae). Only one sequence was unidentifiable, i.e. less than 90% similar to any characterized type.

Arcella

Zoothamnium

Chaos

Cercomonas

Eimeria

Epistylis

Euglypha

Balamuthia

Stenamoeba

Acineta

Tetrahymena

Vorticella
(a relative of Epicarchesium)

Spumella

Hartmannella

   

The authors then spent some effort to prove they could label and recover protist rRNA from this predominantly bacterial ecosystem. They started by seeding 10^4/ml 13C-labeled Tetrahymena (an easy-to-grow lab organism) into their sludge (Fig 1) and showed they could get the corresponding rRNA band in the heavy-isotope fractions. In these gels, they separate RNAs by density in cesium gradients, fractionate these (these are the fraction numbers), then do reverse transcriptase PCR using protist-specific primers and separate the products by DGGE:

They went on to show that they could get heavy-isotope labeling of grazing protist rRNA from labeled Bacteria by seeding 10^8/ml 13C-labeled E. coli into the sludge and allowing grazing for 16 hours (Fig 2). On the DGGE gel, it looks like basically all of the protists were labeled to some extent, but the most heavily labeled (and so the predoninant grazers) were Epicarchesium, Spumella, and Zoothamnium. One type, the common amoeba Hartmannella, seemed to specifically not be labeled.

They go on (see Figs 3, 4, and 5) to show that carbon (carbonate) fixation and acetate utilization in this sludge is limited by ammonium concentration, and that they could increase carbon fixation by the addition of ammonium sulfate. This would be expected in this environment, which is conditioned on the feeding of nitrogen-rich "input". The implication is that ammonia oxidizing Bacteria are growing, and fixing carbon in the process.

The experiment they've been building up to is to test the protists that are labeled when they feed 13CO2 (actually bicarbonate) to the sludge (Fig 6) along with some ammonia:

The only lanes shown are those of the heavy RNA, after 0, 6, and 10 hours of labeling. 13CO2 is being fixed by (presumably) autotrophic ammonia oxidizers, which are being eaten by the protists. The only bands that are heavier in the 13C-labeled lanes are the bands corresponding to Epistylis. The implication is that this ciliate predominates in the grazing on ammonia oxidizing autotrophs.

In contrast, as a control, they feed the sludge 13C-acetate under nitrate-reducing (denitrifying) conditions (lots of nitrate, no oxygen), which presumably would serve as a general carbon source for heterotrophic Bacteria, and found no specific labeling of protists (the 12C and 13C lanes are the same):

The implication is that either their aren't any protists that specifically feed on denitrifiers, or that protist feeding is no longer prevalent under anaerobic condiions (this wouldn't be surprizing).