Microbial responses to various process disturbances in a continuous hydrogen reactor fed with galactose

Kumar, G.,Park, J.H.,Sivagurunathan, P.,Lee, S.H.,Park, H.D.,Kim, S.H. Society for Bioscience and Bioengineering, Japan ; 2017 Journal of bioscience and bioengineering Vol.123 No.2

<P>In this study, microbial responses of a continuous hydrogen reactor fed with galactose have been investigated. Process disturbances reduced H-2 production performance as well as large fluctuations in microbial diversity. The peak values of the hydrogen yield (HY) was not influenced greatly during the steady state period, and accounted as 2.01 +/- 0.05 and 2.14 +/- 0.03 mol/mol galactose(added), while hydraulic retention time (HRT) was at 12 and 8 h, respectively. Microbial community analysis via 454 pyrosequencing revealed that functional redundancy following changes in the microbial community distribution led to the stability of the fermentation performance. The butyrate to acetate (B/A) ratio well correlated with changes in the microbial community. The energy generation rate and energy yield resulted in the peak values of 134 kJ/L-d and 612 kJ/mol(added). (C) 2016, The Society for Biotechnology, Japan. All rights reserved.</P>

Nitrification resilience and community dynamics of ammonia-oxidizing bacteria with respect to ammonia loading shock in a nitrification reactor treating steel wastewater

Cho, K.,Shin, S.G.,Lee, J.,Koo, T.,Kim, W.,Hwang, S. Society for Bioscience and Bioengineering, Japan ; 2016 Journal of bioscience and bioengineering Vol.122 No.2

<P>The aim of this study was to investigate the nitrification resilience pattern and examine the key ammonia-oxidizing bacteria (AOB) with respect to ammonia loading shocks (ALSs) in a nitrification bioreactor treating steel wastewater. The perturbation experiments were conducted in a 4-L bioreactor operated in continuous mode with a hydraulic retention time of 10 d. Three sequential ALSs were given to the bioreactor (120, 180 and 180 mg total ammonia nitrogen (TAN)/L. When the first shock was given, the nitrification process completely recovered after 14 d of further operation. However, the resilience duration was significantly reduced to 1 d after the second and third ALSs. In the bioreactor, Nitrosomonas aestuarii dominated the other AOB species, Nitrosomonas europaea and N. nitrosa, throughout the process. In addition, the population of N. aestuarii increased with ammonia utilization following each ALS; i.e., this species responded to acute ammonia overloadings by contributing to ammonia oxidation. This finding suggests that N. aestuarii could be exploited to achieve stable nitrification in industrial wastewaters that contain high concentrations of ammonia. (C) 2016, The Society for Biotechnology, Japan. All rights reserved.</P>

Mixed-culture H₂ fermentation performance and the relation between microbial community composition and hydraulic retention times for a fixed bed reactor fed with galactose/glucose mixtures

Anburajan, P.,Park, J.H.,Sivagurunathan, P.,Pugazhendhi, A.,Kumar, G.,Choi, C.S.,Kim, S.H. Society for Bioscience and Bioengineering, Japan ; 2017 Journal of bioscience and bioengineering Vol.124 No.3

<P>This study examined the mesophilic continuous biohydrogen fermentation from galactose and glucose mixture with an initial substrate concentration of 15 g/L (galactose 12 g/L and glucose 3 g/L) as a resembling carbon source of pre-treated red algal hydrolyzate. A fixed bed reactor was fed with the sugar mixture at various hydraulic retention times (HRTs) ranging 12 to 1.5 h. The maximum hydrogen production rate of 52.6 L/L-d was found at 2 h HRT, while the maximum hydrogen yield of 2.3 +/- 0.1 mol/mol hexose(added), was achieved at 3 h HRT. Microbial communities and species distribution were analyzed via quantitative polymerase chain reaction (qPCR) and the dominant bacterial population was found as Clostridia followed by Lactobacillus sp. Packing material retained higher 16S rRNA gene copy numbers of total bacteria and Clostridium butyricum fraction compared to fermentation liquor. The finding of the study has demonstrated that H-2 production from galactose and glucose mixture could be a viable approach for hydrogen production. (C) 2017, The Society for Biotechnology, Japan. All rights reserved.</P>

Reduction of d-lactate content in sauerkraut using starter cultures of recombinant Leuconostoc mesenteroides expressing the ldhL gene

Jin, Q.,Li, L.,Moon, J.S.,Cho, S.K.,Kim, Y.J.,Lee, S.J.,Han, N.S. Society for Bioscience and Bioengineering, Japan ; 2016 Journal of bioscience and bioengineering Vol.121 No.5

