Biosynthesis of Polyhydroxyalkanoate Terpolymer from Methanol via the Reverse β-Oxidation Pathway in the Presence of Lanthanide

Methylorubrum extorquens AM1 is the attractive platform for the production of value-added products from methanol. We previously demonstrated that M. extorquens equipped with PHA synthase with broad substrate specificity synthesized polyhydroxyalkanoates (PHAs) composed of (R)-3-hydroxybutyrate and small fraction of (R)-3-hydroxyvalerate (3HV) and (R)-3-hydroxyhexanoate (3HHx) units on methanol. This study further engineered M. extorquens for biosynthesis of PHAs with higher 3HV and 3HHx composition focusing on the EMC pathway involved in C1 assimilation. The introduction of ethylmalonyl-CoA decarboxylase, catalyzing a backward reaction in the EMC pathway, aiming to increase intracellular propionyl/butyryl-CoA precursors did not affect PHA composition. Reverse b-oxidation pathway and subsequent (R)-specific hydration of 2-enoyl-CoA were then enhanced by heterologous expression of four genes derived from Ralstonia eutropha for the conversion of propionyl/butyryl-CoAs to the corresponding (R)-3-hydroxyacyl-CoA monomers. The resulting strains produced PHAs with higher 3HV and 3HHx compositions, while the methylotrophic growth was severely impaired. This growth impairment was interestingly restored by the addition of La3+ without a negative impact on PHA biosynthesis, suggesting the activation of the EMC pathway by La3+. The engineered M. extorquens synthesized PHA terpolymer composed of 5.4 mol% 3HV and 0.9% of 3HHx with 41% content from methanol as a sole carbon source in the presence of La3+.

Biosynthesis of Poly-(3-hydroxybutyrate) under the Control of an Anaerobically Induced Promoter by Recombinant Escherichia coli from Sucrose

Poly-(3-hydroxybutyrate) (PHB) is a polyester with biodegradable and biocompatible characteristics and has many potential applications. To reduce the raw material costs and microbial energy consumption during PHB production, cheaper carbon sources such as sucrose were evaluated for the synthesis of PHB under anaerobic conditions. In this study, metabolic network analysis was conducted to construct an optimized pathway for PHB production using sucrose as the sole carbon source and to guide the gene knockout to reduce the generation of mixed acid byproducts. The plasmid pMCS-sacC was constructed to utilize sucrose as a sole carbon source, and the cascaded promoter P3nirB was used to enhance PHB synthesis under anaerobic conditions. The mixed acid fermentation pathway was knocked out in Escherichia coli S17-1 to reduce the synthesis of byproducts. As a result, PHB yield was improved to 80% in 6.21 g/L cell dry weight by the resulted recombinant Escherichia coli in a 5 L bed fermentation, using sucrose as the sole carbon source under anaerobic conditions. As a result, the production costs of PHB will be significantly reduced.

Characterization of Morganella sp. for its Paraquat Degradation Potential

One of the beneficial roles of the microbial population is their ability to convert toxic herbicides to lesser toxic compounds such as water and carbon (IV) oxide. Paraquat which is an acutely toxic herbicide is used on farmlands and has been found to affect human health. This study was aimed at characterizing bacteria with the potential to degrade paraquat. Previously isolated bacteria from culture collection labelled A-F were screened for their potential to degrade and utilized paraquat as the sole carbon source in Bushnell Hass agar media. Of the six isolates, isolate E (Morganella sp.) was observed to have the highest growth and tolerance to paraquat after 72 h of incubation at 37 ºC. Characterization study revealed that Morganella sp. can utilize and grow with optimum conditions of pH 6.5, the temperature of 30 ºC and can tolerate up to 400 mg/L paraquat concentration with an increase in growth as inoculum size increases. Thus, these findings showed that Morganella sp. can degrade toxic paraquat to a less toxic form and therefore can be a good isolate for the future bioremediation process of the pollutant.

