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Petroleum-hydrocarbon-degrading bacteria were obtained after enrichment on crude oil (as a 'chocolate mousse') in a continuous supply of Indonesian seawater amended with nitrogen, phosphorus and iron nutrients. They were related to Alcanivorax and Marinobacter strains, which are ubiquitous petroleum-hydrocarbon-degrading bacteria in marine environments, and to Oceanobacter kriegii (96.4-96.5 % similarities in almost full-length 16S rRNA gene sequences). The Oceanobacter-related bacteria showed high n-alkane-degrading activity, comparable to that of Alcanivorax borkumensis strain SK2. On the other hand, Alcanivorax strains exhibited high activity for branched-alkane degradation and thus could be key bacteria for branched-alkane biodegradation in tropical seas. Oceanobacter-related bacteria became most dominant in microcosms that simulated a crude oil spill event with Indonesian seawater. The dominance was observed in microcosms that were unamended or amended with fertilizer, suggesting that the Oceanobacter-related strains could become dominant in the natural tropical marine environment after an accidental oil spill, and would continue to dominate in the environment after biostimulation. These results suggest that Oceanobacter-related bacteria could be major degraders of petroleum n-alkanes spilt in the tropical sea. INTRODUCTIONA wide variety of micro-organisms are known to degrade petroleum hydrocarbons (Head et al., 2006;Prince, 2005). Most hydrocarbonoclastic bacteria metabolize either aliphatic or aromatic hydrocarbons, although some bacteria such as strains of Pseudomonas (Whyte et al., 1997) and Rhodococcus (Andreoni et al., 2000) have been shown to degrade both types of hydrocarbons. Among hydrocarbonoclastic bacteria, Alcanivorax (Hara et al., 2003;Kasai et al., 2001;Roling et al., 2004;Yakimov et al., 1998Yakimov et al., , 2005 and Cycloclasticus (Dyksterhouse et al., 1995;Kasai et al., 2002a;Maruyama et al., 2003) strains have been identified as key micro-organisms in the degradation of aliphatic and aromatic hydrocarbons, respectively, in marine environments (Harayama et al., 2004). Alcanivorax strains are distributed in natural marine environments around the world . The ability of Alcanivorax strains to use branched alkanes is high (Hara et al., 2003;McKew et al., 2007), and this could be one of the reasons why these strains predominate in crude-oil-impacted temperate marine environments (Cappello et al., 2007;Hara et al., 2003;Kasai et al., 2001;Roling et al., 2002Roling et al., , 2004Yakimov et al., 2005). Thalassolituus oleivorans has been reported to degrade aliphatic hydrocarbons (Yakimov et al., 2004), and Thalassolituus strains have recently been shown to dominate in n-alkane-containing temperate seawater microcosms (McKew et al., 2007;Yakimov et al., 2005) and in crude-oil-containing temperate estuarine seawater microcosms (Coulon et al., 2007;McKew et al., 2007).In addition to these 'professional' hydrocarbonoclastic bacteria, many 'non-professional' hydrocarbonoclastic bacteria such as Mar...

For a resolution of reducing carbon dioxide emission and increasing food production to respond to the growth of global population, production of biofuels from non-edible biomass is urgently required. Abundant orange wastes, such as peel and strained lees, are produced as by-product of orange juice, which is available non-edible biomass. However, d-limonene included in citrus fruits often inhibits yeast growth and makes the ethanol fermentation difficult. This study demonstrated that isopropanol-butanol-ethanol fermentation ability of Clostridium beijerinckii and cellulosic biomass degrading ability of C. cellulovorans were cultivated under several concentrations of limonene. As a result, C. cellulovorans was able to grow even in the medium containing 0.05% limonene (v/v) and degraded 85% of total sugar from mandarin peel and strained lees without any pretreatments. More interestingly, C. beijerinckii produced 0.046 g butanol per 1 g of dried strained lees in the culture supernatant together with C. cellulovorans.

A ␤-1,3-xylanase gene (txyA) from a marine bacterium, Alcaligenes sp. strain XY-234, has been cloned and sequenced. txyA consists of a 1,410-bp open reading frame that encodes 469 amino acid residues with a calculated molecular mass of 52,256 Da. The domain structure of the ␤-1,3-xylanase (TxyA) consists of a signal peptide of 22 amino acid residues, followed by a catalytic domain which belongs to family 26 of the glycosyl hydrolases, a linker region with one array of DGG and six repeats of DNGG, and a novel carbohydrate-binding module (CBM) at the C terminus. The recombinant TxyA hydrolyzed ␤-1,3-xylan but not other polysaccharides such as ␤-1,4-xylan, carboxymethylcellulose, curdlan, glucomannan, or ␤-1,4-mannan. TxyA was capable of binding specifically to ␤-1,3-xylan. The analysis using truncated TxyA lacking either the N-or C-terminal region indicated that the region encoding the CBM was located between residues 376 and 469. Binding studies on the CBM revealed that the K d and the maximum amount of protein bound to ␤-1,3-xylan were 4.2 M and 18.2 mol/g of ␤-1,3-xylan, respectively. Furthermore, comparison of the enzymatic properties between proteins with and without the CBM strongly indicated that the CBM of TxyA plays an important role in the hydrolysis of ␤-1,3-xylan.

BackgroundKojic acid (5-Hydroxy-2-(hydroxymethyl)-4-pyrone) is one of the major secondary metabolites in Aspergillus oryzae. It is widely used in food, pharmaceuticals, and cosmetics. The production cost, however, is too high for its use in many applications. Thus, an efficient and cost-effective kojic acid production process would be valuable. However, little is known about the complete set of genes for kojic acid production. Currently, kojic acid is produced from glucose. The efficient production of kojic acid using cellulose as an inexpensive substrate would help establish cost-effective kojic acid production.ResultsA kojic acid transcription factor gene over-expressing the A. oryzae strain was constructed. Three genes related to kojic acid production in this strain were transcribed in higher amounts than those found in the wild-type strain. This strain produced 26.4 g/L kojic acid from 80 g/L glucose. Furthermore, this strain was transformed with plasmid harboring 3 cellulase genes. The resultant A. oryzae strain successfully produced 0.18 g/L of kojic acid in 6 days of fermentation from the phosphoric acid swollen cellulose.ConclusionsKojic acid was produced directly from cellulose material using genetically engineered A. oryzae. Because A. oryzae has efficient protein secretion ability and secondary metabolite productivity, an A. oryzae-based cell factory could be a platform for the production of various kinds of bio-based chemicals.