Formulation of high cell density medium for aerobic cultivation of Escherichia coli DH5α
Obtaining high cell yield of uniform characteristics is a key requirement for many areas of applied microbiology research where there is a need to perform large scale characterization and performance tests of the biomass under different scenarios. However, biomass yield from many commercially available growth media remained poor; thereby, resulting in batch-to-batch variation in cell characteristics. Using Escherichia coli DH5α as a model organism, this research seeks to develop a liquid growth medium capable of high cell density aerobic cultivation of the bacterium. Results revealed the formulated medium was capable of increasing the optical density of the culture broth by more than 3 times compared to LB Lennox medium without the generation of growth inhibitory compounds during cultivation. Obtained large population of cells could also be maintained for a longer period compared to commercially available medium such as Tryptic Soy Broth. Additionally, a high capacity phosphate buffer system helped modulate fluctuations in pH during cell cultivation which improved biomass formation. Collectively, a formulated medium capable of high cell density cultivation of E. coli DH5α was developed.
Representative publications
Wenfa Ng and Yen-Peng Ting, “Formulation of a semi-defined medium for high cell density cultivation of Escherichia coli in shake flasks”, PeerJ Preprints, Link to preprint
Wenfa Ng, “High cell density cultivation of Escherichia coli DH5α in shake flasks with a new formulated medium”, PeerJ Preprints, Link to preprint
Effect of wash buffers on zeta potential analysis of bacterial cells
Bacterial cells in contact with a solution such as a growth medium would inevitably adsorb ions and metabolites from the solution. Such nonspecific adsorption of ions and metabolites could thus confer additional electric charges to the cell surface and masks the real surface charge. Thus, various wash buffers of different ionic strength have been used in repeated steps of centrifugation and washing for removing nonspecifically adsorbed ions and metabolites from bacterial cell surface. However, their relative efficacy remained poorly understood. Thus, the goal of this project was in using a systematic study to understand the relative efficacies of different wash buffers in removing nonspecifically adsorbed ions and metabolites from the cell surface. Experiment results from the model system of Escherichia coli DH5α grown in two different growth media that differed in salt content revealed that high ionic strength of wash buffers was correlated with greater removal of nonspecifically adsorbed ions and metabolites. More importantly, a threshold exists beyond which high ionic strength wash buffers could remove ions intrinsic to the cell envelope and thereby alter cell surface characteristics. Depending on the cell wall structure and types and amounts of ions and metabolites in the growth medium, differing amounts of ions and metabolites would nonspecifically adsorb to the cell surface. Thus, the threshold ionic strength for removing nonspecifically adsorbed ions and metabolites is likely bacterial species and growth medium specific. In the case of E. coli DH5α grown in LB Lennox + 2g/L glucose and a formulated medium, the threshold ionic strength was 0.15M (e.g., 9 g/L sodium chloride wash buffer). High ionic strength wash buffers such as 0.6M sodium chloride and 0.1M sodium citrate would result in removal of ions intrinsic to the cell envelope that manifested as changes to the point of zero charge (pHzpc) of the cells. More importantly, the research also revealed that citrate ions could adsorb onto the cell surface and thus wash buffers with high concentration of citrate should not be used in preparing cell samples for zeta potential analysis. Finally, using wash buffer of 0.15M ionic strength (i.e., 9 g/L sodium chloride), the research possibly revealed the real surface charge of E. coli DH5α grown in the two growth media.
Representative publications
Wenfa Ng and Yen-Peng Ting, “Bacterial surface charge in “layers”: revealed by wash buffers of different ionic strength”, PeerJ Preprints, Link to preprint
Wenfa Ng and Yen-Peng Ting, “Zeta potential of bacterial cells: Effect of wash buffers”, PeerJ Preprints, Link to preprint
Colourless agar for enhanced colour contrast between agar and microbial colonies
Solid medium used for the cultivation of microorganisms are typically beige in colour. Given that many microbial colonies are also similar in colour, there is a lack of colour contrast necessary for easy and accurate identification of small microbial colonies on agar plates. Thus, the objective of this research is the development of a colourless agar which when coupled with coloured paper of suitable hue would help increase colour contrast between microbial colonies and agar background. However, reaction between glucose and ammonium compounds would generate coloured compounds during autoclave heat sterilization. By separately sterilizing glucose and ammonium compounds, this project developed a simple protocol for the preparation of colourless agar that remained colourless even with the addition of yeast extraction at 1 g/L. Growth experiments revealed that the colourless agar could cultivate common bacteria such as Escherichia coli, Bacillus subtilis, Pseudomonas protegens, and Pseudomonas aeruginosa to relatively high cell densities. In addition, the colourless agar could also support cell concentration common in viable cell counting of microbes, which opens up possibility in using the colourless agar together with automated image analysis for colony identification and counting. In assessing the microbial flora of deionized and tap water, the colourless agar was also able to support a large variety of different microorganisms. Specifically, colonies of different morphologies, size and pigmentation could be observed on the colourless agar where types of microbes cultivated positively correlated with yeast extract concentration. Overall, a method was developed for the preparation of colourless agar that lends a useful tool for automated image analysis of microbial colonies where accurate identification of colonies occurred through enhanced colour contrast between colonies and agar background.
