JBB : Journal of Bioscience and Bioengineering

            

Journal of Bioscience and Bioengineering vol.122 cover

 



  • Development of a versatile method for targeted gene deletion and insertion by using the pyrF gene in the psychrotrophic bacterium, Shewanella livingstonensis Ac10
    Publication date: December 2016
    Source:Journal of Bioscience and Bioengineering, Volume 122, Issue 6

    Author(s): Tomokazu Ito, Chunjie Gong, Jun Kawamoto, Tatsuo Kurihara

    Shewanella livingstonensis Ac10, a psychrotrophic bacterium isolated from Antarctic seawater, grows well at low temperatures close to 0°C. The bacterium is useful as a host in a low-temperature protein expression system. It is also useful as a model microorganism to investigate the mechanisms of microbial cold-adaptation. Versatile genetic manipulation techniques would be useful to investigate the biology of this bacterium and to develop its applications. In this study, we developed a method for targeted gene deletion and insertion by using the gene coding for orotidine-5′-phosphate decarboxylase (pyrF), which is involved in pyrimidine synthesis. We found that S. livingstonensis Ac10 is sensitive to 5-fluoroorotic acid (5-FOA), which is converted to a highly toxic compound by the product of pyrF. A uracil-auxotrophic strain resistant to 5-FOA was constructed by deleting pyrF, thus allowing the use of a plasmid-borne copy of pyrF for selection of recombinants. We constructed the pyrF complementation suicide plasmid pKKP, which contains pyrF, the R6K replication origin, the mob site of RP4, an antibiotic marker gene, and a multiple cloning site. To demonstrate pyrF-based gene replacement, we deleted the internal region of orf5, the gene coding for an eicosapentaenoic acid (EPA) synthesis enzyme. We also successfully inserted a His6-tag-coding sequence into orf8, the gene coding for another EPA synthesis enzyme. This system allows the markerless deletion and insertion of desired sequences at specific sites in the genome, which remarkably facilitates genetic manipulation of this bacterium.





  • Antimicrobial activity against Porphyromonas gingivalis and mechanism of action of the cationic octadecapeptide AmyI-1-18 and its amino acid-substituted analogs
    Publication date: December 2016
    Source:Journal of Bioscience and Bioengineering, Volume 122, Issue 6

    Author(s): Masayuki Taniguchi, Akihito Ochiai, Kiyoshi Takahashi, Shun-ichi Nakamichi, Takafumi Nomoto, Eiichi Saitoh, Tetsuo Kato, Takaaki Tanaka

    The antimicrobial peptide AmyI-1-18 is a cationic α-helical octadecapeptide derived from α-amylase in rice (Oryza sativa L. japonica) that contains four cationic amino acid residues (two arginines and two lysines). To enhance the antibacterial activity of AmyI-1-18 against Porphyromonas gingivalis (a bacterium associated with periodontal disease), we synthesized 12 analogs bearing substitutions with alanine, leucine, and/or arginine that were designed based on helical wheel projections and investigated their antibacterial properties. The antibacterial properties of four analogs bearing substitution of a single arginine or lysine with alanine were almost similar to those of AmyI-1-18, suggesting that the antibacterial properties depend on the presence of three cationic amino acid residues. Of three single arginine-substituted analogs, AmyI-1-18(G12R) exhibited an antibacterial activity 2.8-fold higher [50% growth-inhibitory concentration (IC50): 4.6 μM] than that of AmyI-1-18 (IC50: 13 μM). Likewise, the antibacterial properties of two single leucine-substituted analogs were significantly enhanced; in particular, AmyI-1-18(N3L) exhibited an antibacterial activity (IC50: 2.5 μM) 5.2-fold higher than that of AmyI-1-18. The hemolytic activity of AmyI-1-18(N3L) against mammalian red blood cells was low (2% at 50 μM). A membrane-depolarization assay using a membrane potential-sensitive fluorescent dye revealed that, similar to AmyI-1-18, the antibacterial activity of AmyI-1-18(N3L) was not dependent on its membrane-disrupting activity. Our results demonstrate that the antibacterial properties of AmyI-1-18 against P. gingivalis are significantly improved, without a significant increase in hemolytic activity, by replacing asparagine with leucine at position 3.





