JBB : Journal of Bioscience and Bioengineering

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Journal of Bioscience and Bioengineering vol.120 cover

Journal of Bioscience and Bioengineering – Recent Articles

  • Comparisons of cell culture medium using distribution of morphological features in microdevice
    Publication date: Available online 3 July 2015
    Source:Journal of Bioscience and Bioengineering

    Author(s): Hiroto Sasaki , Junko Enomoto , Yurika Ikeda , Hiroyuki Honda , Junji Fukuda , Ryuji Kato

    As the number of available cell types grows, it becomes necessary to develop more effective ways to optimize the cell-culture medium for each cell line and culture condition. However, because of the vast number of parameters that must be decided, such as the combination of components, optimization is both laborious and costly. Microdevices are a cost-effective way to perform such evaluations because they use only a small volume of media and enable high-throughput analyses. However, assays performed in microdevices are themselves minimized, and each assay unit (well/chamber) commonly contains an insufficient number of cells for comprehensive evaluations such as gene-expression or flow-cytometry analyses. To address this issue, we introduced image-based analysis in conjunction with microdevice assays; this approach allows quantification of every cell in each assay unit. To quantitatively profile differences in cellular behaviors in a microdevice under different culture media conditions, we developed a non-staining image-based analysis method that utilizes cellular morphology. Our approach combines the structural advantages of microdevices, which can increase the stability of images, and the quantitative advantages of an image-based cell evaluation technique that utilizes time-course population change in several morphological features. Our results demonstrate that cellular changes due to small alterations in the concentration of serum in medium or differences in the basal medium can be profiled using only microscopic images.





  • Impact of zinc supplementation on the improved fructose/xylose utilization and butanol production during acetone–butanol–ethanol fermentation
    Publication date: Available online 3 July 2015
    Source:Journal of Bioscience and Bioengineering

    Author(s): You-Duo Wu , Chuang Xue , Li-Jie Chen , Feng-Wu Bai

    Lignocellulosic biomass and dedicated energy crops such as Jerusalem artichoke are promising alternatives for biobutanol production by solventogenic clostridia. However, fermentable sugars such as fructose or xylose released from the hydrolysis of these feedstocks were subjected to the incomplete utilization by the strains, leading to relatively low butanol production and productivity. When 0.001 g/L ZnSO4·7H2O was supplemented into the medium containing fructose as sole carbon source, 12.8 g/L of butanol was achieved with butanol productivity of 0.089 g/L/h compared to only 4.5 g/L of butanol produced with butanol productivity of 0.028 g/L/h in the control without zinc supplementation. Micronutrient zinc also led to the improved butanol production up to 8.3 g/L derived from 45.2 g/L xylose as sole carbon source with increasing butanol productivity by 31.7%. Moreover, the decreased acids production was observed under the zinc supplementation condition, resulting in the increased butanol yields of 0.202 g/g-fructose and 0.184 g/g-xylose, respectively. Similar improvements were also observed with increasing butanol production by 130.2 % and 8.5 %, butanol productivity by 203.4% and 18.4%, respectively, in acetone–butanol–ethanol fermentations from sugar mixtures of fructose/glucose (4:1) and xylose/glucose (1:2) simulating the hydrolysates of Jerusalem artichoke tubers and corn stover. The results obtained from transcriptional analysis revealed that zinc may have regulatory mechanisms for the sugar transport and metabolism of Clostridium acetobutylicum L7. Therefore, micronutrient zinc supplementation could be an effective way for economic development of butanol production derived from these low-cost agricultural feedstocks.





