Description: The blowout of the Deepwater Horizon in the Gulf of Mexico in 2010 occurred at a depth of 1500 m, corresponding to a hydrostatic pressure of 15 MPa. Up to now, knowledge about the impact of high pressure on oil-degrading bacteria has been scarce. To investigate how the biodegradation of crude oil and its components is influenced by high pressures, like those in deep-sea environments, hydrocarbon degradation and growth of two model strains were studied in high-pressure reactors. The alkane-degrading strain Rhodococcus qingshengii TUHH-12 grew well on n-hexadecane at 15 MPa at a rate of 0.16 h−1, although slightly slower than at ambient pressure (0.36 h−1). In contrast, the growth of the aromatic hydrocarbon degrading strain Sphingobium yanoikuyae B1 was highly affected by elevated pressures. Pressures of up to 8.8 MPa had little effect on growth of this strain. However, above this pressure growth decreased and at 12 MPa or more no more growth was observed. Nevertheless, S. yanoikuyae continued to convert naphthalene at pressure 〉12 MPa, although at a lower rate than at 0.1 MPa. This suggests that certain metabolic functions of this bacterium were inhibited by pressure to a greater extent than the enzymes responsible for naphthalene degradation. These results show that high pressure has a strong influence on the biodegradation of crude oil components and that, contrary to previous assumptions, the role of pressure cannot be discounted when estimating the biodegradation and ultimate fate of deep-sea oil releases such as the Deepwater Horizon event.

Description: Knowledge is scarce about the degradation of ketones in yeasts. For bacteria a subterminal degradation of alkanes to ketones and their further metabolization has been described which always involved Baeyer-Villiger monooxygenases (BVMOs). In addition, the question has to be clarified whether alkenes are converted to ketones, in particular for the oil degrading yeast Candida maltosa little is known. In this study we show the degradation of the aliphatic ketone dodecane-2-one by Candida maltosa and the related yeasts Candida tropicalis, Candida catenulata and Candida albicans as well as Trichosporon asahii and Yarrowia lipolytica. One pathway is initiated by the formation of decyl acetate, resulting from a Baeyer-Villiger-oxidation of this ketone. Beyond this, an initial reduction to dodecane-2-ol by a keto reductase was clearly shown. In addition, two different ways to metabolize dodec-1-ene were proposed. One involved the formation of dodecane-2-one and the other one a conversion leading to carboxylic and dicarboxylic acids. Furthermore the induction of ketone degrading enzymes by dodecane-2-one and dodec-1-ene was shown. Interestingly, with dodecane no subterminal degradation products were detected and it did not induce any enzymes to convert dodecane-2-one.

Description: Several species of white-rot fungi were investigated for their utility in prolonged decolouration of the recalcitrant sulfonated azo dye, amaranth. Trametes pubescens, T. multicolor, T. meyenii and T. versicolor decoloured amaranth azo-dye best on low-nitrogen agar-solidified media whereas Bjerkandera adusta and Phlebia radiata were most effective in low nitrogen medium supplemented with manganese. Trametes cotonea did not decolour effectively under any condition. The decolouring Trametes species were also effective in liquid culture whereas B. adusta and P. radiata were not. Trametes meyenii, T. pubescens and T. multicolor were equal to or better than commonly employed T. versicolor at decolouring amaranth. This is the first study to show the dye decolouration potential of T. meyenii, T. pubescens, and T. multicolor. Supplementing with Mn(II) increased assayable manganese peroxidase activity, but not long-term decolouration, indicating that laccase is the main decolourizing enzyme in these Trametes species. This appears to be because of inadequate Mn3+ chelation required by manganese peroxidase because adding relatively low amounts of malonate enhanced decolouration rates. The ability of Trametes meyenii to simultaneously decolour dye over prolonged periods of time while growing in relatively nutrient-rich medium appears to be unique amongst white-rot fungi, indicating its potential in wastewater bioremediation.

