Isolation and identification of Pantoea agglomerans from the inflated bag with dried hop pellets stored under a modified atmosphere.

AIMS: Isolation, characterization and identification of possible microbial contaminant(s) in the inflated foil bag containing hop pellets packed and stored in a modified atmosphere. METHODS AND RESULTS: Package gas of the inflated foil bag containing hop pellets was analysed by gas chromatography. Compared with the reference modified atmosphere, containing about 16 vol.% of CO2 , the inflated bag atmosphere contained 53 vol.% CO2 , suggesting possible microbial contamination. Therefore, several standard and mineral media, with added hop pellets or hop infusion, were used for cultivation at different temperatures under an anaerobic atmosphere. Cultivation in mineral medium with hop pellets yielded a bacterial isolate that was identified by MALDI-TOF mass spectrometry and verified by partial 16S rRNA gene analysis as Pantoea agglomerans, a known plant epiphyte. CONCLUSIONS: A novel strain of P. agglomerans (designed as DBM 3696) was found to be suspicious of causing inflation of the foil bag containing dried hop pellets packed in modified atmosphere. SIGNIFICANCE AND IMPACT OF THE STUDY: This study suggests that P. agglomerans, probably hop epiphyte, could cause sporadic inflation of bags with hop pellets packed in modified atmosphere causing logistical problems during bags transport.

Comparison of expression of key sporulation, solventogenic and acetogenic genes in C. beijerinckii NRRL B-598 and its mutant strain overexpressing spo0A.

The production of acetone, butanol and ethanol by fermentation of renewable biomass has potential to become a valuable industrial process. Mechanisms of solvent production and sporulation involve some common regulators in some ABE-producing clostridia, although details of the links between the pathways are not clear. In this study, we compare a wild-type (WT) Clostridium beijerinckii NRRL B-598 with its mutant strain OESpo0A, in which the gene encoding Spo0A, an important regulator of both sporulation and solventogenesis, is overexpressed in terms of solvent and acid production. We also compare morphologies during growth on two different media: TYA broth, where the WT culture sporulates, and RCM, where the WT culture does not. In addition, RT-qPCR-based analysis of expression profiles of spo0A, spoIIE, sigG, spoVD, ald and buk1 genes involved in sporulation or solvent production in these strains, were compared. The OESpo0A mutant did not produce spores and butanol titre was lower compared to the WT, but increased amounts of butyric acid and ethanol were produced. The gene spo0A had high levels of expression in the WT under non-sporulating culture conditions while other selected genes for sporulation factors were downregulated significantly. Similar observations were obtained for OESpo0A where spo0A overexpression and downregulation of other sporulation genes were demonstrated. Higher expression of spo0A led to higher expression of buk1 and ald, which could confirm the role of spo0A in activation of the solventogenic pathway, although solvent production was not affected significantly in the WT and was weakened in the OESpo0A mutant.

Development of flow cytometry technique for detection of thinning of peptidoglycan layer as a result of solvent production by Clostridium pasteurianum.

Clostridium pasteurianum forms acetic and butyric acids in an initial growth phase, which is a typical feature of clostridial acetone-butanol fermentation where an initial accumulation of acids is followed by production of solvents 1-butanol, acetone and ethanol. The initiation of the solvent production coupled with endospore formation leads to decrease of cell-wall thickness; thinner cell wall is more resistant against solvents and dyes. These changes can be observed by the method based on adaptation of Gram staining. The cell wall of G+ bacteria allows the entry of hexidium iodide and rhodamine 123, whereas the outer membrane of G- bacteria does not allow the uptake and therefore G+ bacteria are stained with higher fluorescence intensity than G- bacteria. The ratio of fluorescence intensity (FI) to forward scatter (FSC) was determined to correspond to G+ bacteria when clostridia were producing less solvents. The significant drop of the ratio FI to FSC to the level corresponding to G- bacteria is detected after initiation of solvent production.