  • Inhibitory effects of sulfur compounds on methane oxidation by a methane-oxidizing consortium
    Publication date: Available online 2 July 2015
    Source:Journal of Bioscience and Bioengineering

    Author(s): Eun-Hee Lee , Kyung-Eun Moon , Tae Gwan Kim , Sang-Don Lee , Kyung-Suk Cho

    Kinetic and enzymatic inhibition experiments were performed to investigate the effects of methanethiol (MT) and hydrogen sulfide (H2S) on methane oxidation by a methane-oxidizing consortium. In the coexistence of MT and H2S, the oxidation of methane was delayed until MT and H2S were completely degraded. MT and H2S could be degraded, both with and without methane. The kinetic analysis revealed that the methane-oxidizing consortium showed a maximum methane oxidation rate (V max) of 3.7 mmol g-dry cell weight (DCW)−1 h−1 and a saturation constant (K m ) of 184.1 μM. MT and H2S show competitive inhibition on methane oxidation, with inhibition values (K i ) of 1504.8 and 359.8 μM, respectively. MT was primary removed by particulate methane monooxygenases (pMMO) of the consortium, while H2S was degraded by the other microorganisms or enzymes in the consortium. DNA and mRNA transcript levels of the pmoA gene expressions were decreased to ∼106 and 103 pmoA gene copy number g-DCW−1 after MT and H2S degradation, respectively; however, both the amount of the DNA and mRNA transcript recovered their initial levels of ∼107 and 105 pmoA gene copy number g-DCW−1 after methane oxidation, respectively. The gene expression results indicate that the pmoA gene could be rapidly reproducible after methane oxidation. This study provides comprehensive information of kinetic interactions between methane and sulfur compounds.





  • Formation of chitosan-fucoidan nanoparticles and their electrostatic interactions: Quantitative analysis
    Publication date: Available online 2 July 2015
    Source:Journal of Bioscience and Bioengineering

    Author(s): Eun Ju Lee , Kwang-Hee Lim

    The stoichiometric distributions of both positive amino groups and negative sulfate ions loaded in chitosan-fucoidan nanoparticles (CFNs) were predicted quantitatively by correlating the separate yields of loaded chitosan and fucoidan, and a proposed relative charge density model (case 1). In addition, those distributions of both positive amino groups and negative sulfate ions loaded in CFNs were obtained by deriving the expression of their loaded concentrations directly from the experimental data (case 2). Both the model-prediction and experimental derivations were remarkably consistent with each other except at pH 2. The discrepancy between cases 1 and 2 at pH 2 was explained by an increase in the sulfate group loading because of the most intensive electrostatic (specific ion) interactions at pH 2. The ratio of the CFN-free net charge density shielded by counter-ions in the solution entrapped in CFNs to their counter-ion-crosslinking charge density was suggested to be a quantitative criterion for determining the size distribution of CFNs. The formation of CFNs ranked according to size was predicted well and explained reasonably by the suggested criterion, considering both the ionic strength of the entrapped solution in CFNs and the nonspecific binding (interaction) of the positive amino groups among the chitosan molecules. Furthermore, the fraction of nonspecifically-bound positive amino groups causing hysteresis was quantified from the positive net charged amino groups per unit-mass CFN. Thus, its magnitude was predicted to have a strong correlation with the CFN-preparation conditions, such as pH and fucoidan to chitosan mass ratio.





  • Development of a rapid method to isolate polyhydroxyalkanoates from bacteria for screening studies
    Publication date: Available online 2 July 2015
    Source:Journal of Bioscience and Bioengineering

    Author(s): Isaac Vizcaino-Caston , Catherine A. Kelly , Annabel V.L. Fitzgerald , Gary A. Leeke , Mike Jenkins , Tim W. Overton

    We describe a novel method of Polyhydroxyalkanoate (PHA) extraction using dimethyl sulphoxide (DMSO) for use in screening studies. Compared to conventional chloroform extraction, the DMSO method was shown to release comparable quantities of PHA from Cupriavidus necator cells, with comparable properties as determined using Fourier transform infrared spectroscopy and differential scanning calorimetry.





