Thunyarat Pongtharangkul

Bacteria Interface Pickering Emulsions Stabilized by Self-assembled Bacteria–Chitosan Network

An oil-in-water Pickering emulsion stabilized by biobased material based on a bacteria-chitosan network (BCN) was developed for the first time in this study. The formation of self-assembled BCN was possible due to the electrostatic interaction between negatively charged bacterial cells and polycationic chitosan. The BCN was proven to stabilize the tetradecane/water interface, promoting formation of highly stable oil-in-water emulsion (o/w emulsion). We characterized and visualized the BCN stabilized o/w emulsions by scanning electron microscopy (SEM) and laser scanning confocal microscopy (LSCM). Due to the sustainability and low environmental impact of chitosan, the BCN-based emulsions open up opportunities for the development of an environmental friendly new interface material as well as the novel type of microreactor utilizing bacterial cells network.

Evaluation of culture medium for nisin production in a repeated-batch biofilm reactor.

In this study, a biofilm reactor with plastic composite support (PCS), made by high‐temperature extrusion of agricultural products and polypropylene, was evaluated for nisin production using L. lactisstrain NIZO 22186. The high‐biomass density of the biofilm reactor was found to contribute to a significantly shorter lag time of nisin production relative to a suspended‐cell reactor. In comparison to glucose (579 IU/mL), sucrose significantly increased the nisin production rate by 1.4‐fold (1100 IU/mL). However, results revealed that high levels of sucrose (8% w/v) had a suppressing effect on nisin production and a stimulating effect on lactic acid production. A high concentration of MgSO4·7H2O at 0.04% (w/v) was found to reduce the nisin production, while concentrations of KH2PO4 of up to 3% (w/v) did not have any significant effect on growth or nisin production. The best of the tested complex media for nisin production using the PCS biofilm reactor consisted of 4% (w/v) sucrose, 0.02% (w/v) MgSO4·7H2O, and 0.1% (w/v) KH2PO4. Nisin production rate in the biofilm reactor was significantly increased by 3.8‐fold (2208 IU/mL) when using the best complex medium tested.

Characterization of an organic‐solvent‐tolerant Brevibacillus agri strain 13 able to stabilize solvent/water emulsion

Brevibacillus agri strain 13 was isolated and characterized as a Gram-positive organic-solvent-tolerant bacterium able to grow at 45 degrees C. It can tolerate high concentrations (5% and 20%, v/v) of various organic solvents with a broad range of log P(ow) when the organic solvent was provided as a nonaqueous layer. Although it can tolerate a number of aromatic solvents, it cannot utilize them as a sole carbon source. The surface characteristics of cells exposed to organic solvent were investigated using the bacterial adhesion to hydrocarbon test, a contact angle measurement, zeta potential determination, and fluorescence microscopy analysis and compared with that of nonexposed cells. The results showed that although it has a hydrophilic cell surface, it has a unique indigenous cell surface characteristic in which the cells can stabilize solvent-in-water emulsion by adhering to the solvent-water interface of the solvent droplets. The tolerance and predilection of B. agri strain 13 toward organic solvents may suggest its potential application as a whole-cell biocatalyst for the biotransformation process of water-immiscible substrate(s).

Construction of CoA-dependent 1-butanol synthetic pathway functions under aerobic conditions in Escherichia coli

1-Butanol is an important industrial platform chemical and an advanced biofuel. While various groups have attempted to construct synthetic pathways for 1-butanol production, efforts to construct a pathway that functions under aerobic conditions have met with limited success. Here, we constructed a CoA-dependent 1-butanol synthetic pathway that functions under aerobic conditions in Escherichia coli, by expanding the previously reported (R)-1,3-butanediol synthetic pathway. The pathway consists of phaA (acetyltransferase) and phaB (NADPH-dependent acetoacetyl-CoA reductase) from Ralstonia eutropha, phaJ ((R)-specific enoyl-CoA hydratase) from Aeromonas caviae, ter (trans-enoyl-CoA reductase) from Treponema denticola, bld (butylraldehyde dehydrogenase) from Clostridium saccharoperbutylacetonicum, and inherent alcohol dehydrogenase(s) from E. coli. To evaluate the potential of this pathway for 1-butanol production, culture conditions, including volumetric oxygen transfer coefficient (kLa) and pH were optimized in a mini-jar fermenter. Under optimal conditions, 1-butanol was produced at a concentration of up to 8.60gL(-1) after 46h of fed-batch cultivation.

Optimization of zofimarin production by an endophytic fungus, Xylaria sp. Acra L38

To optimize the medium for high zofimarin production, sucrose maltose, glucose, tryptone and peptone were used in an orthogonal array design experiment, where the highest value of zofimarin produced was 25.6 μg/mL. This value was about 3 times higher than that obtained with Czapek yeast extract (CzYE) culture medium. A study with Plackett-Burman design showed that sucrose, maltose, glucose and NaNO3 were significant factors in zofimarin production. Further studies using central composite design (CCD) showed the significance of glucose and the interactions of these critical components affecting zofimarin production. Multiple regression analysis of the data yielded a poor fit as shown by the mismatch of the model with these variable factors. When a polynomial equation was applied, the maximum zofimarin production was predicted to be 201.9 μg/mL. Experimental verification yielded a much lower amount of zofimarin, at around 70 μg/mL. Reconsideration of the CCD data and repetition of some runs with high zofimarin production resulted in reproducible zofimarin yield at 79.7 μg/mL. Even though the amount was lower than the predicted value, the medium optimization study was considered to be quite successful as the yield increased to around 8 times that obtained with the original CzYE culture medium.

