Homologous overexpression of [FeFe] hydrogenase in Enterobacter cloacae IIT-BT 08 to enhance hydroge

The present study investigated the influence of increase in intracellular [FeFe] hydrogenase levels, in Enterobacter cloacae IIT-BT 08, on the formation of molecular hydrogen. The hydA gene from E. cloacae IIT-BT 08 was successfully amplified and cloned downstream of a tac promoter in an Escherichiacoli-Enterobacter reconstructed pGEX-Kan shuttle vector and introduced into E. cloacae. Finally E. cloacae strain carrying multiple copies of pGEX-Kan-hydA vector was developed. Homologous overexpression of the [FeFe] hydrogenase gene increased the hydrogenase activity by1.3-fold as compared to the wild type. SDS–PAGE confirmed the successful expression of the GST-tagged hydA protein. The hydrogen yield and rate of production in recombinant strain were found to be 1.2-fold and 1.6-fold higher, respectively, compared to the wild type strain. This was found to be concomitant with the shift in the metabolic pathway. In addition, feasibility of using cheese whey as a substrate for biohydrogen production and the effect of its supplementation with yeast extract as nitrogen source was studied for both the wild type and the recombinant strain. It was found that supplementation with 0.3% (w/v) yeast extract enhanced hydrogen production from whey. Further, the yield and rate of hydrogen production from the recombinant was found to be more promising as compared to the wild type.

[1]  Debabrata Das,et al.  Continuous hydrogen production by immobilized Enterobacter cloacae IIT-BT 08 using lignocellulosic materials as solid matrices. , 2001 .

[2]  J. Zeikus,et al.  Regulation of hydrogen metabolism in Butyribacterium methylotrophicum by substrate and pH , 1996, Applied Microbiology and Biotechnology.

[3]  Narendra Kumar,et al.  Production and purification of α-amylase from hydrogen producing Enterobacter cloacae IIT-BT 08 , 2000 .

[4]  F. Loewus.  Improvement in Anthrone Method for Determination of Carbohydrates , 1952 .

[5]  Luis Manuel Rosales-Colunga,et al.  Hydrogen production by Escherichia coli ΔhycA ΔlacI using cheese whey as substrate , 2010 .

[6]  Luis Manuel Rosales-Colunga,et al.  Continuous biohydrogen production using cheese whey: Improving the hydrogen production rate , 2009 .

[7]  A. E. Greenberg,et al.  Standard methods for the examination of water and wastewater : supplement to the sixteenth edition , 1988 .

[8]  D. Das,et al.  Improvement of Biohydrogen Production Under Decreased Partial Pressure of H2 by Enterobacter cloacae , 2006, Biotechnology Letters.

[9]  Tatsushi Kawai,et al.  Biological production of hydrogen from cellulose by natural anaerobic microflora , 1995 .

[10]  Mei-Ling Chong,et al.  Biohydrogen production by Clostridium butyricum EB6 from palm oil mill effluent , 2009 .

[11]  Bruce E Logan,et al.  Biological hydrogen production by Clostridium acetobutylicum in an unsaturated flow reactor. , 2006, Water research.

[12]  Byung Hoon Jo,et al.  Production of biohydrogen by recombinant expression of [NiFe]-hydrogenase 1 in Escherichia coli , 2010, Microbial cell factories.

[13]  Marion B. Ansorge-Schumacher,et al.  Influence of hydrogenase overexpression on hydrogen production of Clostridium acetobutylicum DSM 792 , 2010 .

[14]  D. Das,et al.  Purification and characterization of [Fe]-hydrogenase from high yielding hydrogen-producing strain, Enterobacter cloacae IIT-BT08 (MTCC 5373) , 2009 .

[15]  Ruihong Zhang,et al.  Biohydrogen production from cheese processing wastewater by anaerobic fermentation using mixed microbial communities , 2007 .

[16]  Debabrata Das,et al.  Redirection of biochemical pathways for the enhancement of H2 production by Enterobacter cloacae , 2001, Biotechnology Letters.

[17]  R. Zall,et al.  Optimizing alcohol production from whey using computer technology , 1985, Biotechnology and bioengineering.

[18]  Zsófia Kádár,et al.  Yields from glucose, xylose, and paper sludge hydrolysate during hydrogen production by the extreme thermophile Caldicellulosiruptor saccharolyticus , 2004, Applied biochemistry and biotechnology.

[19]  E. Crabbe,et al.  Influence of initial pH on hydrogen production from cheese whey. , 2005, Journal of biotechnology.

[20]  Jun Cheng,et al.  Heterologous expression of a hydrogenase gene in Enterobacter aerogenes to enhance hydrogen gas production , 2009 .

