SummaryPseudomonas CMR12a is a biocontrol strain that produces phenazine antibiotics and as yet uncharacterized cyclic lipopeptides (CLPs). The CLPs of CMR12a were studied by chemical structure analysis and in silico analysis of the gene clusters encoding the non-ribosomal peptide synthetases responsible for CLP biosynthesis. CMR12a produces two different classes of CLPs: orfamides B, D and E, whereby the latter two represent new derivatives of the orfamide family, and sessilins A-C. The orfamides are made up of a 10 amino acid peptide coupled to a β-hydroxydodecanoyl or β-hydroxytetradecanoyl fatty acid moiety, and are related to orfamides produced by biocontrol strain Pseudomonas protegens Pf-5. The sessilins consist of an 18-amino acid peptide linked to a β-hydroxyoctanoyl fatty acid and differ in one amino acid from tolaasins, toxins produced by the mushroom pathogen Pseudomonas tolaasii. CLP biosynthesis mutants were constructed and tested for biofilm formation and swarming motility. Orfamides appeared indispensable for swarming while sessilin mutants showed reduced biofilm formation, but enhanced swarming motility. The interplay between the two classes of CLPs fine tunes these processes. The presence of sessilins in wild type CMR12a interferes with swarming by hampering the release of orfamides and by co-precipitating orfamides to form a white line in agar.

Pseudomonas CMR12a was previously selected as an efficient biocontrol strain producing phenazines and cyclic lipopeptides (CLPs). In this study, biocontrol capacity of Pseudomonas CMR12a against Rhizoctonia root rot of bean and the involvement of phenazines and CLPs in this ability were tested. Two different anastomosis groups (AGs) of Rhizoctonia solani, the intermediately aggressive AG 2-2 and the highly aggressive AG 4 HGI, were included in growth-chamber experiments with bean plants. The wild-type strain CMR12a dramatically reduced disease severity caused by both R. solani AGs. A CLP-deficient and a phenazine-deficient mutant of CMR12a still protected bean plants, albeit to a lesser extent compared with the wild type. Two mutants deficient in both phenazine and CLP production completely lost their biocontrol activity. Disease-suppressive capacity of CMR12a decreased after washing bacteria before application to soil and thereby removing metabolites produced during growth on plate. In addition, microscopic observations revealed pronounced branching of hyphal tips of both R. solani AGs in the presence of CMR12a. More branched and denser mycelium was also observed for the phenazine-deficient mutant; however, neither the CLP-deficient mutant nor the mutants deficient in both CLPs and phenazines influenced hyphal growth. Together, results demonstrate the involvement of phenazines and CLPs during Pseudomonas CMR12a-mediated biocontrol of Rhizoctonia root rot of bean.

Production of biosurfactants is a common feature in bacteria, and in particular in plant-associated species. These bacteria include many plant beneficial and plant pathogenic Pseudomonas spp., which produce primarily cyclic lipopeptide and rhamnolipid type biosurfactants. Pseudomonas-derived biosurfactants are involved in many important bacterial functions. By modifying surface properties, biosurfactants can influence common traits such as surface motility, biofilm formation and colonization. Biosurfactants can alter the bio-availability of exogenous compounds, such as nutrients, to promote their uptake, and of endogenous metabolites, including phenazine antibiotics, resulting in an enhanced biological activity. Antibiotic activity of biosurfactants towards microbes could play a role in intraspecific competition, self-defence and pathogenesis. In addition, bacterial surfactants can affect plants in different ways, either protecting them from disease, or acting as a toxin in a plant-pathogen interaction. Biosurfactants are involved in the biocontrol activity of an increasing number of Pseudomonas strains. Consequently, further insight into the roles and activities of surfactants produced by bacteria could provide means to optimize the use of biological control as an alternative crop protection strategy.

In this study nitrogen removal via nitrite >80% was achieved after approximately 80days in a sequencing batch reactor (SBR) treating pre-treated industrial wastewater originating from the potato industry. Thereafter, SBR performance was investigated during the formation of aerobic nitrite granules (ANG). The first granules appeared after 26days leading to full granulation after 64days. ANG showed excellent settling properties, as the Sludge Volume Index (SVI) went down to 16mL/g and a SVI/SVI=1 was obtained. qPCR analysis showed that slow growing organisms, especially polyphosphate accumulating organisms (PAO) were stimulated by an anaerobic feeding strategy. The average nitrogen removal was 95.3% over the entire operational period, and it mainly followed the "nitrite-route". Moreover, with ANG also phosphorus removal efficiencies up to 65.7% could be achieved. However, it has to be mentioned that nitrous oxide was an important denitrification product, which implies some environmental concerns.

In this study, petrochemical wastewater from the port of Antwerp was used for the development of aerobic granular sludge. Two different reactor setups were used, (1) a completely aerated sequencing batch reactor (SBR) with a feast/famine regime and (2) a sequencing batch reactor operated with an anaerobic feast/aerobic famine strategy (SBR). The seed sludge showed poor settling characteristics with a sludge volume index (SVI) of 285mL.gMLSS and a median particle size by volume of 86.0µm±1.9µm. In both reactors, granulation was reached after 30days with a SVI of 71mL.gMLSS and median granule size of 264.7µm in SBR and a SVI of 56mL.gMLSS and median granule size of 307.4µm in SBR. The chemical oxygen demand (COD) and dissolved organic carbon (DOC) removal was similar in both reactors and above 95%. The anaerobic DOC uptake increased from 0.13% to 43.2% in 60days in SBR.