  • In vitro synthesis of polyhydroxyalkanoates using thermostable acetyl-CoA synthetase, CoA transferase, and PHA synthase from thermotorelant bacteria
    Publication date: December 2016
    Source:Journal of Bioscience and Bioengineering, Volume 122, Issue 6

    Author(s): Kenji Tajima, Xuerong Han, Yoshiki Hashimoto, Yasuharu Satoh, Toshifumi Satoh, Seiichi Taguchi

    Thermostable enzymes are required for the rapid and sustainable production of polyhydroxyalkanoate (PHA) in vitro. The in vitro synthesis of PHA using the engineered thermostable synthase PhaC1SG(STQK) has been reported; however, the non-thermostable enzymes acetyl-CoA synthetase (ACS) and CoA transferase (CT) from mesophilic strains were used as monomer-supplying enzymes in this system. In the present study, acs and ct were cloned from the thermophilic bacteria Pelotomaculum thermopropionicum JCM10971 and Thermus thermophilus JCM10941 to construct an in vitro PHA synthesis system using only thermostable enzymes. ACS from P. thermopropionicum (ACSPt) and CT from T. thermophilus (CTTt) were confirmed to have high thermostability, and their optimal temperatures were around 60°C and 75°C, respectively. The in vitro PHA synthesis was successfully performed by ACSPt, CTTt, PhaC1SG(STQK), and poly(3-hydroxybutyrate) [P(3HB)] was synthesized at 45°C. Furthermore, the yields of P(3HB) and P(lactate-co-3HB) at 37°C were 1.4-fold higher than those of the in vitro synthesis system with non-thermostable ACS and CT from mesophilic strains. Overall, the thermostable ACS and CT were demonstrated to be useful for the efficient in vitro PHA synthesis at relatively high temperatures.





  • Novel synthetic kojic acid-methimazole derivatives inhibit mushroom tyrosinase and melanogenesis
    Publication date: December 2016
    Source:Journal of Bioscience and Bioengineering, Volume 122, Issue 6

    Author(s): Ming-Jen Chen, Chih-Chuan Hung, Yan-Ru Chen, Shih-Ting Lai, Chin-Feng Chan

    In this study, two kojic acid-methimazole (2-mercapto-1-methylimidazole, MMI, 1) derivatives, 5-hydroxy-2-{[(1-methyl-1H-imidazol-2-yl)thio]methyl}-4H-pyran-4-one (compound 4) and 5-methoxy-2-{[(1-methyl-1H-imidazol-2-yl)thio]methyl}-4H-pyran-4-one (compound 5), were synthesized to examine their inhibitory kinetics on mushroom tyrosinase. Compound 4 exhibited a potent inhibitory effect on monophenolase activity in a dose-dependent manner, with an IC50 value of 0.03 mM. On diphenolase activity, compound 4 exhibited a less inhibitory effect (IC50 = 1.29 mM) but was stronger than kojic acid (IC50 = 1.80 mM). Kinetic analysis indicated that compound 4 was both as a noncompetitive monophenolase and diphenolase inhibitor. By contrast, compound 5 exhibited no inhibitory effects on mushroom tyrosinase activity. The IC50 value of compound 4 for the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity was 4.09 mM, being much higher than the IC50 of compound 4 for inhibiting the tyrosinase activity. The results indicated that the antioxidant activity of compound 4 may be partly related to the potent inhibitory effect on mushroom tyrosinase. Compound 4 also exerted a potent inhibitory effect on intracellular melanin formation in B16/F10 murine melanoma cells, and caused no cytotoxicity. Furthermore, compound 4 induced no adverse effects on the Hen's egg test-chorioallantoic membrane (HET-CAM).





