Cultivation-based approach at organophosphonate-degrading microbiota from oligotrophic Lake Constance

Abstract

Understanding the complex relationships between ecosystems, in which trophic states are changing, and the adaptations (resilience) of its microbiota is important. The re-oligotrophication of Lake Constance from the beginning of the 1980s until today may have caused the adaptation of the plankton microbiota to utilize also additional phosphorus (P) sources, specifically organophosphonate (OP). The OP substrates may come from natural sources, such as ciliatine and methylphosphonate (MP), and can be anthropogenic xenobiotic chemicals, such as glyphosate. Bacteria have been reported to effectively degrade various OP species, potentially mobilizing such additional P input for phytoplankton growth and, thus, higher trophic levels. However, the degradation of OP by Lake Constance bacteria, or potentially by microalgae or cyanobacteria directly, and the accessibility of OP to support phytoplankton growth and a potential key role of heterotrophic bacteria in such processes, have not been characterized. In this thesis, these topics were addressed by a laboratory cultivation approach, when using ciliatine, MP and glyphosate as model OPs.

Twelve OP-degrading heterotrophic bacterial strains were successfully enriched from Lake Constance water and isolated. Three representative isolates were characterized in more detail by growth experiment and HPLC-MS analysis. One isolate, Ochrobactrum (Brucella) anthropi DNF1 was found to effectively degrade 0.2 mM ciliatine, glyphosate, and MP as P sources. The draft genome of strain DNF1 was sequenced and annotated (IMG Genome ID: 2963528115). It revealed the presence of a phosphonate utilization (phn) gene cluster and of other members of the phosphate (Pho) regulon, as well as a candidate gene for glycine oxidase, which may be involved in the known a glyphosate degradation II pathway. Candidate genes for adenine phosphoribosyltransferase, phosphoribosyl 1,2-cyclic phosphate phosphodiesterase (phnP-like gene) and inorganic pyrophosphatase which, which are known to be involved in glyphosate degradation III, are present. A candidate gene for phosphonoacetaldehyde dehydrogenase (phnY), which is known to be involved in ciliatine degradation II, is also present. Differential proteomics revealed that proteins associated with the C-P lyase complex (PhnGHIJKLMN) were produced by O. anthropi DNF1 when grown with 0.2 mM OPs compared to orthophosphate as a P source. In glyphosate-grown cells, also the proteins for phosphoribosyl 1,2-cyclic phosphate phosphodiesterase and inorganic pyrophosphatase were also found to be produced. Enzyme assays with cell extracts of O. anthropi DNF1 grown with glyphosate as a P source showed aminomethyl-phosphonate (AMPA) formation, but no sarcosine production was observed. This suggests that O. anthropi DNF1 utilizes glyphosate mainly through C-P lyase complex encoded by the phn gene the cluster (glyphosate degradation III pathway) but also produces AMPA presumably through glycine oxidase (glyphosate II pathway). Furthermore, O. anthropi DNF1 employs C-P lyase also to degrade MP, and it is suggested that ciliatine is utilized through the ciliatine II pathway with phosphonoacetaldehyde dehydrogenase PhnY.

No OP utilization was observed for all tested, lab-grown photoautotrophic phytoplankton monocultures, comprising cyanobacteria and microalgae and their associated bacterial flora (xenic cultures), when grown under P-limited conditions. Notably, axenic cultures of diatom Achnanthidium minutissimum exhibited growth when OPs were added as alternative P sources, but no OP utilization was detectable. Furthermore, the co-inoculation of O. anthropi DNF1 had no effect under the cultivation conditions used. However, phytoplankton-bacteria enrichment cultures inoculated with Lake Constance plankton filtrate showed phytoplankton growth with ciliatine as a sole P source, but not with MP and glyphosate, and the ciliatine cultures grew across sub-cultivations. Based on the results of 18S and 16S rRNA gene fragment amplicon sequencing, it was found that green algae (order Chlorellales, Prasiolales and Sphaeropleales) were the predominant phytoplankton present in the bacteria-phytoplankton culture. The most prominent bacterial groups found in both the culture with ciliatine (family Rhodanobacteraceae and family Rhodospirillaceae) and the control (uncultured Alphaproteobacteria) were Alpha- and Gammaproteobacteria. This suggests that specific phytoplankton-associated heterotrophic bacteria are needed to facilitate the utilization of OPs by the photoautotrophic enrichment cultures, as none of the cultures of lab-cultivated phytoplankton monocultures and their associated bacterial flora showed OP utilization any more.

This thesis offers a more comprehensive understanding of the relationship between heterotrophic bacteria and phytoplankton of Lake Constance, particularly in their utilization of OP, and introduces novel bacterial strains isolated from the lake capable of metabolizing ciliatine, glyphosate and MP.