<P>The n-form of lactate, which causes metabolic stress upon excessive dietary intake, is mainly produced by Leuconostoc sp., the predominant species in sauerkraut. To shift the metabolic flux of D-lactate from pyruvate to L-lactate, we expressed the L-lactate dehydrogenase (ldhL) gene in Leuconostoc mesenteroides ATCC 8293. The IdhL gene from Lactobacillus plantarum was introduced into L. mesenteroides using the shuttle vectors pLeuCM and pLeuCM42. To elevate the expression level of IdhL in L. mesenteroides, the nucleotides for pyruvate kinase promoter were fused to IdhL and cloned into above vectors to construct pLC18pkL and pLC42pkL. As results, introduction of pLC42pkL in L. mesenteroides significantly improved both L-LDH activity and L-lactate productivity during fermentation, decreasing the D-/L-lactate ratio. When used as a starter culture for sauerkraut fermentation, recombinant L. mesenteroides harboring pLC42pkL increased L-lactate concentration and decreased D-lactate concentration compared to the wild type strain. We newly developed a recombinant L. mesenteroides which has high L-lactate dehydrogenase activity and applied this strain to minimize the harmful effect of D-lactate during the sauerkraut fermentation. To the best of our knowledge, we demonstrate for the first time the effective use of recombinant Leuconostoc sp. for quality improvement of fermented foods. (C) 2015, The Society for Biotechnology, Japan. All rights reserved.</P>

Characterization of a trehalose-degrading enzyme from the hyperthermophilic archaeon Sulfolobus acidocaldarius

Moon, J.H.,Lee, W.,Park, J.,Choi, K.H.,Cha, J. Society for Bioscience and Bioengineering, Japan 2016 Journal of bioscience and bioengineering Vol. No.

<P>We purified a cytosolic trehalase (TreH) from a thermoacidophilic archaeon Sulfolobus acidocaldarius. Enzyme activity in cell-free extracts indicated that trehalose degradation in the cell occurred via the hydrolytic activity of TreH, and not via TreP (phosphorolytic activity) or TreT (transfer activity). TreH was purified to near-homogeneity by DEAE anion exchange chromatography, followed by size exclusion and HiTrap Q anion-exchange chromatography, and its molecular mass was estimated as 40 kDa. Maximum activity was observed at 85 degrees C and pH 4.5. The half-life of TreH was 53 and 41 min at 90 degrees C and 95 degrees C, respectively. TreH was highly specific for trehalose and was inhibited by glucose with a K-i of 0.05 mM. Compared with TreH from other trehalases, TreH from S. acidocaldarius is the most thermostable trehalase reported so far. Furthermore, this is the first trehalase characterized in the Archaea domain. (C) 2015 The Society for Biotechnology, Japan. All rights reserved.</P>

Formation of chitosan-fucoidan nanoparticles and their electrostatic interactions: Quantitative analysis

Lee, E.J.,Lim, K.H. Society for Bioscience and Bioengineering, Japan ; 2016 Journal of bioscience and bioengineering Vol.121 No.1

<P>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. (C) 2015, The Society for Biotechnology, Japan. All rights reserved.</P>

Modified harvest system for enhancing Factor VIII yield in alternating tangential flow perfusion culture

Kim, S.C.,An, S.,Kim, H.K.,Park, B.S.,Na, K.H.,Kim, B.G. Society for Bioscience and Bioengineering, Japan ; 2016 Journal of bioscience and bioengineering Vol.121 No.5

<P>This study describes the development and experimental verification of a modified harvest system to enhance Factor VIII (FVIII) yield in an alternating tangential flow (ATF) perfusion culture. The main innovation of the modified harvest system is the use of check and pinch valves, eliminating the need of a peristaltic pump for harvest. The system was applied to perfusion cultures of Chinese hamster ovary cells, which co-express both recombinant human FVIII (rhFVIII) and von Willebrand factor (vWF). The modified harvest system showed comparable cell growth with the conventional harvest system using a peristaltic pump. The perfusion rate was successfully controlled using the system. In addition, the modified harvest system achieved an approximately 13.6-fold increase in the final concentration yield of FVIII activity and a 1.47-fold increase in the production yield of FVIII activity compared with a peristaltic pump. Enhancement of the yield of FVIII activity resulted from the reduction of FVIII antigen (FVIII:Ag) retention. As a result of transmembrane pressure (TMP) measurement, the reduction of the retained FVIII:Ag was due to the increased TMP, which was caused by the characteristic function of a check valve, compared with a peristaltic harvest system. The modified harvest system developed in this study could be useful to enhance the production yield of other recombinant proteins in ATF perfusion culture. (C) 2015, The Society for Biotechnology, Japan. All rights reserved.</P>

Characterization of a thermostable glycoside hydrolase family 36 α-galactosidase from Caldicellulosiruptor bescii