[21]  Gustavo Davila-Vazquez,et al.  Fermentative hydrogen production in batch experiments using lactose, cheese whey and glucose: Influence of initial substrate concentration and pH , 2008 .

[22]  Sang-Eun Oh,et al.  Biohydrogen gas production from food processing and domestic wastewaters , 2005 .

[23]  T. I. Hedrick,et al.  Nutritional Composition of Sweet- and Acid-type Dry Wheys. II. Vitamin, Mineral, and Calorie Contents , 1977 .

[24]  F. Kosikowski,et al.  IMPROVING ALCOHOL FERMENTATION IN CONCENTRATED ULTRAFILTRATION PERMEATES OF COTTAGE CHEESE WHEY , 1978 .

[25]  Giorgio Valle,et al.  Functional insights from the structural modelling of a small Fe-hydrogenase. , 2006, Biochemical and biophysical research communications.

[26]  C. Lin,et al.  Biohydrogen production by mesophilic fermentation of food wastewater. , 2004, Water science and technology : a journal of the International Association on Water Pollution Research.

[27]  Bruce E Logan,et al.  Inhibition of biohydrogen production by undissociated acetic and butyric acids. , 2005, Environmental science & technology.

[28]  C-C. Chen,et al.  Kinetics of hydrogen production with continuous anaerobic cultures utilizing sucrose as the limiting substrate , 2001, Applied Microbiology and Biotechnology.

[29]  K. Korkmaz,et al.  Comparative Evaluation of Bio-Hydrogen Production From Cheese Whey Wastewater Under Thermophilic and Mesophilic Anaerobic Conditions , 2009 .

[30]  Debabrata Das,et al.  Improvement of biohydrogen production by Enterobacter cloacae IIT-BT 08 under regulated pH. , 2011, Journal of biotechnology.

[31]  A. J. Mawson,et al.  Bioconversions for whey utilization and waste abatement , 1994 .

[32]  Michael Kornaros,et al.  Biohydrogen and methane production from cheese whey in a two-stage anaerobic process , 2008 .

[33]  L. Borzacconi,et al.  Feasibility of biohydrogen production from cheese whey using a UASB reactor: Links between microbial community and reactor performance , 2009 .

[34]  D. Lee,et al.  Molecular characterization and homologous overexpression of [FeFe]-hydrogenase in Clostridium tyrobutyricum JM1 , 2010 .

[35]  Gerasimos Lyberatos,et al.  Production of Gaseous Biofuels and Electricity from Cheese Whey , 2011 .

[36]  H Yokoi,et al.  Microbial hydrogen production from sweet potato starch residue. , 2001, Journal of bioscience and bioengineering.

[37]  Michael Kornaros,et al.  Using cheese whey for hydrogen and methane generation in a two-stage continuous process with alternative pH controlling approaches. , 2009, Bioresource technology.

[38]  Tong Zhang,et al.  Biohydrogen production from starch in wastewater under thermophilic condition. , 2003, Journal of environmental management.

[39]  D. Das,et al.  Molecular cloning, characterization, and overexpression of a novel [Fe]-hydrogenase isolated from a high rate of hydrogen producing Enterobacter cloacae IIT-BT 08. , 2004, Biochemical and biophysical research communications.

[40]  Hang-Sik Shin,et al.  Biohydrogen production by anaerobic fermentation of food waste , 2004 .

[41]  Hang-Sik Shin,et al.  Effect of substrate concentration on hydrogen production and 16S rDNA-based analysis of the microbial community in a continuous fermenter , 2006 .

[42]  Han-Qing Yu,et al.  Hydrogen production from rice winery wastewater in an upflow anaerobic reactor by using mixed anaerobic cultures , 2002 .

[43]  Jean-Paul Schwitzguébel,et al.  Hydrogen production by Clostridium thermolacticum during continuous fermentation of lactose , 2004 .

[44]  Y. Ueno,et al.  Hydrogen Production from Industrial Wastewater by Anaerobic Microflora in Chemostat Culture , 1996 .

[45]  Yinguang Chen,et al.  Mesophilic Biohydrogen Production from Food Waste , 2008, 2008 2nd International Conference on Bioinformatics and Biomedical Engineering.

[46]  Kenji Morimoto,et al.  Overexpression of a hydrogenase gene in Clostridium paraputrificum to enhance hydrogen gas production. , 2005, FEMS microbiology letters.

[47]  D. Das,et al.  Microbial hydrogen production with Bacillus coagulans IIT-BT S1 isolated from anaerobic sewage sludge. , 2007, Bioresource technology.

[48]  Hisham Hafez,et al.  Biological Hydrogen Production from Corn-Syrup Waste Using a Novel System , 2009 .