  • Constitutive expression of recombinant human hyaluronidase PH20 by Pichia pastoris
    Publication date: December 2016
    Source:Journal of Bioscience and Bioengineering, Volume 122, Issue 6

    Author(s): Kuan-Jung Chen, Sabrina Sabrina, Nermeen S. El-Safory, Guan-Chiun Lee, Cheng-Kang Lee

    PH20 is known as sperm adhesion molecule 1 (SPAM1) and also has hyaluronidase function to preferentially hydrolyze the glycosidic linkage of hyaluronic acid (HA). A DNA fragment containing core domain of human PH20 gene was cloned into a constitutive expression plasmid (pGAPZαC) of Pichia pastoris to produce a fusion protein with α factor signal in the N-terminus and 6 × His as well as c-Myc tags in the C-terminus. The resulting plasmid pGAPZαC-PH20 was integrated into the genome of P. pastoris strain GS115. Functional recombinant human PH20 (rHuPH20) was successfully expressed and secreted by the recombinant P. pastoris transformant. Highest hyaluronidase activity of 2 mU/mL could be obtained at 3 day in an YPD culture. After purified by phenylboronic acid resin adsorption, rHuPH20 with a specific activity of 230 mU/mg was obtained. Via periodic acid-Schiff staining and zymogram analysis, the partially purified rHuPH20 was determined to be highly glycosylated to various extents with molecular mass in the range of 100–300 kDa. The enzyme showed a maximal activity at pH 5.0 but no appreciable activity at pH ≤3 and pH ≥8. The hyaluronidase activity could be stably maintained at 4°C but lost 40% after incubating at 30°C for 4 h. Both N-acetyl cysteine and glutathione showed a half maximal inhibitory concentration (IC50) of 8 mM against rHuPH20.





  • Characterization of an O-methyltransferase specific to guaiacol-type benzenoids from the flowers of loquat (Eriobotrya japonica)
    Publication date: December 2016
    Source:Journal of Bioscience and Bioengineering, Volume 122, Issue 6

    Author(s): Takao Koeduka, Mami Kajiyama, Takumi Furuta, Hideyuki Suzuki, Tomohiko Tsuge, Kenji Matsui

    Volatile benzenoids, including methyl p-methoxybenzoate, p-anisaldehyde, and p-anisalcohol, are responsible for the sweet and characteristic fragrance of loquat (Eriobotrya japonica, Rosaceae) flowers. Although the full pathway of volatile benzenoid synthesis has yet to be elucidated, their chemical structures suggest that O-methyltransferases are present in loquat and function in the methylation of the para-OH groups. In the present study, we used RNA-sequencing to identify four loquat genes (EjOMT1, EjOMT2, EjOMT3, and EjOMT4) that encode O-methyltransferases. We found that EjOMT1 was highly expressed in floral tissues, with an expression pattern that coincided with changes in intracellular volatile benzenoids during flower development. Recombinant EjOMT1 protein expressed in Escherichia coli showed the highest activity towards guaiacol with a K m value of 35 μM. Furthermore, the protein also showed lesser activities towards guaiacol-type benzenoids including eugenol, isoeugenol, vanillin, and ferulic acid, in addition to much weaker activities towards catechol and p-hydroxybenzenoid derivatives. However, no activity was shown towards phenylpropenes without m-methoxy substitution, t-anol and chavicol. Taken together, our findings indicate that EjOMT1 has a broad substrate specificity towards compounds with both para-OH and meta-OCH3 groups, unlike previously characterized O-methyltransferases for volatile benzenoid/phenylpropanoid biosynthesis.





  • Effect of NADH kinase on poly-3-hydroxybutyrate production by recombinant Escherichia coli
    Publication date: December 2016
    Source:Journal of Bioscience and Bioengineering, Volume 122, Issue 6

    Author(s): Peng-Hui Hong, Jie Zhang, Xiao-Jie Liu, Tian-Wei Tan, Zheng-Jun Li

    The cofactor NADPH participates in a variety of anabolic reactions and its availability is considered to play a critical role in biotransformation processes. NADH kinase (Pos5) from Saccharomyces cerevisiae catalyzes the phosphorylation of NADH to generate NADPH. To investigate the effect of NADH kinase on poly-3-hydroxybutyrate (PHB) production, pos5 was co-expressed with PHB synthetic operon phbCAB in Escherichia coli. The recombinant strain carrying pos5 and phbCAB co-expression plasmid reached 5.96 g/L cell dry weight with 64.1% PHB accumulation in 72 h shake flask cultivation, while the control strain without pos5 yielded 3.93 g/L cell dry weight with 58.5% PHB content. PHB production titer was enhanced from 2.30 g/L to 3.82 g/L. Intracellular cofactor concentration analysis revealed that the ratio of NADP/NAD in pos5 overexpression strain was two times more compared with that of the control without pos5. The results showed that NADH kinase could be employed as an effective metabolic manipulation target to improve PHB synthesis.





