Maximizing diesel removal from contaminated sand using Scirpus mucronatus and assessment of rhizobacteria addition effect.

Phytoremediation is one of the green technologies that is friendly to nature, utilizes fewer chemicals, and exhibits good performance. In this study, phytoremediation was used to treat diesel-contaminated sand using a local aquatic plant species, Scirpus mucronatus, by analyzing the amount of total petroleum hydrocarbons (TPHs). Optimization of diesel removal was performed according to Response Surface Methodology (RSM) using Box-Behnken Design (BBD) under pilot-scale conditions. The quadratic model showed the best fit to describe the obtained data. Actual vs. predicted values from BBD showed a total of 9.1 % error for the concentration of TPH in sand and 0 % error for the concentration of TPH in plants. Maximum TPH removal of 42.3 ± 2.1 % was obtained under optimized conditions at a diesel initial concentration of 50 mg/kg, an aeration rate of 0.48 L/min, and a retention time of 72 days. The addition of two species of rhizobacteria (Bacillus subtilis and Bacillus licheniformis) at optimum conditions increased the TPH removal to 51.9 ± 2.6 %. The obtained model and optimum condition can be adopted to treat diesel-contaminated sand within the same TPH range (50-3000 mg/kg) in sand.

Partial Purification and Characterization of the Recombinant Benzaldehyde Dehydrogenase from Rhodococcus ruber UKMP-5M.

BACKGROUND: Benzaldehyde dehydrogenase (BZDH) is encoded by the xylC that catalyzes the conversion of benzaldehyde into benzoate in many pathways such as toluene degradation. OBJECTIVES: In this study, the xylC gene from Rhodococcus ruber UKMP-5M was expressed in Escherichia coli, purified, and characterized. MATERIALS AND METHODS: The xylC was amplified and cloned in E. coli. The recombinant plasmid pGEMT-xylC was digested by NdeI and HindIII to construct plasmid pET28b-xylC and transformed in E. coli BL21 (DE3). Expression of the recombinant protein was induced by 1 mM isopropyl ß-D-thiogalactoside (IPTG) at 37°C. The BZDH was purified by ion exchange chromatography, in which the product was an NAD-dependent enzyme using benzaldehyde as a substrate for enzyme characterization. The end metabolite was identified via gas chromatography mass spectrometry (GC-MS). RESULTS: The recombinant BZDH is 27 kDa, purified by ion exchange chromatography. The activity of BZDH was 9.4 U.µL-1 The optimum pH and temperature were 8.5 and 25ºC, respectively. The Michaelis constant (Km) and maximum velocity (Vmax) were 4.2 mM and 19.7 U.mL-1, respectively. The metabolite of BZDH was benzene carboxylic acid as determined by GC-MS analysis. CONCLUSIONS: BZDH has the ability to degrade benzaldehyde to less toxic compounds. The BZDH is a critical enzyme for the degradation of aromatic hydrocarbons in Rhodococcus sp. The BZDH from R. ruber UKMP-5M is showed similar function with other aldehyde dehydrogenases.

Expression, purification and kinetic characterization of recombinant benzoate dioxygenase from Rhodococcus ruber UKMP-5M.

In this study, benzoate dioxygenase from Rhodococcus ruber UKMP-5M was catalyzed by oxidating the benzene ring to catechol and other derivatives. The benzoate dioxygenase (benA gene) from Rhodococcus ruber UKMP-5M was then expressed, purified, characterized, The benA gene was amplified (642 bp), and the product was cloned into a pGEM-T vector. The recombinant plasmid pGEMT-benA was digested by double restriction enzymes BamHI and HindIII to construct plasmid pET28b-benA and was then ligated into Escherichia coli BL21 (DE3). The recombinant E. coli was induced with 0.5 mM isopropyl ß-D-thiogalactoside (IPTG) at 22˚C to produce benzoate dioxygenase. The enzyme was then purified by ion exchange chromatography after 8 purification folds. The resulting product was 25 kDa, determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting. Benzoate dioxygenase activity was found to be 6.54 U/mL and the optimal pH and temperature were 8.5 and 25°C, respectively. Maximum velocity (Vmax) and Michaelis constant (Km) were 7.36 U/mL and 5.58 µM, respectively. The end metabolite from the benzoate dioxygenase reaction was cyclohexane dione, which was determined by gas chromatography mass spectrometry (GC-MS).

Growth enhancement of effective microorganisms for bioremediation of crude oil contaminated waters.

The bioremediation of polluted groundwater, wastewater aeration pond and biopond sites was investigated using bacteria isolated from these sites located at the oil refinery Terengganu Malaysia. Out of 62 isolates, only 16 isolates from groundwater (8) and wastewater aeration pond (3) and biopond (5) were chosen based on growth medium containing 1% (v/v) Tapis crude oil. Only four isolates; Acinetobacter faecalis, Staphylococcus sp., Pseudomonas putida and Neisseria elongata showed percentage biodegradation of crude oil more than 50% after 5 days using Mineral Salts Medium (MSM). The effect of physical parameters (temperature, pH and agitation) on growth by all four strains showed a maximum growth in MSM medium with 1% Tapis crude oil at 37 degrees C with pH 7 and agitation of 130 rpm.

Cloning of high activity xylanase gene from Bacillus pumilus PJ19.

The xylanase gene from Bacillus pumilus PJ19 amplified by polymerase chain reaction (PCR) was cloned into pCRII vector and transformed into Escherichia coli strain INValphaF'. Starting from an ATG as an initiator codon, an open reading frame coding for 202 amino acids was obtained. The recombinant xylanase sequence showed a 96% homology with the xylanase sequence from B. pumilus IPO strain and had an estimated molecular weight of 22,474. Xylanase activity expressed by E. coli INValphaF' harboring the cloned gene was located primarily in the cytoplasmic fraction.