  • Characterization of a novel thermostable N-acylhomoserine lactonase from the thermophilic bacterium Thermaerobacter marianensis
    Publication date: July 2015
    Source:Journal of Bioscience and Bioengineering, Volume 120, Issue 1

    Author(s): Tomohiro Morohoshi , Yoshiaki Tominaga , Nobutaka Someya , Tsukasa Ikeda

    Thermaerobacter marianensis is an extremely thermophilic bacterium, which was isolated from the Mariana Trench, with an optimal growth temperature of approximately 75°C. N-Acylhomoserine lactone (AHL) is a quorum-sensing signal molecule used by many gram-negative bacteria. Here, we report the identification of an AHL-degrading gene homolog (designated aiiT) in the genome of T. marianensis JCM 10246. AiiT has 59.7%, 21.2%, and 11.2% identity to AhlS from Solibacillus silvestris, AiiA from Bacillus cereus, and AidC from Chryseobacterium sp., respectively. Homologs of aiiT were also found in Thermaerobacter nagasakiensis, T. composti, and T. subterraneus. A purified AiiT-maltose binding fusion showed high AHL-degrading activity against N-hexanoyl-l-homoserine lactone, N-octanoyl-l-homoserine lactone, and N-decanoyl-l-homoserine lactone at temperatures ranging from 40 to 80°C. HPLC analysis revealed that AiiT functions as an AHL-lactonase that catalyzes AHL ring opening by hydrolyzing lactones. AiiT displayed maximal activity at high temperatures (60–80°C) and showed higher thermostability than other AHL lactonases.





  • Improving xylitol production through recombinant expression of xylose reductase in the white-rot fungus Phanerochaete sordida YK-624
    Publication date: July 2015
    Source:Journal of Bioscience and Bioengineering, Volume 120, Issue 1

    Author(s): Sho Hirabayashi , Jianqiao Wang , Hirokazu Kawagishi , Hirofumi Hirai

    We generated an expression construct consisting of the xylose reductase (XR) gene (xr) from Phanerochaete chrysosporium. Transformant X7 exhibited increased xylitol production and markedly higher XR activities than the wild-type strain. RT-PCR analysis demonstrated that the increased XR activity was associated with constant expression of the recombinant xr gene.





  • Glucose oxidase adsorption performance of carbonaceous mesocellular foams prepared with different carbon sources
    Publication date: July 2015
    Source:Journal of Bioscience and Bioengineering, Volume 120, Issue 1

    Author(s): Fengjiao Zhou , Ximing Pu , Dapeng Luo , Guangfu Yin , Kai Zhuang , Xiaoming Liao , Zhongbin Huang , Xianchun Chen , Yadong Yao

    Several carbonaceous mesocellular foams (C-MCFs) were prepared with MCF-silica as template using the carbon precursors of sucrose, furfuryl alcohol and lab-made phenolic resin, and the corresponding C-MCFs were named as C-MCF-Suc, C-MCF-FA and C-MCF-PR, respectively. The results of SEM, transmission electron microscopy, N2 adsorption–desorption and energy-dispersive X-ray measurements indicated that the C-MCFs prepared from different carbon source appeared morphologically with different degree of order and different pore distribution. The C-MCF-FA exhibited the highest ordered structure and the smallest pore distribution among the foams. The optimum conditions for adsorption of C-MCFs on glucose oxidase (GOD) were also studied, and the maximum adsorbance was determined. The adsorption of GOD on C-MCF-FA was performed at different pH with different GOD concentrations. The maximum adsorption (423.3 mg g−1) was observed near the isoelectric point of the GOD (pI ≈ 5.0) with a GOD concentration of 6.0 mg mL−1, suggesting that the GOD adsorption on C-MCFs might be affected strongly by the electric repulsion between the GOD molecules. Moreover, GOD adsorption performances on different C-MCFs revealed that both the pore size and the pore volume played important roles in the adsorption process, and the window size of C-MCFs dominated the residual immobilized amounts of GOD. Compared to the other two C-MCFs, the C-MCF-FA with a smaller window pore (10 nm) and higher volume (1.40 cm3 g−1) exhibited the highest GOD adsorption and catalytic activity. Furthermore, the immobilized GOD exhibited improved thermal and storable stabilities. Thus the C-MCF-FA could be served as the prospective GOD carrier material used in enzymatic fuel cells.





