Saovanee Dharmsthiti

Biodegradation of lipid-rich wastewater by a mixed bacterial consortium

Abstract A mixed bacterial culture comprising Pseudomonas aeruginosa LP602, Bacillus sp. B304 and Acinetobacter calcoaceticus LP009 for use in treatment of lipid-rich wastewater was formulated. In our tests, 1 l of wastewater was treated with a mixed inoculum of 6.1×108 CFU of LP 602 , 4.8×10 8 CFU B304 and 8.9×107 CFU of LP009 in 20 ml of basal salt solution ( 1.5 mg / ml Na 2 HPO 4 , 0.6 mg / ml KH 2 PO 4 , 40 μg / ml MgSO 4 and 4 μg / ml FeSO 4 ) supplemented with 1% (w/v) CaCl2. The intended role of B304 was that of a protease and amylase producer while those of LP602 and LP009 were those of lipase producers. The BOD value and lipid content were reduced from ∼3500 and 20,000 mg / l , respectively, to mg / l within 12 days under aerobic conditions.

Purification and characterization of lipase from psychrophilic Acinetobacter calcoaceticus LP009

A lipase-producing bacterium, Acinetobacter calcoacetius LP009, was isolated from raw milk. The optimum conditions for growth and lipase production by A. calcoaceticus LP009 were 15 degrees C with shaking at 200 rpm in LB supplemented with 1.0% (v/v) Tween 80. The crude lipase was purified to homogeneous state by ultrafiltration and gel filtration chromatography on Sephadex G-100. Its molecular weight determined by SDS-PAGE was 23 kDa and it exhibited maximum activity at pH 7.0 and 50 degrees C. It was stable over the pH range of 4.0 to 8.0 and at temperatures lower than 45 degrees C. It was a metalloenzyme that is positionally non-specific and had the ability to improve fat hydrolysis in soybean meal and in premixed animals feed.

Novel roles of ohrR-ohr in Xanthomonas sensing, metabolism, and physiological adaptive response to lipid hydroperoxide.

Lipid hydroperoxides are highly toxic to biological systems. Here, the Xanthomonas campestris pv. phaseoli sensing and protective systems against linoleic hydroperoxide (LOOH) were investigated by examining the phenotypes, biochemical and regulatory characteristics of various Xanthomonas mutants in known peroxide resistance pathways. Analysis of LOOH resistance levels indicates that both alkyl hydroperoxide reductase (AhpC) and organic hydroperoxide resistance enzyme (Ohr) have important and nonredundant roles in the process. Nonetheless, inactivation of ohr leads to a marked reduction in LOOH resistance levels. The regulatory characteristics of an ohr mutant add further support to its primary role in LOOH protection. Northern analysis shows that LOOH had differential effects on induction of ahpC and ohr expression with the latter being more sensitive to the inducer. Analysis of the ahpC and ohr promoters confirmed that the LOOH-dependent induction of these promoters is mediated by the transcription regulators OxyR and OhrR, respectively. Using the in vivo promoter assays and the in vitro gel mobility shift assay, we show that LOOH directly oxidized OhrR at the sensing residue Cys-22 leading to its inactivation. In addition, physiological analysis shows that pretreatment of X. campestris pv. phaseoli with a sublethal dose of LOOH induced high levels of resistance to subsequent exposure to lethal concentrations of LOOH. This novel LOOH-induced adaptive response requires a functional ohrR-ohr operon. These data illustrate an important novel physiological role for the ohrR-ohr system in sensing and inactivating lipid hydroperoxides.

Evaluation of the roles that alkyl hydroperoxide reductase and Ohr play in organic peroxide-induced gene expression and protection against organic peroxides in Xanthomonas campestris

Alkyl hydroperoxide reductase (ahpC) and organic hydroperoxide resistance (ohr) are distinct genes, structurally and regulatory, but have similar physiological functions. In Xanthomonas campestris pv. phaseoli inactivation of either gene results in increased sensitivity to killing with organic peroxides. An ahpC1-ohr double mutant was highly sensitive to both growth inhibition and killing treatment with organic peroxides. High level expression of ahpC or ohr only partially complemented the phenotype of the double mutant, suggesting that these genes function synergistically, but through different pathways, to protect Xanthomonas from organic peroxide toxicity. Functional analyses of Ohr and AhpC abilities to degrade organic hydroperoxides revealed that both Ohr and AhpC could degrade tert-butyl hydroperoxide (tBOOH) while the former was more efficient at degrading cumene hydroperoxide (CuOOH). Expression analysis of these genes in the mutants showed no compensatory alterations in the levels of AhpC or Ohr. However, CuOOH induced expression of these genes in the mutants was affected. CuOOH induced ahpC expression was higher in the ohr mutant than in the parental strain; in contrast, the ahpC mutation has no effect on the level of induced ohr expression. These analyses reveal complex physiological roles and expression patterns of seemingly functionally similar genes.

Production and Immobilization of Lipase from Aeromonas sobria Harboring a Heterologous Gene

Abstract Aeromonas sobria LP004 harboring the Acinetobacter calcoaceticus lipase gene was designated as strain LP094. Lipase production of LP094 in WYGS medium [Lotrakul and Dharmsthiti, World J. Microbiol. Biotechnol., 13 : 163–166, 1997] was scaled up to a 50- l volume. LP094 lipase immobilized by ionic binding to either IR120 (Na + ) or IRC50 (H + ) Amberlite resins was found to retain more than 90% activity after 15 d storage at room temperature (≈ 25 to 30°C) or at 4°C. After 5 repeated lipid hydrolysis reactions, the activities of both immobilized preparations remained higher than 65%.