  • Ophthalmic acid accumulation in an Escherichia coli mutant lacking the conserved pyridoxal 5′-phosphate-binding protein YggS
    Publication date: December 2016
    Source:Journal of Bioscience and Bioengineering, Volume 122, Issue 6

    Author(s): Tomokazu Ito, Ayako Yamauchi, Hisashi Hemmi, Tohru Yoshimura

    Escherichia coli YggS is a highly conserved pyridoxal 5′-phosphate (PLP)-binding protein whose biochemical function is currently unknown. A previous study with a yggS-deficient E. coli strain (ΔyggS) demonstrated that YggS controls l-Ile- and l-Val-metabolism by modulating 2-ketobutyrate (2-KB), l-2-aminobutyrate (l-2-AB), and/or coenzyme A (CoA) availability in a PLP-dependent fashion. In this study, we found that ΔyggS accumulates an unknown metabolite as judged by amino acid analyses. LC/MS and MS/MS analyses of the compound with propyl chloroformate derivatization, and co-chromatography analysis identified this compound as γ-l-glutamyl-l-2-aminobutyryl-glycine (ophthalmic acid), a glutathione (GSH) analogue in which the l-Cys moiety is replaced by l-2-AB. We also determine the metabolic consequence of the yggS mutation. Absence of YggS initially increases l-2-AB availability, and then causes ophthalmic acid accumulation and CoA limitation in the cell. The expression of a γ-glutamylcysteine synthetase and a glutathione synthetase in a ΔyggS background causes high-level accumulation of ophthalmic acid in the cells (∼1.2 nmol/mg cells) in a minimal synthetic medium. This opens the possibility of a first fermentative production of ophthalmic acid.





  • Expression and characterization of alkaline protease from the metagenomic library of tannery activated sludge
    Publication date: December 2016
    Source:Journal of Bioscience and Bioengineering, Volume 122, Issue 6

    Author(s): Selvaraju Gayathri Devi, Anwar Aliya Fathima, Mary Sanitha, Sellamuthu Iyappan, Wayne R. Curtis, Mohandass Ramya

    Metagenomics has the potential to facilitate the discovery of novel enzymes; however, to date, only a few alkaline proteases have been characterized from environmentally-sourced DNA. We report the identification and characterization of an alkaline serine protease designated as Prt1A from the metagenomic library of tannery activated sludge. Sequence analysis revealed that Prt1A is closely related to S8A family subtilisins with a catalytic triad of Asp143, His173, and Ser326. The putative protease gene (prt-1A) was subcloned in pET 28a (+) vector and overexpressed in Escherichia coli BL21(DE3)pLysS cells. This 38.8 KDa recombinant protease was purified to homogeneity by nickel affinity chromatography and exhibited optimal enzyme activity at elevated pH (11.0) and temperature (55°C). The enzyme activity was enhanced by the addition of 5 mM Ca2+ ions, and was stable in the presence of anionic detergent, oxidizing agent and various organic solvents. The enzyme displayed high affinity and catalytic efficiency for casein under standard assay conditions (V max = 279 U/mg/min, K m = 1.70 mg/mL) and was also compatible with commercial detergents. These results suggest that Prt1A protease could act as an efficient enzyme in various industrial applications.





