The effect of organic solvents on biofilm formation of the thermoacidophilic Archaeon Sulfolobus acidocaldarius

Abstract

Sulfolobus acidocaldarius is a thermoacidophilic Crenarchaeon (78°C and pH 2-3) with established genetic systems. With these properties, S. acidocaldarius offers high potential for biotechnological applications as alternative host for the production of bulk and fine chemicals, solvents and second generation biofuels, which are, however, often toxic to cells. Biofilms, the dominant lifestyle of microorganisms, were shown to confer higher resistance towards solvent exposure and hence turn more and more into research focus for biotechnological applications. Although S. acidocaldarius is known to form biofilms, the development of suitable incubation systems is still in its beginning and also the influence of organic solvents on S. acidocaldarius has only rarely been studied. In this work, incubation systems for the cultivation of submerse biofilms of S. acidocaldarius cells on glass and polystyrene surfaces in microtiter plates, μ- and Petri-dishes as well as a flow-through system were successfully developed and optimized. These newly developed methods enabled detailed microscopic analyses and also yielded sufficient amounts of biofilms for isolation and analyses of extracellular polymeric substances (EPS) as well as for application of various -omics methods. The analyses were performed in presence and absence of organic solvents (with special respect to 1-butanol) and the results were compared to shaking cultures. S. acidocaldarius formed biofilms even in shaking cultures in Erlenmeyer flasks at the liquid/glass/air interface as a (stress)response to sublethal concentration of different solvents. In accordance with shaking cultures, in a 96-well plate static incubation system, the tendency of enhanced biofilm formation as a response to sublethal concentrations of 1-butanol (<1.5% (v/v)) was visible. By the use of an adapted resazurin assay, indicating respiratory activity, the viability of biofilm cells was confirmed colorimetrically. Furthermore, in static incubation systems e.g. on glass slides, S. acidocaldarius formed monolayered biofilms of homogenously distributed cells even in the absence of solvents as shown by crystal violet staining and subsequent light microscopy. In the presence of solvents these biofilms appeared thicker and showed an altered morphology with a heterogenous distribution and cell aggregate formation, indicated by enhanced crystal violet binding. Additionally, visualised by scanning electron microscopy, under 1.5% (v/v) 1-butanol exposure the aggregated cells were surrounded by extracellular material, probable EPS. Furthermore, the cell morphology was altered compared to the control experiments showing a diversified cell envelope with holes. In the presence of 1% (v/v) 1-butanol, less changes in biofilm morphology and no changes in cell morphology were observed by the application of atomic force microscopy. Lectin binding and subsequent confocal laser-scanning microscopy of submersed biofilms showed an increased amount of carbohydrate structures, especially α-mannopyranosyl- and α-glucopyranosyl residues. The enhanced EPS formation was also confirmed by EPS isolation and quantitative determination using colorimetric assays, which showed a 5-fold increase of carbohydrates within the extracted EPS. The amount of proteins was even 19-fold higher compared to the control without butanol, indicating that EPS formation in biofilms is significantly increased in the presence of toxic solvent concentration. Beside the influence of 1-butanol on changes in biofilm morphology and EPS formation, the response of S. acidocaldarius towards changes in lifestyle and 1-butanol was investigated by transcriptomic and proteomic studies. For this, S. acidocaldarius was cultivated as shaking culture in Erlenmeyer flasks without 1-butanol exposure and statically in Petri-dishes for biofilm and planktonic growth in the absence and presence of 1-butanol. Statically grown biofilm and planktonic cells in Petri-dishes showed only less differentially regulated genes and proteins. Lots of differentially regulated genes and proteins were found in cells of shaking cultures and statically incubated planktonic cells. In the presence of 1% (v/v) 1-butanol, genes and proteins are predominantly down-regulated, especially proteins containing transmembrane helices, confirmed by transcriptomic analyses. In planktonic as well as biofilm cells in the presence of 1-butanol, the archaellum operon, responsible for cell motility, was down-regulated. Generally, in response to 1-butanol and different lifestyles, changes in cell division and vesicle formation, stress response systems like the CRISPR-Cas and toxin-antitoxin system, transcriptional regulators and protein kinases and phosphatases (signal transduction) were observed. In conclusion, this study gives an overview of the stress response of S. acidocaldarius towards organic solvents and 1-butanol in detail. For the first time the influence of organic solvents on biofilm formation was described and investigated by various microscopic methods and EPS isolation and quantification. In addition, transcriptomic and proteomic studies were performed to investigate genome-wide and cellular responses towards different lifestyles and 1-butanol.