  • Enzyme-assisted supercritical carbon dioxide extraction of black pepper oleoresin for enhanced yield of piperine-rich extract
    Publication date: July 2015
    Source:Journal of Bioscience and Bioengineering, Volume 120, Issue 1

    Author(s): Sayantani Dutta , Paramita Bhattacharjee

    Black pepper (Piper nigrum L.), the King of Spices is the most popular spice globally and its active ingredient, piperine, is reportedly known for its therapeutic potency. In this work, enzyme-assisted supercritical carbon dioxide (SC-CO 2 ) extraction of black pepper oleoresin was investigated using α-amylase (from Bacillus licheniformis) for enhanced yield of piperine-rich extract possessing good combination of phytochemical properties. Optimization of the extraction parameters (without enzyme), mainly temperature and pressure, was conducted in both batch and continuous modes and the optimized conditions that provided the maximum yield of piperine was in the batch mode, with a sample size of 20 g of black pepper powder (particle diameter 0.42 ± 0.02 mm) at 60°C and 300 bar at 2 L/min of CO2 flow. Studies on activity of α-amylase were conducted under these optimized conditions in both batch and continuous modes, with varying amounts of lyophilized enzyme (2 mg, 5 mg and 10 mg) and time of exposure of the enzyme to SC-CO2 (2.25 h and 4.25 h). The specific activity of the enzyme increased by 2.13 times when treated in the continuous mode than in the batch mode (1.25 times increase). The structural changes of the treated enzymes were studied by 1 H NMR analyses. In case of α-amylase assisted extractions of black pepper, both batch and continuous modes significantly increased the yields and phytochemical properties of piperine-rich extracts; with higher increase in batch mode than in continuous.





  • Cholesterol oxidase with high catalytic activity from Pseudomonas aeruginosa: Screening, molecular genetic analysis, expression and characterization
    Publication date: July 2015
    Source:Journal of Bioscience and Bioengineering, Volume 120, Issue 1

    Author(s): Noriyuki Doukyu , Shyou Nihei

    An extracellular cholesterol oxidase producer, Pseudomonas aeruginosa strain PA157, was isolated by a screening method to detect 6β-hydroperoxycholest-4-en-3-one-forming cholesterol oxidase. On the basis of a putative cholesterol oxidase gene sequence in the genome sequence data of P. aeruginosa strain PAO1, the cholesterol oxidase gene from strain PA157 was cloned. The mature form of the enzyme was overexpressed in Escherichia coli cells. The overexpressed enzyme formed inclusion bodies in recombinant E. coli cells grown at 20°C and 30°C. A soluble and active PA157 enzyme was obtained when the recombinant cells were grown at 10°C. The purified enzyme was stable at pH 5.5 to 10 and was most active at pH 7.5–8.0, showing optimal activity at pH 7.0 and 70°C. The enzyme retained about 90% of its activity after incubation for 30 min at 70°C. The enzyme oxidized 3β-hydroxysteroids such as cholesterol, β-cholestanol, and β-sitosterol at high rates. The K m value and V max value for the cholesterol were 92.6 μM and 15.9 μmol/min/mg of protein, respectively. The V max value of the enzyme was higher than those of commercially available cholesterol oxidases. This is the first report to characterize a cholesterol oxidase from P. aeruginosa.