Isolation and Characterization of Biosurfactant-producing Alcaligenes sp. YLA11 and its Diesel Degradation Potentials

This study aimed to isolate and identify biosurfactant producing and diesel alkanes degrading bacteria. For this reason, bacteria isolated from the diesel contaminated site were screened for their potential to produce biosurfactants and degrade diesel alkanes. Primary selection of diesel degraders was carried out by using conventional enrichment culture technique where 12 bacterial strains were isolated based on their ability to grow on minimal media supplemented with diesel as sole carbon source, which was followed by qualitative screening methods for potential biosurfactant production. Isolate B11 was the only candidate that shows positive signs for drop collapse, foaming, haemolytic test, oil displacement of more than 22 ± 0.05 mm, and emulsification (E24) of 14 ± 0.30%. The effect of various culture parameters (incubation time, diesel concentration, nitrogen source, pH and temperature) on biodegradation of diesel was evaluated. The optimum incubation time was confirmed to be 120 days for isolates B11, the optimum PH was confirmed as 8.0 for the isolate, Similarly, the optimum temperature was confirmed as 35oC. In addition, diesel oil was used as the sole carbon source for the isolates. The favourable diesel concentration was 12.5 % (v/v) for the isolate. The isolate has shown degradative ability towards Tridecane (C13), dodecane, 2, 6, 10-trimethyl- (C15), Tetradecane (C14), 2,6,10-Trimethyltridecane (C16), Pentadecane (C15). It degraded between 0.27% - 9.65% individual diesel oil alkanes. The strain has exhibited the potential of degrading diesel oil n-alkanes and was identified as Alcaligenes species strain B11 (MZ027604) using the 16S rRNA sequencing.

Characterization of Aniline Degradation by A Previously Isolated Molybdenum-reducing Pseudomonas sp.

Microorganisms play an integral role in detoxification and removal of toxic compounds from the environment. Aniline is the simplest aromatic amine, consisting of a phenyl group attached to an amino group that is used as herbicide to control weeds. Aniline is detrimental to both environment and health. In this research, six previously isolated bacteria (isolate A-F) were screened on Bushnell Hass media for their potential to grow and utilize aniline as a sole carbon source. Isolate A (Pseudomonas sp.) was found to tolerate and grow best with aniline sole source of carbon. Optimum conditions for aniline degradation by this isolate were found to be pH 6.0, temperature between 30 and 37 °C, inoculums size of 600 μL, aniline concentration of 200 mg/L and incubation time of 96 h. The capacity of this isolate to reduce toxic aniline to less toxic form is novel and makes the bacterium important instrument for bioremediation of this pollutant.

Characterization of Butachlor Degradation by A Molybdenum-Reducing and Aniline-degrading Pseudomonas sp.

Butachlor is a chloroacetanilide herbicide that is selective in action and commonly used for pre-emergence control of weeds. It is believed to have range of toxicity from acute to chronic and also can cause DNA strand breaks and chromosomal aberrations in humans. This study was aimed at characterizing the potential of previously isolated bacteria for butachlor degradation. The isolates from culture collection, labelled A-F were screened for butachlor degradation on Bushnell Hass agar media with butachlor as a sole carbon source. Isolate A (molybdenum-reducing and aniline-degrading Pseudomonas sp.) was observed to grow best and tolerated the highest concentration of butachlor supplemented in the media after 72 h of incubation at 37 ℃. Characterization study revealed that the Pseudomonas sp. can utilize and grow with butachlor at optimum pH between 6.0 - 6.5, temperature between 30 – 37 ℃ and can tolerate up to 600 mg/L butachlor concentration with increase in growth as inoculum size increases. Additionally, this bacterial strain shows no lag phase regardless of the concentration of the herbicide used and reach its maximum growth after 24 h of incubation. The ability of this isolate to tolerate and utilize butachlor as sole carbon source makes it suitable for future bioremediation of this toxicant.