Representative publications
Wenfa Ng, “Colourless agar for enhancing colour contrast between microbial colonies and agar”, PeerJ Preprints, Link to preprint
Wenfa Ng, “Microbes in deionized and tap water: Implications for maintenance of laboratory water production system”, PeerJ Preprints, Link to preprint
Ammonium interference in adsorption of copper by formaldehyde crosslinked seaweed from low concentration solutions
Ammonium ions co-exist with heavy metal ions in many environmental water matrixes and could possibly influence the removal of the heavy metal ions by various water treatment techniques. One such possibility is adsorption on seaweed such as Sargassum sp. which has been shown to be useful for adsorbing a variety of heavy metals such as copper, lead, cadmium and chromium. Given that ammonium ions exist as positively charged NH4+ ions at circumneutral pH of around 5-6, they could possibly interfere with the adsorption of cationic heavy metals on seaweed. Thus, the objective of this project is to understand the possible interference effect of ammonium ion on the adsorption of copper from low concentration solutions (4-20 ppm Cu2+) by formaldehyde crosslinked Sargassum sp. (modified seaweed). Modification of seaweed through formaldehyde crosslinking helped reduced organic leaching but the residual leaching remained at 4 ppm total organic carbon. Batch kinetic and equilibrium experiment results revealed that ammonium ion interfered with copper adsorption in a concentration-dependent manner. Specifically, beyond a threshold concentration of 50 ppm NH4+-N, ammonium ion reduced the uptake capacity of modified seaweed as well as increased the final equilibrium copper concentration. Overall, ammonium ion interfered with the uptake of copper by modified seaweed through possibly competing for the same functional groups on the seaweed surface. In addition, leaching of organics from the modified seaweed meant that the biosorbent could not be used in drinking water treatment applications.
Representative publication
Wenfa Ng, “Ammonium interference reduced copper uptake by formaldehyde-crosslinked Sargassum seaweed”, PeerJ Preprints, Link to preprint
Feasibility of formaldehyde-crosslinked Sargassum sp. seaweed in adsorbing cationic dyes
Utilization of waste resource for beneficial uses has been a dominant theme in environmental protection in the past decades. One effort in this direction is the use of local seaweed for the adsorption of heavy metals in industrial wastewater treatment. This comes about due to the favourable electrostatic interactions between negatively charged seaweed cell surface and cationic heavy metal ions. But, could use of seaweed in environmental remediation be expanded to organic molecules such as dyes? An initial study in this direction confirmed that Sargassum sp. seaweed obtained from the coast of Singapore could adsorb cationic but not anionic dyes. Further tests elucidated that formaldehyde crosslinking enhanced seaweed biosorption performance for the model cationic dye, Basic Yellow 11 compared to untreated seaweed or those presaturated with H+ ions. Systematic experimentation further deduced that the seaweed biosorbent is sensitive to ionic strength from sodium chloride, and competitive binding effect from copper and nickel ions. The latter is of particular relevance given the presence of copper and nickel heavy metal ions in wastewater from dyeing operations. Finally, mechanistic studies with seaweed preadsorbed with sodium, potassium, magnesium and calcium ions strongly suggest that ion exchange played a major role in mediating adsorption of Basic Yellow 11 onto binding sites on the seaweed surface.
Representative publication
Wenfa Ng, “Evaluating the feasibility of sorption of Basic Yellow 11 on Sargassum sp. marine seaweed”, figshare, Link to presentation
Developing ethanol-based NADH regeneration in Escherichia coli expressing ethanol utilization pathway
Many biotransformation reactions of relevance to industry are catalyzed by oxidoreductase that require expensive cofactors for proper enzymatic function. Such cofactor either need to be supplied stoichiometrically or could be regenerated by a second cofactor regeneration reaction coupled to the main biotransformation reaction. Efforts in NADH regeneration for biocatalysis have mainly revolved around glucose dehydrogenase (GDH), but the system suffers from low NADH yield per glucose molecule. This project sought to explore the utility of a two-gene ethanol utilization pathway (EUP) in enabling regeneration of NADH in support of biocatalytic reactions. Able to regenerate two NADH per ethanol molecule, EUP is theoretically more efficient than GDH, but experimental tests are needed to verify predicted efficiency and help identify bottlenecks that hamper greater NADH regeneration. Results in resting cells experiment confirmed that EUP was more efficient than GDH in supporting a ketone to alcohol biotransformation in Escherichia coli. More importantly, with the EUP pathway terminating in acetyl-CoA, growing cells was shown to help generate a driving force that pull carbon flux from ethanol to acetyl-CoA, which helped regenerate more NADH in support of biotransformation reaction. Despite the promising results, further engineering of EUP is needed to help solve a deficiency in ethanol substrate consumption under anaerobic conditions that prevents the full potential of the system in cofactor regeneration from being exploited.
Representative publications
Wenfa Ng, “Characterizing ethanol-sensitive promoters and understanding the evolutionary forces acting on them in common biotechnology chassis”, figshare, Link to preprint
Wenfa Ng, “Developing an ethanol responsive regulon in Escherichia coli for biotechnology applications”, figshare, Link to preprint
Wenfa Ng, “Developing an ethanol utilisation pathway based NADH regeneration system in Escherichia coli”, bioRxiv, Link to preprint
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