BTEX- and naphthalene-degrading bacterium Microbacterium esteraromaticum strain SBS1-7 isolated from estuarine sediment

In this study, a non-pathogenic, BTEX-degrading Microbacterium esteraromaticum SBS1-7 was isolated from estuarine sediment in Thailand via an enrichment technique. M. esteraromaticum SBS1-7 was able to degrade all six BTEX components, in both liquid medium and soil slurry system, when BTEX was supplied as an individual component or a mixture. It exhibited a high level of tolerance towards a wide range of hydrocarbons and also utilized alkanes and naphthalene. Detection of metabolites produced during BTEX and naphthalene degradation revealed highly extensive biodegradation pathways used by M. esteraromaticum SBS1-7. Toluene was metabolized via activities of both monooxygenase (toluene 4-monooxygenase or T4MO) and dioxygenases (toluene dioxygenase or TDO and naphthalene 1,2-dioxygenase or NDO). Benzene was metabolized via phenol, possibly by an activity of T4MO. Ethylbenzene was converted into styrene and 1-phenethyl alcohol by a well-documented activity of NDO. Dioxidation of ethylbenzene, possibly by ethylbenzene dioxygenase or EBDO, was also found. All xylene isomers were converted into their corresponding alcohols via an activity of NDO while naphthalene was metabolized via dioxidation reaction by the same enzyme. This study is, by far, the first direct evidence of BTEX biodegradation by a non-pathogenic, rhizosphere bacterium M. esteraromaticum.

Turning hydrophilic bacteria into biorenewable hydrophobic material with potential antimicrobial activity via interaction with chitosan

Alteration of a bacteriocin-producing hydrophilic bacterium, Lactococcus lactis IO-1, into a hydrophobic material with potential antimicrobial activity using chitosan was investigated and compared with five other bacterial species with industrial importance. The negatively charged bacterial cells were neutralized by positively charged chitosan, resulting in a significant increase in the hydrophobicity of the bacterial cell surface. The largest Gram-positive B. megaterium ATCC 14581 showed a moderate response to chitosan while the smaller E. coli DH5α, L. lactis IO-1 and P. putida F1 exhibited a significant response to an increase in chitosan concentration. Because L. lactis IO-1 is a good source for natural peptide lantibiotic that is highly effective against several strains of food spoilage organisms and pathogens, hydrophobic material derived from L. lactis IO-1 and chitosan is a promising novel material with antimicrobial activity for the food and pharmaceutical industries.

Optimization of medium and pH control profile in biofilm fermentation for nisin production

In this study, biofilm reactor with Plastic Composite Support (PCS), made by high temperature extrusion of agricultural products and polypropylene, has been evaluated on nisin production using L. lactis NIZO 22186. High-biomass density characteristic of biofilm reactor was found to contribute in a significantly shorter lag time of nisin production. In comparison to glucose, sucrose significantly increased nisin production rate by 1.4-fold. However, obtained data also revealed a suppressing effect of high level of sucrose on nisin production and its stimulating effect on lactic acid production. High concentration of MgSO4.7H2O at 0.04% was found to reduce the nisin production, while concentration of KH2PO4 of up to 3% did not have any significant effect on growth or nisin production. The optimal complex media for nisin production using PCS biofilm reactor consisted of 4% sucrose, 0.02% MgSO4.7H2O, and 0.1% KH2PO4. Nisin production rate in biofilm reactor was significantly increased by 3.8-fold when using an optimized medium. Higher nisin could be obtained with a simple adjustment in the pH-control profile by allowing the pH to drop freely via autoacidification after 4 h of fermentation.

Hydroxypropyl methylcellulose enhances the stability of o/w Pickering emulsions stabilized with chitosan and the whole cells of Lactococcus lactis IO-1

Hydroxypropyl methylcellulose (HPMC) was used as a co-emulsifier with chitosan and the whole cells of bacteriocin-producing Lactococcus lactis IO-1 (L. lactis IO-1) for the preparation of bacteria interface Pickering emulsion. The obtained emulsion exhibited a high stability against centrifugation force, ionic strength and low temperature, with the whole cells of L. lactis IO-1 located at the oil/water interface. Because L.lactis IO-1 was found to produce peptide lantibiotic against several strains of Gram-positive food pathogen, the highly stable emulsion demonstrated in this study exhibited high potential as an antimicrobial emulsion with several health benefits of chitosan and HPLC useful for food industry.

Engineering of Corynebacterium glutamicum as a prototrophic pyruvate-producing strain: Characterization of a ramA-deficient mutant and its application for metabolic engineering

ABSTRACT To construct a prototrophic Corynebacterium glutamicum strain that efficiently produces pyruvate from glucose, the effects of inactivating RamA, a global regulator responsible for activating the oxidative tricarboxylic acid (TCA) cycle, on glucose metabolism were investigated. ΔramA showed an increased specific glucose consumption rate, decreased growth, comparable pyruvate production, higher formation of lactate and acetate, and lower accumulation of succinate and 2-oxoglutarate compared to the wild type. A significant decrease in pyruvate dehydrogenase complex activity was observed for ΔramA, indicating reduced carbon flow to the TCA cycle in ΔramA. To create an efficient pyruvate producer, the ramA gene was deleted in a strain lacking the genes involved in all known lactate- and acetate-producing pathways. The resulting mutant produced 161 mM pyruvate from 222 mM glucose, which was significantly higher than that of the parent (89.3 mM; 1.80-fold). Graphical Abstract Engineering of Corynebacterium glutamicum as a prototrophic pyruvate-producing strain: Characterization of a ramA-deficient mutant and its application for metabolic engineering