Browsing by Author "Lin, Hsiao-Hsien"

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Analysis of microbial diversity and optimal conditions for enhanced biogas production from swine waste anaerobic digestion
(2013-09-01)
Chung, Ying-Chien
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Chang, Yu-Cheng
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Chen, Yu-Pei
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Lin, Wei-Chih
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Lin, Hsiao-Hsien
Swine wastewater pretreated by solid-liquid separation was optimized for biogas production and water purification. Dynamic diversity of the bacterial community in the anaerobic plug flow reactor was investigated under various temperatures and hydraulic retention times (HRT). Results of batch experiments indicated that under optimal operating conditions, biogas production, methane production rate, and chemical oxygen demand (COD) removal efficiencies increased up to 26%, 52%, and 44%, respectively. In the continuous experiments, optimal operating conditions for the plug flow operator were found to be HRT of 8 d and temperature of 35 degrees C, under which relatively high CH4 concentration (67%), CH4 production rate (4.6 l/d), and total COD removal (79%) were achieved. A minimum of 12 bands was observed in the denaturing gradient gel electrophoresis profile. Phylogenetic analysis revealed the predominance of phyla Firmicutes, Proteobacteria, and Euryarchaeota in the anaerobic plug-flow reactor. However, the occurrence and prevalence of specific bacterial species varied with HRT and temperature. This study establishes the optimal operating parameters for the anaerobic plug-flow digestion of swine wastewater, which can be employed in a full-scale reactor to maximize biogas production and to purify water quality. (C) 2013 AIP Publishing LLC.
High glucose enhances cAMP level and extracellular signal-regulated kinase phosphorylation in Chinese hamster ovary cell: Usage of Br-cAMP in foreign protein beta-galactosidase expression
(2017-07-01)
Lin, Hsiao-Hsien
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Lee, Tsung-Yih
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Liu, Ting-Wei
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Tseng, Ching-Ping
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生物科技學系
Glucose is a carbon source for Chinese hamster ovary (CHO) cell growth, while low growth rate is considered to enhance the production of recombinant proteins. The present study reveals that glucose concentrations higher than 1 g/L reduce the growth rate and substantially increase in cAMP (similar to 300%) at a high glucose concentration (10 g/L). High glucose also enhances the phosphorylation of extracellular signal-regulated kinase (ERK) and p27(kiP) by Western blot analysis. To determine whether the phosphorylation of ERK is involved in the mechanism, a cyclic-AMP dependent protein kinase A (PICA) inhibitor (H-8) or MEK (MAPKK) inhibitor (PD98059) was added to block ERK phosphorylation. We show that both the high glucose-induced ERIC phosphorylation and growth rate return to baseline levels. These results suggest that the cAMP/PKA and MAP signaling pathways are involved in the above mentioned mechanism. Interestingly, the direct addition of 8-bromo-cAMP (Br-cAMP), a membrane-permeable cAMP analog, can mimic the similar effects produced by high glucose. Subsequently Br-cAMP could induce beta-galactosidase (beta-Gal) recombinant protein expression by 1.6-fold. Furthermore, Br-cAMP can additionally enhance the (beta-Gal production (from 2.8- to 4.5 fold) when CHO cells were stimulated with glycerol, thymidine, dimethyl sulfoxide, pentanoic acid, or sodium butyrate. Thus, Br-cAMP may be used as an alternative agent in promoting foreign protein expression for CHO cells. (C) 2017, The Society for Biotechnology, Japan. All rights reserved.
High Growth Rate Downregulates fumA mRNA Transcription but Is Dramatically Compensated by Its mRNA Stability in Escherichia coli
(2012-05-01)
Lin, Hsiao-Hsien
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Lin, Ching-Hsueh
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Hwang, Shiaw-Min
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Tseng, Ching-Ping
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生物科技學系
Little is known about the association among the transcription, post-transcription, and protein production of the fumA gene. This study demonstrates that increasing growth rate (k) from 0.24/h to 0.96/h causes a marked eightfold reduction in fumA transcription as assessed using the beta-galactosidase activity from fumA promoter fused with a lacZ reporter. It was further confirmed using Northern blot analysis. Most interestingly, the FumA protein levels remained unchanged over the growth rate, as indicated by Western blot analysis. Therefore, whether the reduced fumA mRNA expression under the high growth rate can be overcome by increasing the stability of the fumA mRNA was tested. The half-life of fumA mRNA was established to significantly increase by fivefold when the growth rate was increased to 0.96/h. This finding suggests that the cells could turn down the expression of fumA mRNA because of increased stability of its mRNA under the high growth rate. This notion indicates that mRNA stability plays an essential role in maintaining a critical cellular level of a given protein when the mRNA transcript is downregulated by a metabolic event.