  • Extracellular mercury sequestration by exopolymeric substances produced by Yarrowia spp.: Thermodynamics, equilibria, and kinetics studies
    Publication date: December 2016
    Source:Journal of Bioscience and Bioengineering, Volume 122, Issue 6

    Author(s): Ganiyu Oladunjoye Oyetibo, Keisuke Miyauchi, Hitoshi Suzuki, Satoru Ishikawa, Ginro Endo

    Exopolymeric substances (EPS) produced by highly mercury-resistant strains of the yeast Yarrowia spp. (Idd1 and Idd2) were isolated and studied for their mercury binding potential. Excellent yield (approximately 0.3 g EPS per gram biomass) of soluble EPS in medium with 3% glucose was observed in the Yarrowia cultures 7 day post-inoculation. A gram dry weight of the EPS consists mainly of carbohydrates (0.4 g), protein (0.3–0.4 g), uronic acid (0.02 g), and nucleic acids (0.002 g). Mercury interactions with the biopolymer were measured as uptake kinetics from a simulated aquatic system and modelled with thermodynamics and calculated mass action equilibria. The EPS forms a complex with Hg2+ in water with small activation energy (≤2 kJ mol−1), achieving about 30 mg Hg2+ adsorption per gram dry weight of EPS. The adsorption models confirmed complexation of Hg2+ by the EPS via heterogeneous multilayer adsorption that obey second-order kinetics at constant rate of 4.0 and 8.1 mg g−1 min−1. The EPS used chemisorption as rate-limiting step that controls the uptake of Hg2+ from aquatic systems during micro-precipitation as bio-removal strategy. The EPS are promising biotechnological tools to design bioreactors for treatment of mercury-rich industrial wastewater.





  • 16S rRNA gene-based comprehensive analysis of microbial community compositions in a full-scale leachate treatment system
    Publication date: December 2016
    Source:Journal of Bioscience and Bioengineering, Volume 122, Issue 6

    Author(s): Hoang T.N. Dao, Kyohei Kuroda, Nozomi Nakahara, Tsuyoshi Danshita, Masashi Hatamoto, Takashi Yamaguchi

    In this study, we performed a comprehensive analysis of microbial community compositions in leachate and leachate treatment system (14 processes) during dry and rainy seasons (from February to September and from October to January, respectively), at Khanh Son landfill site, Danang City, Vietnam. In this study, raw leachate in dry and rainy seasons was predominated by Arcobacter, Clostridia, Thermotogales, Methanobacteriaceae, and Methanosaeta. During the two seasons, the system had different microbial community compositions. Orders Methanobacteriales, Clostridiales, MBA08 (order-level clone cluster), and Thermotogales predominated the influent, anaerobic pond, and anoxic pond during the dry season, while Campylobacterales and Pseudomonadales orders were predominant in the anaerobic/anoxic systems during the rainy season. In the facultative pond, aerated ponds, sediment tanks, and polishing ponds, predominant orders during the dry season included Actinomycetales, “Saprospirales”, Flavobacteriales, Rhizobiales, Rhodospirillales, Burkholderiales, and Alteromonadales; during the rainy season: Sphingobacteriales, Rickettsiales, Sphingomonadales, and Pseudomonadales. In the final post treatment (polishing ponds with vegetation), significant removal of organic matter, total nitrogen, and colour occurred, while nitrogen-fixing and root-associated or related organisms predominated. This suggested that the vegetation in the ponds was essential to achieve the sufficient leachate treatment.





  • Heterologous expression of a new manganese-dependent peroxidase gene from Peniophora incarnata KUC8836 and its ability to remove anthracene in Saccharomyces cerevisiae
    Publication date: December 2016
    Source:Journal of Bioscience and Bioengineering, Volume 122, Issue 6

    Author(s): Aslan Hwanhwi Lee, Chang-Min Kang, Young Min Lee, Hanbyul Lee, Cheol-Won Yun, Gyu-Hyeok Kim, Jae-Jin Kim

    The white rot fungus Peniophora incarnata KUC8836 has received an attention as the greatest degrader of polycyclic aromatic hydrocarbons (PAHs), which are hazardous xenobiotics and recalcitrant pollutants. To characterize the mechanisms through which MnP degrades PAHs, heterologous expression of manganese-dependent peroxidase (MnP) gene pimp1 was performed in Saccharomyces cerevisiae BY4741 via the pGEM-T Easy vector, resulting in the recombinant plasmid pESC-URA/pimp1 containing the MnP signal peptide. MnP was significantly secreted into the culture medium with galactose as an active protein with higher efficiency (3.58 U mL−1) by transformants than by the wild-type S. cerevisiae. The recombinant MnP protein was shown to have a molecular weight of 44 kDa by western blotting analysis. With regard to enhancing the bioremediation of PAHs in the environment, anthracene was effectively degraded by the MnP encoded by pimp1, with a degradation rate of 6.5% when Tween 80 was added. In addition, the MnP activity of the transformant exhibited the highest efficiency (2.49 U mL−1) during the degradation. These results show that pimp1 might be useful for biodegradation and gene expression technologies at a transcriptional level, and genetic approaches can be improved by incorporating the highly ligninolytic gene pimp1 and the fungus P. incarnata KUC8836.





