Methodology development for environmental microbiology: Medium formulation and surface charge analysis
Creators: name: Ng, Wenfa
Publisher: name: figshare
Year: 2016
Subjects: subject: Biological Engineering
subject: 60501 Bacteriology
subject: FOS: Biological sciences
subject: FOS: Biological sciences
subject: Biological Techniques
subject: Biotechnology

description: Lack of adequate cell mass hampers many fields allied to biotechnology. Besides providing sufficient material for ensuring minimum levels of analytical accuracy, high cell mass buttresses against batch-to-batch variation in analysis as well as enabling proof-of-concept experiments at larger scales. Conventional methods for increasing cellular material revolves around scaling up fermentation using increasing sizes of bioreactors; however, high cost and complexity of operation precludes the widespread adoption of fermenters in universities laboratories, where the workhorse for cell production and experimentation is the shake-flask. While useful and amenable to linear scale-up through parallel cultivation, shake-flask’s main bugbear lies in the relatively low cell density achievable during cultivation of various bacteria in commercial growth media. Why? Strong indications suggest that reliance on fed-batch culture with provision of supplemental carbon sources reduce motivation for developing growth medium suitable for batch culture. This thesis reports a formulated medium capable of providing 3 times higher optical density for <i>Escherichia coli</i> DH5α (ATCC 53868) compared to LB Lennox medium under aerobic culture at 37 <sup>o</sup>C and 230 rpm. Cell count data via the aerobic spread plate method further reveals that the formulated medium was able to support a larger population of viable cells over a longer period of time relative to Tryptic soy broth. Provision of large number of viable cells, however, solves only one part of the “materials” problem for many areas of applied and environmental microbiology. What remains is the more important problem of ensuring the cells have characteristics suited for investigating the specific angle of a problem. Using the field of colloid science in general, and the more specific subfield of bacterial cell surface characterization and its applications in water and wastewater treatment as an example, the second part of the thesis focuses on evaluating the efficacy of various wash buffers in removing non-specifically adsorbed ions and biomolecules from the surface of <i>Escherichia coli</i> DH5α, with zeta potential-pH profile as proxy readout. Experimental results reveal and confirms a deeper truth about experimental design: the need to choose the appropriate wash buffer for preparing the cell surface for the specific solvent conditions that the cells would be used in. For example, results strongly suggest that sodium citrate is capable of removing both the nonspecifically adsorbed and native ions from the cell surface or envelope, the latter set of ions being important for maintaining cell membrane and envelope stability. Hence, essentiality of using sodium citrate as wash buffer rests on the target conditions at which the cells would be used. Microbes, microscopic though they may be, can be visualized easily via the solid medium culture method (primitive but important), and by the quintessential technique of cell biology: microscopy. However, current commercialized solid agar media hamper the direct visualization of specific colonies on agar, especially if their pigmentation has low colour contrast with the agar’s colour. Scanning across the colour spectrum, no colour, except colourless, is suitable for providing the “base colour” necessary for increasing the colour contrast between colonies and agar. In achieving the goal of developing a simple and effective method for preparing colourless agar, the problem of generation of coloured compounds from the heat-induced reaction between glucose and nitrogen-containing compounds was verified and its resolution enabled by the separation of glucose and nitrogen sources in separate solutions. After autoclave sterilization and cooling to ~48 <sup>o</sup>C, the two solutions were mixed for reconstituting the agar medium, which when poured into standard 100 mm Petri dishes, delivered agar of near homogenous consistency and high optical transparency. For culturing microbes deficient in vitamin biosynthesis, yeast extract (up to 1 g/L) could be added during agar reconstitution, and continue to yield a colourless agar medium. Inoculation of deionized water (freshly prepared by filter and ion-exchange resins treatment) on the colourless agar generated observations indicative of the need to conduct regular maintenance of the deionized water production system given the plethora of colonies observed (but not identified via 16S rRNA sequencing). The observation also speaks of the importance of autoclave sterilization (121 <sup>o</sup>C and 14.7 psi above air pressure at sea level) in ensuring sterility in prepared medium, which points to regular upkeep (maintenance and cleaning) of autoclave system as critical to quality control of experiments. More importantly, presence of microbes in deionized water meant that all labs (biology or non-biology oriented) need to account for possible interference of viable microorganisms in their experiments; for example, slight increase in ionic strength due to cell lysis leading to local fluctuations in ion concentration that manifests into unpredictability of crystallization conditions.