  • Requirement of carbon dioxide for initial growth of facultative methylotroph, Acidomonas methanolica MB58
    Publication date: July 2015
    Source:Journal of Bioscience and Bioengineering, Volume 120, Issue 1

    Author(s): Ryoji Mitsui , Hiroko Katayama , Mitsuo Tanaka

    The facultative methylotrophic bacterium Acidomonas methanolica MB58 can utilize C1 compounds via the ribulose monophosphate pathway. A large gene cluster comprising three components related to C1 metabolism was found in the genome. From upstream, the first was an mxa cluster encoding proteins for oxidation of methanol to formaldehyde; the second was the rmp cluster encoding enzymes for formaldehyde fixation; and the third was the cbb gene cluster encoding proteins for carbon dioxide (CO2) fixation. Examination of CO2 requirements for growth of A. methanolica MB58 cells demonstrated that it did not grow on any carbon source under CO2-free conditions. Measurement of ribulose-1,5-bisphosphate carboxylase activity and RT-PCR analysis demonstrated enzymatic activity was detected in A. methanolica MB58 at growth phase, regardless of carbon sources. However, methanol dehydrogenase and 3-hexlose-6-phosphate synthase expression was regulated by methanol or formaldehyde; it were detected during growth and apparently differed from ribulose-1,5-bisphosphate carboxylase expression. These results suggested that A. methanolica MB58 may be initially dependent on autotrophic growth and that carbon assimilation was subsequently coupled with the ribulose monophosphate pathway at early- to mid-log phases during methylotrophic growth.





  • Enhancement of l-phenylalanine production by engineered Escherichia coli using phased exponential l-tyrosine feeding combined with nitrogen source optimization
    Publication date: July 2015
    Source:Journal of Bioscience and Bioengineering, Volume 120, Issue 1

    Author(s): Peipei Yuan , Weijia Cao , Zhen Wang , Kequan Chen , Yan Li , Pingkai Ouyang

    Nitrogen source optimization combined with phased exponential l-tyrosine feeding was employed to enhance l-phenylalanine production by a tyrosine-auxotroph strain, Escherichia coli YP1617. The absence of (NH4)2SO4, the use of corn steep powder and yeast extract as composite organic nitrogen source were more suitable for cell growth and l-phenylalanine production. Moreover, the optimal initial l-tyrosine level was 0.3 g L−1 and exponential l-tyrosine feeding slightly improved l-phenylalanine production. Nerveless, l-phenylalanine production was greatly enhanced by a strategy of phased exponential l-tyrosine feeding, where exponential feeding was started at the set specific growth rate of 0.08, 0.05, and 0.02 h−1 after 12, 32, and 52 h, respectively. Compared with exponential l-tyrosine feeding at the set specific growth rate of 0.08 h−1, the developed strategy obtained a 15.33% increase in l-phenylalanine production (l-phenylalanine of 56.20 g L−1) and a 45.28% decrease in l-tyrosine supplementation.





  • Selection of suitably non-repressing carbon sources for expression of alcohol oxidase isozyme promoters in the methylotrophic yeast Pichia methanolica
    Publication date: July 2015
    Source:Journal of Bioscience and Bioengineering, Volume 120, Issue 1

    Author(s): Tomoyuki Nakagawa , Keishi Wakayama , Takashi Hayakawa

    In this work, we aimed to select suitable non-repressing carbon sources for the expression of promoters derived from the alcohol oxidase (AOD) isozyme genes, P MOD1 and P MOD2 , during the growth of Pichia methanolica. Our results revealed that xylose is the best non-repressing carbon source for heterologous gene expression using both P MOD1 and P MOD2 , and that glycerol is also a suitable carbon source with by which the on/off of P MOD2 expression can be controlled.