  • Comparative evaluation of short-term stress of Cd(II), Hg(II), Pb(II), As(III) and Cr(VI) on anammox granules by batch test
    Publication date: December 2016
    Source:Journal of Bioscience and Bioengineering, Volume 122, Issue 6

    Author(s): Cheng Yu, Yu-Xia Song, Li-Yuan Chai, Cheng-Shan Duan, Chong-Jian Tang, Mohammad Ali, Cong Peng

    Batch experiments were conducted to investigate the short-term effects of Cd(II), Hg(II), Pb(II), As(III) and Cr(VI) on anammox activity, which are considered to be the top 5 toxic heavy metals in China. The IC50 values of Cd(II), Hg(II), Pb(II) and Cr(VI) were calculated to be 7.00, 2.33, 10.40 and 9.84 mg/L, respectively, while As(III) caused only 29.67% decrease in SAA even at the dosage of 60 mg/L. The evaluation of metal concentrations in liquid and sludge revealed that anammox biomass hold a high heavy-metal accumulation ability, which was hypothesized to be the key reason of activity inhibition. The functional groups possessed by anammox biomass and the extracellular polymeric substance (EPS) might contribute to the attachment of heavy metals. Especially, microbial reduction of Cr(VI) to Cr(III) inside the granules was discovered, which was considered to relieve the Cr(VI) inhibition under concentrations lower than 8.96 mg/L.





  • Styrene production from a biomass-derived carbon source using a coculture system of phenylalanine ammonia lyase and phenylacrylic acid decarboxylase-expressing Streptomyces lividans transformants
    Publication date: December 2016
    Source:Journal of Bioscience and Bioengineering, Volume 122, Issue 6

    Author(s): Ryosuke Fujiwara, Shuhei Noda, Tsutomu Tanaka, Akihiko Kondo

    To produce styrene from a biomass-derived carbon source, Streptomyces lividans was adopted as a host strain. The gene encoding ferulic acid decarboxylase from Saccharomyces cerevisiae (FDC1) was introduced into S. lividans, and the resulting S. lividans transformant successfully expressed FDC1 and converted trans-cinnamic acid (CA) to styrene. A key factor in styrene production using microbes is the recovery of volatile styrene. In the present study, we selected polystyrene resin beads XRD-4 as the absorbent agent to recover styrene produced using S. lividans transformants, which enabled recovery of styrene from the culture broth. For styrene production from biomass-derived carbon sources, S. lividans/FDC1 was cultured together with S. lividans/p-encP, which we previously reported as a CA-producing S. lividans strain. This coculture system combined with the recovery of styrene using XAD-4 allowed the production of styrene from glucose, cellobiose, or xylo-oligosaccharide, respectively.





  • Novel spectrophotometric approach for determination of validamycin A in fermentation of Streptomyces hygroscopicus
    Publication date: December 2016
    Source:Journal of Bioscience and Bioengineering, Volume 122, Issue 6

    Author(s): Wei Li, Jinsong Feng, Yan Liu, Jing Jiang, Xiaodong Zheng, Wen-Wen Zhou

