In many legumes, the nitrogen fixing root nodules produce H2 gas that diffuses into soil. It has been demonstrated that such exposure of soil to H2 can promote plant growth. To assess whether this may be due to H2-oxidizing microorganisms, bacteria were isolated from soil treated with H2 under laboratory conditions and from soils collected adjacent to H2 producing soybean nodules. Nineteen isolates of H2-oxidizing bacteria were obtained and all exhibited a half-saturation coefficient (Ks) for H2 of about 1 ml l(-1). The isolates were identified as Variovorax paradoxus, Flavobacterium johnsoniae and Burkholderia spp. using conventional microbiological tests and 16S rRNA gene sequence analysis. Seventeen of the isolates enhanced (57-254%) root elongation of spring wheat seedlings. Using an Arabidopsis thaliana bioassay, plant biomass was increased by 11-27% when inoculated by one of four isolates of V. paradoxus or one isolate of Burkholderia that were selected for evaluation. The isolates of V. paradoxus found in both H2-treated soil and in soil adjacent to soybean nodules had the greatest impact on plant growth. The results are consistent with the hypothesis that H2-oxidizing bacteria in soils have plant growth promoting properties.

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Baeyer-Villiger monooxygenases (BVMOs) are biocatalysts that offer the prospect of high chemo-, regio-, and enantioselectivity in the organic synthesis of lactones or esters from a variety of ketones. In this study, we have cloned, sequenced, and overexpressed in Escherichia coli a new BVMO, cyclopentadecanone monooxygenase (CpdB or CPDMO), originally derived from Pseudomonas sp. strain HI-70. The 601-residue primary structure of CpdB revealed only 29% to 50% sequence identity to those of known BVMOs. A new sequence motif, characterized by a cluster of charged residues, was identified in a subset of BVMO sequences that contain an N-terminal extension of ϳ60 to 147 amino acids. The 64-kDa CPDMO enzyme was purified to apparent homogeneity, providing a specific activity of 3.94 mol/min/mg protein and a 20% yield. CPDMO is monomeric and NADPH dependent and contains ϳ1 mol flavin adenine dinucleotide per mole of protein. A deletion mutant suggested the importance of the N-terminal 54 amino acids to CPDMO activity. In addition, a Ser261Ala substitution in a Rossmann fold motif resulted in an improved stability and increased affinity of the enzyme towards NADPH compared to the wild-type enzyme (K m ‫؍‬ 8 M versus K m ‫؍‬ 24 M). Substrate profiling indicated that CPDMO is unusual among known BVMOs in being able to accommodate and oxidize both large and small ring substrates that include C 11 to C 15 ketones, methyl-substituted C 5 and C 6 ketones, and bicyclic ketones, such as decalone and ␤-tetralone. CPDMO has the highest affinity (K m ‫؍‬ 5.8 M) and the highest catalytic efficiency (k cat /K m ratio of 7.2 ؋ 10 5 M ؊1 s ؊1 ) toward cyclopentadecanone, hence the Cpd designation. A number of whole-cell biotransformations were carried out, and as a result, CPDMO was found to have an excellent enantioselectivity (E > 200) as well as 99% S-selectivity toward 2-methylcyclohexanone for the production of 7-methyl-2-oxepanone, a potentially valuable chiral building block. Although showing a modest selectivity (E ‫؍‬ 5.8), macrolactone formation of 15-hexadecanolide from the kinetic resolution of 2-methylcyclopentadecanone using CPDMO was also demonstrated.

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Upgrading of bio-oil from biomass pyrolysis over Cu-modified -zeolite catalyst in a down-draft fixed-bed reactor, in which the pyrolysis and upgrading processes are integrated, is investigated in details. It is found that high silica -zeolite has high selectivity to the hydrocarbon during the upgrading process. When it is modified by a small amount of Cu, the selectivity can be obviously promoted. Especially, when 0.50 wt% of Cu is loaded on it, almost only hydrocarbons can be detected in the light oil of upgraded bio-oil and its activity can be remained for several reuses even without regeneration treatment. However, if more Cu is loaded, the selectivity decreases to some extent. Interestingly, low Cu loading on -zeolite results in the increase of surface area as well as the formation of more micropores. The surface area reaches the maximum in the case of 0.50 wt% of Cu doping. Based on XRD analysis, when the loading amount is over 1.00 wt%, Cu species aggregate on the surface of zeolite, resulting the blockage of zeolite pores and the decrease of surface area. Doping of Cu decreases the coke deposit on spent catalyst but overloading of Cu results in the increase of coking and the decrease of activity and selectivity. These results indicate that the synergetic effect between the doped metal sites and the protonic sites on the zeolite structure should be benefit for the promising catalytic performance and thus, a proper loading amount is very important for this kind of catalyst.

Recombinant Escherichia coli whole-cell biocatalysts harboring either a Baeyer-Villiger monooxygenase or ferulic acid decarboxylase were employed in organic-aqueous two-phase bioreactor systems. The feasibility of the bioproduction of water-insoluble products, viz., lauryl lactone from cyclododecanone and 4-vinyl guaiacol from ferulic acid were examined. Using hexadecane as the organic phase, 10∼16 g of lauryl lactone were produced in a 3-l bioreactor that operated in a semicontinuous mode compared to 2.4 g of product in a batch mode. For the decarboxylation of ferulic acid, a new recombinant biocatalyst, ferulic acid decarboxylase derived from Bacillus pumilus, was constructed. Selected solvents as well as other parameters for in situ recovery of vinyl guaiacol were investigated. Up to 13.8 g vinyl guaiacol (purity of 98.4%) were obtained from 25 g of ferulic acid in a 2-l working volume bioreactor by using octane as organic phase. These selected examples highlight the superiority of the two-phase biotransformations systems over the conventional batch mode.

No abstract

A green method is developed to increase the yield and quality of bio-oil by ultrasonic pretreatment of biomass followed by in situ catalytic upgrading of bio-oil over metal (Cu, Fe and/or Zn)/g-Al 2 O 3 . It is found that the yield of bio-oil is increased up to 10 wt% after cedar is pretreated by ultrasound before pyrolysis. Various metals (Cu, Fe and Zn) are loaded on g-Al 2 O 3 and applied for upgrading the bio-oil derived from the pyrolysis of pretreated cedar. It is found that the catalysts promote the conversion of oxygenated compounds into aromatic and aliphatic hydrocarbons. In particular, production of monocyclic aromatic hydrocarbons such as benzene and toluene is favored. The best catalytic activity is achieved by using 2.5 wt% Zn/g-Al 2 O 3 with a maximum hydrocarbon yield of 80.3%. The catalyst is reused for up to four cycles. The results show that the catalysts after regeneration by calcination at 550 C for 30 min exhibit long-term stability for upgrading of bio-oil.