  • Evaluation of the effects of amyloid β aggregation from seaweed extracts by a microliter-scale high-throughput screening system with a quantum dot nanoprobe
    Publication date: July 2015
    Source:Journal of Bioscience and Bioengineering, Volume 120, Issue 1

    Author(s): Toshiki Ogara , Tomohito Takahashi , Hajime Yasui , Koji Uwai , Kiyotaka Tokuraku

    Inhibitors of amyloid β (Aβ) aggregation have the potential to serve as lead compounds for anti-Alzheimer's disease (AD) agents because Aβ aggregation is a key step in AD pathogenesis. Recently, we developed a novel microliter-scale high-throughput screening (MSHTS) system for Aβ aggregation inhibitors that applied fluorescence microscopic analysis with quantum dot nanoprobes, and attempted to comprehensively screen the inhibitors from spices using this system (Ishigaki et al., PLoS One, 8, e72992, 2013). In this study, we tried to evaluate the inhibitory activities of 11 seaweed extracts on Aβ aggregation using the MSHTS system. The half-maximal effective concentration (EC50) of the ethanolic extracts from all seaweeds exceeded 4.9 mg/ml, indicating that the extracts inhibit Aβ aggregation although this activity was significantly lower than that displayed by members of the Lamiaceae, a family of herbal spices that showed highest activity among 52 spices tested in our 2013 study. On the other hand, the EC50 of boiling water extracts was 0.013–0.42 mg/ml which was comparable with the EC50 of the extracts from the Lamiaceae family. These results suggest that the extraction efficiency of the inhibitors by boiling water extraction was higher than that by ethanolic extraction. Moreover, analysis of fluorescence micrographs, which were obtained from the MSHTS system, revealed that the morphology of the Aβ aggregates coincubated with boiling water extracts differed from control aggregates, suggesting that the MSHTS system is also useful for screening substances that affect the morphology of aggregates.





  • Anaerobic co-digestion of kitchen waste and pig manure with different mixing ratios
    Publication date: July 2015
    Source:Journal of Bioscience and Bioengineering, Volume 120, Issue 1

    Author(s): Hailin Tian , Na Duan , Cong Lin , Xue Li , Mingzhu Zhong

    Anaerobic co-digestion of kitchen waste (KW) and pig manure (PM) with seven different PM to KW total solids (TS) ratios of 1:0, 5:1, 3:1, 1:1, 1:3, 1:5 and 0:1 was conducted at mesophilic temperature (35 ± 1°C) to investigate the feasibility and process performance. The co-digestion of PM and KW was found to be an available way to enhance methane production compared with solo-digestion of PM or KW. The ratio of PM to KW of 1:1 got the highest biodegradability (BDA) of 85.03% and a methane yield of 409.5 mL/gVS. For the co-digestion of KW and PM, there was no obvious inhibition of ammonia nitrogen because it was in an acceptable range from 1380 mg/L to 2020 mg/L in the whole process. However, severe methane inhibition and long lag phase due to the accumulation of volatile fatty acids (VFAs) was observed while the KW content was over 50%, and in the lag phase, propionic acid and butyric acid made up the major constituents of the total VFAs. The technical digestion time (T80: the time it takes to produce 80% of the digester's maximum gas production) of the above 7 ratios was 15, 21, 22, 27, 49, 62 and 61 days, respectively. In this study, a mixing ratio of 1:1 for PM and KW was found to maximize BDA and methane yield, provided a short digestion time and stable digestion performance and was therefore recommended for further study and engineering application.





  • Characterization of water-soluble dark-brown pigment from Antarctic bacterium, Lysobacter oligotrophicus
    Publication date: July 2015
    Source:Journal of Bioscience and Bioengineering, Volume 120, Issue 1

    Author(s): Tomomi Kimura , Wakao Fukuda , Tomoe Sanada , Tadayuki Imanaka

    Lysobacter oligotrophicus strain 107-E2T isolated from Antarctica produces dark-brown colored water-soluble pigment, in addition to hydrolases and lytic enzymes. The production of pigment is a common characteristic among members of the genus Lysobacter, but the identity of the pigments has been unknown. In this study, we identified the pigment from L. oligotrophicus as melanin pigment (Lo-melanin) by chemical and spectroscopic analyses. Although melanin is generally insoluble in both aqueous and organic solvents, the results in this study revealed that Lo-melanin shows water-solubility by means of the added polysaccharide chain. Lo-melanin production of L. oligotrophicus was increased by ultraviolet (UV) exposure, and survival rate of Escherichia coli under UV-irradiated condition was increased by the addition of Lo-melanin to the medium.