    Validamycin A (Val-A), produced by Streptomyces hygroscopicus 5008 in industrial fermentation, is one of the most widely used anti-fungal agro-antibiotics in Asia and high performance liquid chromatography (HPLC) assay is usually used to determine the production of Val-A. A new approach to determine Val-A by spectrophotometer is developed. During the fermentation of S. hygroscopicus 5008, a pigment secretion was found along with the Val-A biosynthesis. There was a stable relationship between the concentration of Val-A and spectral absorption (SA) value of this pigment at 450 nm, even in different fermentation cultures or conditions. Using SA value as interior label, a rapid spectrophotometric method for determining Val-A production was established. In comparing Val-A productivity by HPLC method with that by SA method, the relative standard deviation (R.S.D.) was 0.007 (less than 0.05, no variation) and the conditional probability [Pr(T < t)] was 0.3491 (greater than 0.05, no difference) at the optimal time point of Val-A fermentation, which demonstrated SA method was as stable and accurate as standard HPLC method. It was applied successfully to finding positive strains with high Val-A productivity and short fermentation time. SA assay is an accurate and cost-effective method for measuring Val-A and screening high-producing strains, and this work provides a new insight for rapid quantitative analysis of antibiotics in fermentation of pigment-producing strains.





  • Activation of CO2-reducing methanogens in oil reservoir after addition of nutrient
    Publication date: December 2016
    Source:Journal of Bioscience and Bioengineering, Volume 122, Issue 6

    Author(s): Guang-Chao Yang, Lei Zhou, Serge Maurice Mbadinga, Jing You, Hua-Zhen Yang, Jin-Feng Liu, Shi-Zhong Yang, Ji-Dong Gu, Bo-Zhong Mu

    Nutrient addition as part of microbial enhanced oil recovery (MEOR) operations have important implications for more energy recovery from oil reservoirs, but very little is known about the in situ response of microorganisms after intervention. An analysis of two genes as biomarkers, mcrA encoding the key enzyme in methanogenesis and fthfs encoding the key enzyme in acetogenesis, was conducted during nutrient addition in oil reservoir. Clone library data showed that dominant mcrA sequences changed from acetoclastic (Methanosaetaceae) to CO2-reducing methanogens (Methanomicrobiales and Methanobacteriales), and the authentic acetogens affiliated to Firmicutes decreased after the intervention. Principal coordinates analysis (PCoA) and Jackknife environment clusters revealed evidence on the shift of the microbial community structure among the samples. Quantitative analysis of methanogens via qPCR showed that Methanobacteriales and Methanomicrobiales increased after nutrient addition, while acetoclastic methanogens (Methanosaetaceae) changed slightly. Nutrient treatment activated native CO2-reducing methanogens in oil reservoir. The high frequency of Methanobacteriales and Methanomicrobiales (CO2-reducers) after nutrient addition in this petroleum system suggested that CO2-reducing methanogenesis was involved in methane production. The nutrient addition could promote the methane production. The results will likely improve strategies of utilizing microorganisms in subsurface environments.





  • Mechanoporation of living cells for delivery of macromolecules using nanoneedle array
    Publication date: December 2016
    Source:Journal of Bioscience and Bioengineering, Volume 122, Issue 6

    Author(s): Daisuke Matsumoto, Ayana Yamagishi, Megumi Saito, Ramachandra Rao Sathuluri, Yaron R. Silberberg, Futoshi Iwata, Takeshi Kobayashi, Chikashi Nakamura

    Efficient and rapid delivery of macromolecule probes, such as quenchbodies and other large biomarkers that cannot readily pass through the plasma membrane, is necessary for live-cell imaging and other intracellular analyses. We present here an alternative, simple method for delivery of macromolecules into live cells. In this method, which we term here mechanoporation, a nanoneedle array is used for making transient pores in the plasma membrane to allow access of desired macromolecules into thousands of live cells, simultaneously. This rapid, 3-step method facilitates an efficient delivery by adding macromolecules into the medium, inserting nanoneedles into the cells and oscillating the nanoneedle array, a process that takes no more than 5 min in total. In addition, we demonstrate here how this method can repeatedly and reproducibly deliver molecules into specifically-selected locations on a given cell culture dish. The results presented here show how this unique mechanoporation method enables rapid and high-throughput bio-macromolecule delivery and live-cell imaging.