Description: Rhodococcus opacus R7 is a Gram-positive bacterium isolated from a polycyclic aromatic hydrocarbon contaminated soil for its versatile metabolism; indeed the strain is able to grow on naphthalene, o-xylene, and several long- and medium-chain n-alkanes. In this work we determined the degradation of n-alkanes in Rhodococcus opacus R7 in presence of n-dodecane (C12), n-hexadecane (C16), n-eicosane (C20), n-tetracosane (C24) and the metabolic pathway in presence of C12. The consumption rate of C12 was 88%, of C16 was 69%, of C20 was 51% and of C24 it was 78%. The decrement of the degradation rate seems to be correlated to the length of the aliphatic chain of these hydrocarbons. On the basis of the metabolic intermediates determined by the R7 growth on C12, our data indicated that R. opacus R7 metabolizes medium-chain n-alkanes by the primary alcohol formation. This represents a difference in comparison with other Rhodococcus strains, in which a mixture of the two alcohols was observed. By GC-MSD analysis we also identified the monocarboxylic acid, confirming the terminal oxidation.Moreover, the alkB gene cluster from R. opacus R7 was isolated and its involvement in the n-alkane degradation system was investigated by the cloning of this genomic region into a shuttle-vector E. coli-Rhodococcus to evaluate the alkane hydroxylase activity. Our results showed an increased biodegradation of C12 in the recombinant strain R. erythropolis AP (pTipQT1-alkR7) in comparison with the wild type strain R. erythropolis AP. These data supported the involvement of the alkB gene cluster in the n-alkane degradation in the R7 strain.

Description: Two haloalkane dehalogenases, LinBUT and LinBMI, each with 296 amino acid residues, exhibit only seven amino acid residue differences between them, but LinBMI’s catalytic performance towards β-hexachlorocyclohexane (β-HCH) is considerably higher than LinBUT’s. To elucidate the molecular basis governing this difference, intermediate mutants between LinBUT and LinBMI were constructed and kinetically characterized. The activities of LinBUT-based mutants gradually increased by cumulative mutations into LinBUT, and the effects of the individual amino acid substitutions depended on combination with other mutations. These results indicated that LinBUT’s β-HCH degradation activity can be enhanced in a stepwise manner by the accumulation of point mutations.

Description: Viable bacterial cells impaled with a single particle of a nano-sized acicular material formed when a mixture containing the cells and the material was exposed to a sliding friction field between polystyrene and agar gel; hereafter, we refer to these impaled cells as penetrons. We have used nano-sized acicular material to establish a novel method for bacterial transformation. Here, we generated penetrons that carried antisense DNA adsorbed on nano-sized acicular material (α-sepiolite) by providing sliding friction onto the surface of agar gel; we then investigated whether penetron formation was applicable to gene silencing techniques. Antisense DNA was artificially synthesized as 15 or 90mer DNA oligonucleotides based on the sequences around the translation start codon of target mRNAs. Mixtures of bacterial cells with antisense DNA adsorbed on α-sepiolite were stimulated by sliding friction on the surface of agar gel for 60 s. Upon formation of Escherichia coli penetrons, β-lactamase and β-galactosidase expression was evaluated by counting the numbers of colonies formed on LB agar containing ampicillin and by measuring β-galactosidase activity respectively. The numbers of ampicillin resistant colonies and the β-galactosidase activity derived from penetrons bearing antisense DNA (90mer) was repressed to 15% and 25%, respectively, of that of control penetrons which lacked antisense DNA. Biphenyl metabolite, ring cleavage yellow compound produced by Pseudomonas pseudoalcaligenes penetron treated with antisense oligonucleotide DNA targeted to bphD increased higher than that lacking antisense DNA. This result indicated that expression of bphD in P. pseudoalcaligenes penetrons was repressed by antisense DNA that targeted bphD mRNA. Sporulation rates of Bacillus subtilis penetrons treated with antisense DNA (15mer) targeted to spo0A decreased to 24.4% relative to penetrons lacking antisense DNA. This novel method of gene silencing has substantial promise for elucidation of gene function in bacterial species that have been refractory to experimental introduction of exogenous DNA.