  • Polyphasic characterization of an anaerobic hexachlorobenzene-dechlorinating microbial consortium with a wide dechlorination spectrum for chlorobenzenes
    Publication date: July 2015
    Source:Journal of Bioscience and Bioengineering, Volume 120, Issue 1

    Author(s): Xue Zhou , Chunfang Zhang , Dongdong Zhang , Takanori Awata , Zhixing Xiao , Qi Yang , Arata Katayama

    An anaerobic consortium that was capable of reductively dechlorinating hexachlorobenzene (HCB) to benzene was enriched from contaminated sediment. The consortium was capable of dechlorinating all chlorobenzene isomers except 1,4-dichlorobenzene. Singly and doubly flanked chlorines, as well as unflanked meta-substituted chlorines, were dechlorinated, although doubly flanked chlorines were preferred. Formate, acetate and lactate (but not ethanol) could be utilized as optimum electron donors for reductive dechlorination. Alternative electron acceptors, including nitrate and sulfate, completely inhibited HCB degradation, whereas amorphous iron oxide (FeOOH) did not suppress dechlorination activity. No degradation was found in chloramphenicol-treated consortium; however, vancomycin, molybdate, and 2-bromoethanesulfonate did not inhibit HCB dechlorination. The results of inhibitory treatments suggested that the dechlorinators were non-sulfate-reducing gram-negative or vancomycin resistant gram-positive bacteria. In addition to physiological characterization, analyses of 16S rRNA gene library of the consortium and quantitative PCR of 16S rRNA genes suggested that Dehalococcoides sp. was involved in the reductive dechlorination of HCB, and Geobacter sp. may serve as a dechlorinating candidate.





  • Genome sequence determination and metagenomic characterization of a Dehalococcoides mixed culture grown on cis-1,2-dichloroethene
    Publication date: July 2015
    Source:Journal of Bioscience and Bioengineering, Volume 120, Issue 1

    Author(s): Masafumi Yohda , Osami Yagi , Ayane Takechi , Mizuki Kitajima , Hisashi Matsuda , Naoaki Miyamura , Tomoko Aizawa , Mutsuyasu Nakajima , Michio Sunairi , Akito Daiba , Takashi Miyajima , Morimi Teruya , Kuniko Teruya , Akino Shiroma , Makiko Shimoji , Hinako Tamotsu , Ayaka Juan , Kazuma Nakano , Misako Aoyama , Yasunobu Terabayashi , Kazuhito Satou , Takashi Hirano

    A Dehalococcoides-containing bacterial consortium that performed dechlorination of 0.20 mM cis-1,2-dichloroethene to ethene in 14 days was obtained from the sediment mud of the lotus field. To obtain detailed information of the consortium, the metagenome was analyzed using the short-read next-generation sequencer SOLiD 3. Matching the obtained sequence tags with the reference genome sequences indicated that the Dehalococcoides sp. in the consortium was highly homologous to Dehalococcoides mccartyi CBDB1 and BAV1. Sequence comparison with the reference sequence constructed from 16S rRNA gene sequences in a public database showed the presence of Sedimentibacter, Sulfurospirillum, Clostridium, Desulfovibrio, Parabacteroides, Alistipes, Eubacterium, Peptostreptococcus and Proteocatella in addition to Dehalococcoides sp. After further enrichment, the members of the consortium were narrowed down to almost three species. Finally, the full-length circular genome sequence of the Dehalococcoides sp. in the consortium, D. mccartyi IBARAKI, was determined by analyzing the metagenome with the single-molecule DNA sequencer PacBio RS. The accuracy of the sequence was confirmed by matching it to the tag sequences obtained by SOLiD 3. The genome is 1,451,062 nt and the number of CDS is 1566, which includes 3 rRNA genes and 47 tRNA genes. There exist twenty-eight RDase genes that are accompanied by the genes for anchor proteins. The genome exhibits significant sequence identity with other Dehalococcoides spp. throughout the genome, but there exists significant difference in the distribution RDase genes. The combination of a short-read next-generation DNA sequencer and a long-read single-molecule DNA sequencer gives detailed information of a bacterial consortium.