  • Vessel-like channels supported by poly-l-lysine tubes
    Publication date: December 2016
    Source:Journal of Bioscience and Bioengineering, Volume 122, Issue 6

    Author(s): Nobuhito Mori, Yuya Morimoto, Shoji Takeuchi

    Vessel-like channels fabricated by embedding sacrificial structures in three-dimensional (3D) cellular constructs and then removing the sacrificial structures have been proposed as a means of providing nutrition to the cells. Alginate gel fibers have been used in the design of such channels owing to their flexibility. However, these channels are closed during culture due to extensive shrinkage of the hydrogel structures when they contain certain cell types such as fibroblasts. Here, we describe a method for fabricating vessel-like channels supported by semi-permeable poly-l-lysine-alginate membrane tubes (PLL-tubes) in a collagen gel. PLL-coated alginate gel fibers were embedded in collagen gel and the inner alginate gel was removed. We were able to form channels in various designs—including branched structures—owing to the flexibility of the alginate gel fibers. Moreover, channels supported by PLL-tubes remained open without shrinkage of the collagen gel containing fibroblasts. These results demonstrate that 3D cellular constructs can be fabricated for culturing cells that would normally induce shrinkage of hydrogel structures.





  • Novel perfusion-decellularized method to prepare decellularized ureters for ureteral tissue-engineered repair
    Publication date: December 2016
    Source:Journal of Bioscience and Bioengineering, Volume 122, Issue 6

    Author(s): Shu-wei Xiao, Peng-chao Wang, Wei-jun Fu, Zhong-xin Wang, Gang Li, Xu Zhang

    As the endoscopic technique is widely used in the diagnosis and treatment of diseases, the incidence of ureteral injuries increases annually. The classical surgical therapies are not always satisfactory. With the constant development of the tissue engineering technology in the field of urinary reconstruction, the ureteral reconstruction has become possible technology. In this study, a novel perfusion-decellularized protocol, which combined a perfusion system with the commonly used physical and chemical methods, was used to prepare the decellularized ureters for ureteral reconstruction and the urinary tract-derived cells (UDCs) were seeded on the surface of the perfusion-decellularized ureters (PDUs) in order to observe the cells survival, adhesion, proliferation and distribution. The data of H&E staining, DAPI staining, and the agarose gel electrophoresis demonstrated that the cellular components of PDUs were removed, and the decellularized time was shorter than previous study. In addition, compared with the native ureters, the DNA content of the PDUs was significantly decreased just two percent residue (P <0.05). Scanning electron microscopy, collagen and glycosaminoglycan content assay showed that the three-dimensional (3D) ultrastructure and the compositions of the extracellular matrix (ECM) of PDUs were well preserved. When the UDCs were seeded onto the PDUs, the UDCs formed multilayer structure on the surface of the PDUs, infiltrated into the deep layer of the decellularized ureters and then formed laminated structure. In conclusion, the decellularized ureters prepared by the novel perfusion-decellularized method may be the potential surrogate for ureteral tissue-engineered repair.





  • Acoustic vibration can enhance bacterial biofilm formation
    Publication date: December 2016
    Source:Journal of Bioscience and Bioengineering, Volume 122, Issue 6

    Author(s): Mark F. Murphy, Thomas Edwards, Glyn Hobbs, Joanna Shepherd, Frederic Bezombes

    This paper explores the use of low-frequency–low-amplitude acoustic vibration on biofilm formation. Biofilm development is thought to be governed by a diverse range of environmental signals and much effort has gone into researching the effects of environmental factors including; nutrient availability, pH and temperature on the growth of biofilms. Many biofilm-forming organisms have evolved to thrive in mechanically challenging environments, for example soil yet, the effects of the physical environment on biofilm formation has been largely ignored. Exposure of Pseudomonas aeruginosa to vibration at 100, 800 and 1600 Hz for 48 h, resulted in a significant increase in biofilm formation compared with the control, with the greatest growth seen at 800 Hz vibration. The results also show that this increase in biofilm formation is accompanied with an increase in P. aeruginosa cell number. Acoustic vibration was also found to regulate the spatial distribution of biofilm formation in a frequency-dependent manner. Exposure of Staphylococcus aureus to acoustic vibration also resulted in enhanced biofilm formation with the greatest level of biofilm being formed following 48 h exposure at 1600 Hz. These results show that acoustic vibration can be used to control biofilm formation and therefore presents a novel and potentially cost effective means to manipulate the development and yield of biofilms in a range of important industrial and medical processes.