Lee, A.,Choi, K.H.,Yoon, D.,Kim, S.,Cha, J. Society for Bioscience and Bioengineering, Japan ; 2017 Journal of bioscience and bioengineering Vol.124 No.3

<P>The putative gene cluster involved in the degradation of the raffinose family oligosaccharides (RFO) was identified in Caldicellulosiruptor bescii. Within the cluster, the gene encoding a putative alpha-galactosidase (CbAga36) was cloned and expressed in Escherichia coli. Size exclusion chromatography of the purified rCbAga36 indicated that the native form was a tetramer. Its primary sequence was similar to the family of glycoside hydrolase 36. The purified recombinant CbAga36 (rCbAga36) was optimally active at pH 5.0 and 70 degrees C and had a half-life of 15 h and 10 h at 70 degrees C and 80 degrees C, respectively. rCbAga36 showed high activity with the artificial substrate (p-nitrophenyl cc-D-galactopyranoside, pNPaGal) exhibiting lower Km and higher kat than natural substrates such as melibiose and raffinose. Although rCbAga36 demonstrated preferential activity toward the hydrolysis of RFO such as raffinose and stachyose, it did not degrade the polymeric galactomannans. Our results imply that CbAga36 may play a role in the degradation of RFO, transported into the cytoplasm via a transporter into galactose, which is further utilized as an energy source in C. bescii. Furthermore, its ability to synthesize novel oligosaccharides by transglycosylation renders this enzyme potentially useful for the production of dietary oligosaccharides with novel function. (C) 2017, The Society for Biotechnology, Japan. All rights reserved.</P>

Characteristics of human cell line, F2N78, for the production of recombinant antibody in fed-batch and perfusion cultures

Seo, J.S.,Min, B.S.,Kwon, Y.B.,Lee, S.Y.,Cho, J.M.,Park, K.H.,Yang, Y.J.,Maeng, K.E.,Chang, S.J.,Kim, D.I. Society for Bioscience and Bioengineering, Japan ; 2016 Journal of bioscience and bioengineering Vol.121 No.3

<P>A human hybrid cell line, F2N78, was developed by somatic fusion of HEK293 and Namalwa cells for the production recombinant biopharmaceutical proteins. In this study, we performed perfusion culture to verify its potential in culture process used for human cell expression platform. Cell viability could be maintained over 90% and high viable cell density was obtained at higher than 1.0 x 10(7) cells/mL by bleeding process in perfusion culture. The cells were adapted well in both culture modes, but there were apparent differences in protein quality. Compared to fed-batch culture, degalactosylated forms such as G0F and G0 as well as Man5 showed no significant increases in perfusion culture. In terms of charge variants, acidic peaks increased, whereas main peaks constantly decreased according to the length of culture period in both methods. (C) 2015, The Society for Biotechnology, Japan. All rights reserved.</P>

Alleviation of temperature-sensitive secretion defect of Pseudomonas fluorescens ATP-binding cassette (ABC) transporter, TliDEF, by a change of single amino acid in the ABC protein, TliD

Eom, G.T.,Oh, J.Y.,Park, J.H.,Lim, H.J.,Lee, S.J.,Kim, E.Y.,Choi, J.E.,Jegal, J.,Song, B.K.,Yu, J.H.,Song, J.K. Society for Bioscience and Bioengineering, Japan ; 2016 Journal of bioscience and bioengineering Vol.122 No.3

<P>An ABC transporter, TliDEF, from Pseudomonas fluorescens SIK W1, mediates the secretion of its cognate lipase, TliA, in a temperature-dependent secretion manner; the TliDEF-mediated secretion of TliA was impossible at the temperatures over 33 degrees C. To isolate a mutant TliDEF capable of secreting TliA at 35 degrees C, the mutagenesis of ABC protein (TliD) was performed. The mutated tliD library where a random point mutation was introduced by error-prone PCR was coexpressed with the wild-type WE, tliF and tliA in Escherichia con. Among approximately 10,000 colonies of the tliD library, we selected one colony that formed transparent halo on LB-tributyrin plates at 35 degrees C. At the growth temperature of 35 degrees C, the selected mutant TliD showed 1.75 U/ml of the extracellular lipase activity, while the wild-type TliDEF did not show any detectable lipase activity in the culture supernatant of E. coli. Moreover, the mutant TliD also showed higher level of TliA secretion than the wild-type TliDEF at other culture temperatures, 20 degrees C, 25 degrees C and 30 degrees C. The mutant TliD had a single amino acid change (Ser287Pro) in the predicted transmembrane region in the membrane domain of TliD, implying that the corresponding region of TliD was important for causing the temperature-dependent secretion of TliDEF. These results suggested that the property of ABC transporter could be changed by the change of amino acid in the ABC protein. (C) 2016, The Society for Biotechnology, Japan. All rights reserved.</P>