The Overexpression of YALI0B07117g Results in EnhancedErythritol Synthesis from Glycerol by the Yeast Yarrowia lipolytica

The unconventional yeast Yarrowia lipolytica is used to produce erythritol from glycerol. In this study, the role of the erythrose reductase (ER) homolog YALI0B07117g in erythritol synthesis was analyzed. The deletion of the gene resulted in an increased production of mannitol (308%) and arabitol (204%) before the utilization of these polyols began. The strain overexpressing the YALI0B07117g gene was used to increase the erythritol yield from glycerol as a sole carbon source in batch cultures, resulting in a yield of 0.4 g/g. The specific consumption rate (qs) increased from 5.83 g/g/L for the WT strain to 8.49 g/g/L for the modified strain and the productivity of erythritol increased from 0.28 g/(L h) for the A101 strain to 0.41 g/(L h ) for the modified strain. The application of the research may prove positive for shortening the cultivation time due to the increased rate of consumption of the substrate combined with the increased parameters of erythritol synthesis.

A biosurfactant-producing yeast Rhodotorula sp.CC01 utilizing landfill leachate as nitrogen source and its broad degradation spectra of petroleum hydrocarbons

In this study, a biosurfactant producing strain, Rhodotorula sp. CC01 was isolated using landfill leachate as nitrogen source, while olive oil was determined as the best sole carbon source for producing biosurfactants. The biosurfactant produced by Rhodotorula sp. CC01 was characterized as glycolipids with a critical micelle concentration of 70 mg/L, which showed stability over a wide range of pH (2–12), salinity (0–100%), and temperature (20–100°C). During the cultivation process, the surface tension decreased from 51.87 to 28.20 mN/m in 15 h, and the removal efficiency of NH4+-N reached 84.2% after 75 h cultivation with a maximum NH4+-N removal rate of 3.92 mg·L-1·h−1. In addition, Rhodotorula sp. CC01 has proven to be of great potential in remediating petroleum hydrocarbons, as revealed by chromogenic assays. The findings of this study prove a cost-effective strategy for the production of BS by yeast through the utilization of landfill leachate.

Nitrogen (N2) fixation activity under different oxygen concentration of methanotrophic bacteria isolated from rice fields and their molecular identification

Rice fields are a significantly sources of atmospheric methane. Methanotrophic bacteria are unique in their ability to utilize methane as a sole carbon source and their ability to fix N2. This research successfully characterized N2 fixation activity under different oxygen concentrations of methanotrophic bacteria isolated from rice fields. From 19 tested isolates, four isolates performed activity to fix N2. They could fix N2 on different concentration of air saturation (10 % up to 100%). The growth of methanotrophs is not directly corelated with the N2 fixation activity, and their N2 fixation activities are affected by O2 concentrations. The BGM 3 and BGM 9 isolates had very good N2 fixation activity. Their activities were increased by increasing air saturation up to 50% (approximately 10% O2), but then decrease by increasing air saturation from 50% (approximately 10% O2) to 100% (approximately 20% O2). However, the highest N2 fixation activity was performed by the BGM 9 isolate at 30% air saturation (approximately 6% O2), and the isolate was identified as Methylococcus capsulatus. This information can support application of the isolates to achieve sustainable and environmentally friendly agricultural system.

Biodegradation and metabolic pathway of sulfamethoxazole by Sphingobacterium mizutaii

Sulfamethoxazole (SMX) is the most commonly used antibiotic in worldwide for inhibiting aquatic animal diseases. However, the residues of SMX are difficult to eliminate and may enter the food chain, leading to considerable threats on human health. The bacterial strain Sphingobacterium mizutaii LLE5 was isolated from activated sludge. This strain could utilize SMX as its sole carbon source and degrade it efficiently. Under optimal degradation conditions (30.8 °C, pH 7.2, and inoculum amount of 3.5 × 107 cfu/mL), S. mizutaii LLE5 could degrade 93.87% of 50 mg/L SMX within 7 days. Four intermediate products from the degradation of SMX were identified and a possible degradation pathway based on these findings was proposed. Furthermore, S. mizutaii LLE5 could also degrade other sulfonamides. This study is the first report on (1) degradation of SMX and other sulfonamides by S. mizutaii, (2) optimization of biodegradation conditions via response surface methodology, and (3) identification of sulfanilamide, 4-aminothiophenol, 5-amino-3-methylisoxazole, and aniline as metabolites in the degradation pathway of SMX in a microorganism. This strain might be useful for the bioremediation of SMX-contaminated environment.