Inhibitory Effect of Excessive Glucose on Its Biochemical Pathway and the Growth of Chinese Hamster Ovary (CHO) Cells
(2015-01-02)
Lee, Tsung-Yih
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Lin, Hsiao-Hsien
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Chen, Chien-Lung
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Hwang, Shiaw-Min
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Tseng, Ching-Ping
In this study, we found the specific growth rate of CHO cells being reduced when glucose increased from 1 to 10g/L. Under high glucose, we show a 41% increase in lactate dehydrogenase activity consistent with a 70% rate increase in lactate production. The pyruvate kinase activity was significantly increased. However, the activities of citrate synthase, isocitrate dehydrogenase, and malate dehydrogenase involved in the TCA cycle were attenuated. We propose that pyruvate produced from glycolysis is utilized in a lesser extent over the TCA cycle under high-glucose conditions. Thus, the pyruvate is largely converted to lactate, which is toxic to the cells.
Negative Effect of Glucose on ompA mRNA Stability: a Potential Role of Cyclic AMP in the Repression of hfq in Escherichia coli
(2011-10-01)
Lin, Hsiao-Hsien
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Hsu, Chi-Cheng
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Yang, Chi-Dung
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Ju, Yih-Wei
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Chen, Yi-Pei
Glucose is a carbon source that is capable of modulating the level of cyclic AMP (cAMP)-regulated genes. In the present study, we found that the stability of ompA mRNA was reduced in Escherichia coli when glucose (40 mM) was present in Luria-Bertani (LB) medium. This effect was associated with a low level of cAMP induced by the glucose. The results were confirmed with an adenylyl cyclase mutant with low levels of cAMP that are not modulated by glucose. Northern blot and Western blot analyses revealed that the host factor I (Hfq) (both mRNA and protein) levels were downregulated in the presence of cAMP. Furthermore, we showed that a complex of cAMP receptor protein (CRP) and cAMP binds to a specific P3(hfq) promoter region of hfq and regulates hfq expression. The regulation of the hfq gene was confirmed in vivo using an hfq-deficient mutant transformed with an exogenous hfq gene containing the promoter. These results demonstrated that expression of hfq was repressed by the CRP-cAMP complex. The presence of glucose resulted in increased Hfq protein levels, which decreased ompA mRNA stability. An additional experiment showed that cAMP also increased the stability of fur mRNA. Taken together, these results suggested that the repression of Hfq by cAMP may contribute to the stability of other mRNA in E. coli.
Regulatory Role of cAMP Receptor Protein over Escherichia coli Fumarase Genes
(2012-06-01)
Chen, Yu-Pei
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Lin, Hsiao-Hsien
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Yang, Chi-Dung
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Huang, Shin-Hong
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Tseng, Ching-Ping
Escherichia coli expresses three fumarase genes, namely, fumA, fumB, and fumC. In the present study, catabolite repression was observed in the fumA-lacZ and fumC-lacZ fusion strains, but not in the fumB-lacZ fusion strain. The Crp-binding sites in fumA and fumC were identified using an electrophoretic mobility shift assay and footprint analysis. However, the electrophoretic mobility shift assay did not detect band shifts in fumB. Fnr and ArcA serve as transcription regulators of fumarase gene expression. In relation to this, different mutants, including Delta cya, Delta crp, Delta fnr, and Delta arcA, were used to explore the regulatory role of Crp over fumA and fumC. The results show that Crp is an activator of fumA and fumC gene expression under various oxygen conditions and growth rates. ArcA was identified as the dominant repressor, with the major repression occurring at 0-4% oxygen. In addition, Fnr was confirmed as a repressor of fumC for the first time. This study elucidates the effects of Crp on fumarase gene expression.