Olive mill wastewater (OMW), a highly polluting waste from the olive oil industry, was utilized as sole carbon source for the production of polyhydroxyalkanoate (PHA) by extremely halophilic Haloferax Mediterranei (H. mediterranei) in a one stage cultivation step. H. mediterranei showed remarkable cell growth and tolerated the inhibitory effect of polyphenols present in medium containing 25% of OMW. H. mediterranei cultivation conditions were optimized in medium containing 15% OMW by investigating several parameters that affect the production of PHA. The highest polymer yield (0.2g/L) and PHA content (43% PHA/cell dry mass) were achieved at 37°C, 170rpm and 22% salt concentration. Analysis of the produced PHA revealed the production of copolyester poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBHV) containing 6.5mol% 3-hydroxyvalerate (3HV). The production of PHBHV was observed without the need for fermentation step or adding external carbon source. The PHBHV displayed reduced melting points at 140.1°C and 154.4°C when compared to homopolymer polyhydroxybutyrate.

Haloarchaeal alcohol dehydrogenases are exciting biocatalysts with potential industrial applications. In this study, two alcohol dehydrogenase enzymes from the extremely halophilic archaeon Haloferax volcanii (HvADH1 and HvADH2) were homologously expressed and subsequently purified by immobilized metal-affinity chromatography. The proteins appeared to copurify with endogenous alcohol dehydrogenases, and a double Δadh2 Δadh1 gene deletion strain was constructed to prevent this occurrence. Purified HvADH1 and HvADH2 were compared in terms of stability and enzymatic activity over a range of pH values, salt concentrations, and temperatures. Both enzymes were haloalkaliphilic and thermoactive for the oxidative reaction and catalyzed the reductive reaction at a slightly acidic pH. While the NAD(+)-dependent HvADH1 showed a preference for short-chain alcohols and was inherently unstable, HvADH2 exhibited dual cofactor specificity, accepted a broad range of substrates, and, with respect to HvADH1, was remarkably stable. Furthermore, HvADH2 exhibited tolerance to organic solvents. HvADH2 therefore displays much greater potential as an industrially useful biocatalyst than HvADH1.

Enzymatic synthesis of enantiopure aromatic secondary alcohols (including substituted, hetero-aromatic and bicyclic structures) was carried out using halophilic alcohol dehydrogenase ADH2 from Haloferax volcanii (HvADH2). This enzyme showed an unprecedented substrate scope and absolute enatioselectivity. The cofactor NADPH was used catalytically and regenerated in situ by the biocatalyst, in the presence of 5% ethanol. The efficiency of HvADH2 for the conversion of aromatic ketones was markedly influenced by the steric and electronic factors as well as the solubility of ketones in the reaction medium. Furthermore, carbonyl stretching band frequencies ν (C[double bond, length as m-dash]O) have been measured for different ketones to understand the effect of electron withdrawing or donating properties of the ketone substituents on the reaction rate catalyzed by HvADH2. Good correlation was observed between ν (C[double bond, length as m-dash]O) of methyl aryl-ketones and the reaction rate catalyzed by HvADH2. The enzyme catalyzed the reductions of ketone substrates on the preparative scale, demonstrating that HvADH2 would be a valuable biocatalyst for the preparation of chiral aromatic alcohols of pharmaceutical interest.

Alcohol dehydrogenase from halophilic archaeon Haloferax volcanii (HvADH2) was successfully covalently immobilized on metal-derivatized epoxy Sepabeads. The immobilization conditions were optimized by investigating several parameters that affect the halophilic enzyme-support interaction. The highest immobilization efficiency (100 %) and retention activity (60 %) were achieved after 48 h of incubation of the enzyme with Ni-epoxy Sepabeads support in 100 mM Tris-HCl buffer, pH 8, containing 3 M KCl at 5 °C. No significant stabilization was observed after blocking the unreacted epoxy groups with commonly used hydrophilic agents. A significant increase in the stability of the immobilized enzyme was achieved by blocking the unreacted epoxy groups with ethylamine. The immobilization process increased the enzyme stability, thermal activity, and organic solvents tolerance when compared to its soluble counterpart, indicating that the immobilization enhances the structural and conformational stability. One step purification-immobilization of this enzyme has been carried out on metal chelate-epoxy Sepabeads, as an efficient method to obtain immobilized biocatalyst directly from bacterial extracts.

The effect of various organic solvents on the catalytic activity, stability and substrate specificity of alchohol dehydrogenase from Haloferax volcanii (HvADH2) was evaluated. The HvADH2 showed remarkable stability and catalysed the reaction in aqueous-organic medium containing dimethyl sulfoxide (DMSO) and methanol (MeOH). Tetrahydrofuran and acetonitrile were also investigated and adversely affected the stability of the enzyme. High concentration of salt, essential to maintain the enzymatic activity and structural integrity of the halophilic enzyme under standard conditions may be partially replaced by DMSO and MeOH. The presence of organic solvents did not induce gross changes in substrate specificity. DMSO offered a protective effect for the stability of the enzyme at nonoptimal pHs such as 6 and 10. Salt and solvent effects on the HvADH2 conformation and folding were examined through fluorescence spectroscopy. The fluorescence findings were consistent with the activity and stability results and corroborated the denaturing properties of some solvents. The intrinsic tolerance of this enzyme to organic solvent makes it highly attractive to industry.

The largest wastewater treatment plant in Jordan was monitored in the summer to determine the removal of pharmaceuticals and personal care products (PPCPs). Grab samples were collected from the influent and effluent of As-Samra Wastewater Treatment Plant (WWTP). Liquid chromatography and tandem mass spectrometry (LC–MS/MS) were utilized to determine the concentrations of 18 compounds of pharmaceuticals and personal care products (PPCPs). The results showed that 14 compounds were detected in the collected samples from the influent and effluent of As-Samra WWTP. These compounds are 1,7-dimethylxanthine, amphetamine, acetaminophen, caffeine, carbamazepine, cimetidine, cotinine, diphenhydramine, methylenedioxymethamphetamine (MDMA), morphine, phenazone, sulfamethazine, sulfamethoxazole, thiabendazole, and trimethoprim. However, four compounds were below the detection limit (<0.005 µg/L), namely cimetidine, methylenedioxyamphetamine (MDA), methamphetamine, and sulfachloropyridazine. Among PPCPs, the highest estimated average concentrations in raw wastewater were caffeine, acetaminophen, 1,7-dimethylxanthine, cotinine, and carbamazepine sampled during the summer, at an estimated concentration of 155.6 µg/L, 36.7 µg/L, 10.49 µg/L, and 1.104 µg/L, respectively. However, the highest estimated average concentrations in treated wastewater were for carbamazepine, sulfamethoxazole, caffeine, cotinine, and acetaminophen, at 0.856 µg/L, 0.096 µg/L, 0.086 µg/L, 0.078 µg/L, and 0.041 µg/L, respectively. In general, the results showed that some compounds in the collected samples of wastewater in Jordan have concentrations exceeding the values reported in the literature. The removal efficiency rates of 1,7-dimethylxanthine, acetaminophen, caffeine, cotinine, morphine, and trimethoprim were higher than 95%, while those of carbamazepine, sulfamethazine, and sulfamethoxazole were lower than 22.5%. Moreover, diphenhydramine and thiabendazole had negative removal efficiency rates. The removal efficiency rates of the PPCPs in As-Samra WWTP were generally consistent with those of indicator compounds reported in the literature for conventional WWTPs.

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