  • Optimization of chemically defined feed media for monoclonal antibody production in Chinese hamster ovary cells
    Publication date: July 2015
    Source:Journal of Bioscience and Bioengineering, Volume 120, Issue 1

    Author(s): Shohei Kishishita , Satoshi Katayama , Kunihiko Kodaira , Yoshinori Takagi , Hiroki Matsuda , Hiroshi Okamoto , Shinya Takuma , Chikashi Hirashima , Hideki Aoyagi

    Chinese hamster ovary (CHO) cells are the most commonly used mammalian host for large-scale commercial production of therapeutic monoclonal antibodies (mAbs). Chemically defined media are currently used for CHO cell–based mAb production. An adequate supply of nutrients, especially specific amino acids, is required for cell growth and mAb production, and chemically defined fed-batch processes that support rapid cell growth, high cell density, and high levels of mAb production is still challenging. Many studies have highlighted the benefits of various media designs, supplements, and feed addition strategies in cell cultures. In the present study, we used a strategy involving optimization of a chemically defined feed medium to improve mAb production. Amino acids that were consumed in substantial amounts during a control culture were added to the feed medium as supplements. Supplementation was controlled to minimize accumulation of waste products such as lactate and ammonia. In addition, we evaluated supplementation with tyrosine, which has poor solubility, in the form of a dipeptide or tripeptide to improve its solubility. Supplementation with serine, cysteine, and tyrosine enhanced mAb production, cell viability, and metabolic profiles. A cysteine–tyrosine–serine tripeptide showed high solubility and produced beneficial effects similar to those observed with the free amino acids and with a dipeptide in improving mAb titers and metabolic profiles.





  • Characterization of bovine serum albumin partitioning behaviors in polymer-salt aqueous two-phase systems
    Publication date: July 2015
    Source:Journal of Bioscience and Bioengineering, Volume 120, Issue 1

    Author(s): Yin Hui Chow , Yee Jiun Yap , Chin Ping Tan , Mohd Shamsul Anuar , Bimo Ario Tejo , Pau Loke Show , Arbakariya Bin Ariff , Eng-Poh Ng , Tau Chuan Ling

    In this paper, a linear relationship is proposed relating the natural logarithm of partition coefficient, ln K for protein partitioning in poly (ethylene glycol) (PEG)-phosphate aqueous two-phase system (ATPS) to the square of tie-line length (TLL 2 ). This relationship provides good fits (r 2  > 0.98) to the partition of bovine serum albumin (BSA) in PEG (1450 g/mol, 2000 g/mol, 3350 g/mol, and 4000 g/mol)-phosphate ATPS with TLL of 25.0–50.0% (w/w) at pH 7.0. Results also showed that the plot of ln K against pH for BSA partitioning in the ATPS containing 33.0% (w/w) PEG1450 and 8.0% (w/w) phosphate with varied working pH between 6.0 and 9.0 exhibited a linear relationship which is in good agreement (r 2  = 0.94) with the proposed relationship, ln K = α′ pH + β′. These results suggested that both the relationships proposed could be applied to correlate and elucidate the partition behavior of biomolecules in the polymer-salt ATPS. The influence of other system parameters on the partition behavior of BSA was also investigated. An optimum BSA yield of 90.80% in the top phase and K of 2.40 was achieved in an ATPS constituted with 33.0% (w/w) PEG 1450 and 8.0% (w/w) phosphate in the presence of 8.5% (w/w) sodium chloride (NaCl) at pH 9.0 for 0.3% (w/w) BSA load.