Bildung, Isolierung und Nachweis der mikrobiell gebildeten Speicherstoffe Polyphosphat und Polyhydroxybuttersäure bei ausgewählten Mikroorganismen

  • Formation, isolation and proof of the microbes produced storage substances polyphosphate and polyhydroxybutyric with selected microorganisms

Schacht, Petra Yvonne; Dott, Wolfgang (Thesis advisor); Blank, Lars Mathias (Thesis advisor)

Aachen (2016)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2016

Abstract

The present paper is aimed at investigating to what extent the production of the microbial reserve substances polyphosphate and polyhydroxybutyrate can be enhanced and whether they can be isolated. In addition, the possibility of utilising these substances beyond their biological function in the field of environmentally friendly products and technologies is addressed. Owing to the comprehensive method development and the examination of both reserve substances, the present paper consists of three parts, namely methods of detecting the polymer compounds polyphosphate and polyhydroxybutyrate, the microbial production of polyphosphate and the microbial production of polyhydroxybutyrate. Laboratory-scale test series were performed to produce the reserve substances polyphosphate and polyhydroxybutyrate through selective enrichment of microorganisms. The methods for the isolation as well as the qualitative and quantitative detection of the reserve substances were developed and/or established. The produced polyphosphates were detected qualitatively and quantitatively by means of microscopy, fluorescence photometry and gel electrophoresis. The polyphosphates were quantified after extraction by measurement as orthophosphate. Due to a lack of uniform standards, however, the detection methods involve methodological difficulties regarding the determination of the individual chain lengths of the polyphosphates and their quantification, which is why the results should be seen with a critical eye and the detection methods require optimisation. Nevertheless, a simultaneous look at the process flow of all employed detection methods reveals consistent results, which is why the combination of the employed methods can be regarded as purposeful. The produced polyhydroxybutyrate was isolated from the cell and detected qualitatively and quantitatively. The employed detection methods included microscopy, enzyme-mediated assay and gas chromatography. The detection methods for polyhydroxybutyrate yielded satisfactory results, given the availability of suitable standard substances. The phosphate uptake and phosphate accumulation was examined on the basis of the microorganisms in sewage sludge, giving special regard to the fungus species Cunninghamella elegans. It was found that phosphate is accumulated in the cell predominantly as polyphosphate through the activity of microorganisms. At unfavourable culture conditions, such as a change in the pH value, directly or indirectly induced precipitation reactions, such as the chemical precipitation of struvite (magnesium ammonium phosphate) occurred additionally. The carbon/nitrogen/phosphorus ratio in the employed medium was taken as a basis for the assessment of the test results. Compared to the microorganisms in the sewage sludge, the fungus species Cunninghamella elegans was found to be capable of removing from the medium and accumulating in the cell as polyphosphate the tenfold amount of phosphate. Cunninghamella elegans was able to fix a maximum of 10% phosphate in the biomass, whereas the sewage sludge biocoenosis only fixed 3% of phosphate at maximum. Cunninghamella elegans produced a promising biomass yield and accumulated large amounts of phosphate within a short period of time. However, the technical utilisation of polyphosphate production is impracticable due to the necessity of separating the polyphosphates from the biomass, which is not possible without hydrolysis. The microbial production and accumulation of polyhydroxybutyrate was examined on the basis of the bacterial species Paracoccus denitrificans and Cupriavidus necator. Despite varying culture conditions and very high carbon consumption, it was not possible to achieve a high yield of biomass and polyhydroxybutyrate, which is why the method requires optimisation. The highest yield of 3-hydroxybutyrate with Paracoccus denitrificans and Cupriavidus necator amounted to 50% of the dry weight at maximum. In view of the test results achieved at laboratory scale, a transfer to industrial scale should be considered and investigated. The economical utilisation of the reserve substances requires a high cell density of microorganisms as well as a high yield. The tested microorganisms were therefore selected individually for each reserve substance. The further processing of industrial wastewater appears to be a starting point for reducing the production costs of polymers. As part of the tests, industrial process water was added to the employed media as a source of phosphate. Despite some progress in the research into reserve substances, the isolation of the produced reserve substances from microorganisms and their quantification is still difficult, since losses may occur in the course of hydrolysis.At present, the production and extraction of the microbial reserve substances polyphosphate and polyhydroxybutyrate is not yet economical and the method’s technical feasibility is limited.