Description: Microbial biotechnology and biotransformations promise to diversify the scope of the biorefinery approach for the production of high-value products and biofuels from industrial, rural and municipal waste feedstocks. In addition to bio-based chemicals and metabolites, microbial biomass itself constitutes an obvious but overlooked by-product of existing biofermentation systems which warrants fuller attention. The probiotic yeast Saccharomyces boulardii is used to treat gastrointestinal disorders and marketed as a human health supplement. Despite its relatedness to S. cerevisiae that is employed widely in biotechnology, food and biofuel industries, the alternative applications of S. boulardii are not well studied. Using a biorefinery approach, we compared the bioethanol and biomass yields attainable from agriculturally-sourced grass juice using probiotic S. boulardii (strain MYA-769) and a commercial S. cerevisiae brewing strain (Turbo yeast). Maximum product yields for MYA-769 (39.18 [±2.42] mg ethanol mL−1 and 4.96 [±0.15] g dry weight L−1) compared closely to those of Turbo (37.43 [±1.99] mg mL−1 and 4.78 [±0.10] g L−1, respectively). Co-production, marketing and/or on-site utilisation of probiotic yeast biomass as a direct-fed microbial to improve livestock health represents a novel and viable prospect for rural biorefineries. Given emergent evidence to suggest that dietary yeast supplementations might also mitigate ruminant enteric methane emissions, the administration of probiotic yeast biomass could also offer an economically feasible way of reducing atmospheric CH4.

Description: β-xylosidases catalyse the hydrolysis of short chain xylooligosaccharides from their non-reducing ends into xylose. In this study we report the heterologous expression of Aspergillus oryzae β-xylosidase (XylA) in Pichia pastoris under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter. The recombinant enzyme was optimally active at 55°C and pH 4.5 with Km and Vmax values of 1.0 mM and 250 μmol min−1 mg−1 respectively against 4-nitrophenyl β-xylopyranoside. Xylose was a competitive inhibitor with a Ki of 2.72 mM, whereas fructose was an uncompetitive inhibitor reducing substrate binding affinity (Km) and conversion efficiency (Vmax). The enzyme was characterised to be an exo-cutting enzyme releasing xylose from the non-reducing ends of β-1,4 linked xylooligosaccharides (X2, X3 and X4). Catalytic conversion of X2, X3 and X4 decreased (Vmax and kcat) with increasing chain length.

Description: Higher initial glycerol loadings (620 mM) have a negative effect on growth and 1,3-propanediol (1,3-PDO) synthesis in Clostridium butyricum DSM 10702 relative to lower initial glycerol concentrations (170 mM). To help understand metabolic shifts associated with elevated glycerol, protein expression levels were quantified by LC/MS/MS analyses. Thirty one (31) proteins involved in conversion of glycerol to 1,3-PDO and other by-products were analyzed by multiple reaction monitoring (MRM). The analyses revealed that high glycerol concentrations reduced cell growth. The expression levels of most proteins in glycerol catabolism pathways were down-regulated, consistent with the slower growth rates observed. However, at high initial glycerol concentrations, some of the proteins involved in the butyrate synthesis pathways such as a putative ethanol dehydrogenase (CBY_3753) and a 3-hydroxybutyryl-CoA dehydrogenase (CBY_3045) were up-regulated in both exponential and stationary growth phases. Expression levels of proteins (CBY_0500, CBY_0501 and CBY_0502) involved in the reductive pathway of glycerol to 1,3-PDO were consistent with glycerol consumption and product concentrations observed during fermentation at both glycerol concentrations, and the molar yields of 1,3-PDO were similar in both cultures. This is the first report that correlates expression levels of glycerol catabolism enzymes with synthesis of 1,3-PDO in C. butyricum. The results revealed that significant differences in the expression of a small subset of proteins were observed between exponential and stationary growth phases at both low and high glycerol concentrations.

Description: Fungi play a major role in various biogeochemical cycles of terrestrial and marine ecosystems. However, fungi in marine environments remain to be one of the most under-studied microbial groups. This study investigates the diversity of planktonic fungi from the coastal habitat off Pearl River Delta (China) using culture-dependent approach. A total of 22 fungi and 9 yeast isolates were recovered from 30 seawater and 2 sediment samples. Microscopic and ITS rRNA gene sequence analyses revealed that most of the fungi belonged to the phylum Ascomycota and Basidiomycota with a very small percentage (3%) of the subphylum Mucoromycotina of the Phylum Zygomycota. Most of these fungal isolates exhibited considerable production of extracellular enzymes, cellulase, lipase and laccase. Fungal isolates of two genera Mucor and Aspergillus sp. demonstrated pelletization capability over a wide range of pH, suggesting them as potential agents towards algae harvesting and wastewater treatment.