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Research Article

High-Yield Hydrogen Production from Starch and Water by a Synthetic Enzymatic Pathway

  • Y.-H. Percival Zhang mail,

    To whom correspondence should be addressed. E-mail: ypzhang@vt.edu

    Affiliation: Biological Systems Engineering Department, Virginia Tech, Blacksburg, Virginia, United States of America

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  • Barbara R. Evans,

    Affiliation: Chemical Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, United States of America

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  • Jonathan R. Mielenz,

    Affiliation: Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, United States of America

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  • Robert C. Hopkins,

    Affiliation: Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia, United States of America

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  • Michael W.W. Adams

    Affiliation: Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia, United States of America

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  • Published: May 23, 2007
  • DOI: 10.1371/journal.pone.0000456
  • Published in PLOS ONE

Reader Comments (4)

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Comments in relation to papers by Woodward, Yoshida and energetic analysis

Posted by Patrik_Jones on 25 Sep 2007 at 05:55 GMT

Dear Zhang and co-workers,

I find this work and topic interesting as I am myself in the same field. Some comments;

1) The old work of Woodward et al was stimulating but I guessed it was unlikely to be practical given that their data showed very slow H2-production rate and transient yield-optimum. From the perspective of micro-scale energy-production (or any other scale) it is interesting to compare this approach with the recent papers of Yoshida et al in collaboration with Yukawa at RITE (http://aem.asm.org/cgi/co... and later publications), where the applied aim may be similar? They achieved very high rates of production using whole cells (approximately 10,000 times higher than what is shown in Fig. 2, 11.8 moles H2/L/hr compared to ~1 mmol H2/L/hr, unless I've mixed the numbers up..?), although the yield of formate-dependent H2-production obviously will be limited to 2 at a maximum. A discussion of practical limits with respect to production rates would have been interesting in your paper, particularly with respect to micro-scale application such as mobile phones, etc..? Yoshida et al discussed their rates in relation to a system needed to drive 1 kW PEMFC.

2) The materials and methods says helium was used to sparge the system, although Fig. 5 shows N2 as a carrier gas?? Regardless of what gas is used, I think the question of removal of H2 from any H2-producing reaction obviously will be of applied importance, as it will influence the overall reaction rate. This may be one of the obvious advantages of the cell-free system, since metabolic co-factor couples do not need to be tightly constrained by limitations set by other metabolic reactions to a particular "fixed" steady-state levels. The only research papers that I have found so far that considers removal of H2 from whole-cell production systems seems to be Dutch researchers such as Stams, Claassen, and Bussmann. In a papaer by Van Groenestijn et al (Int. J. Hydrogen Energy 27, 1141-), they estimated the energetic and economic costs of H2-removal on a large production scheme, and it appeared to have a big influence on overall cost of production. A discussion of these issues and aspects would have been interesting.

3) Following on from the above, and from your energy diagram, the reaction you studied obviously is an uphill reaction, energetically, in the absence of the connected fuel cell. Where then does this energy come from in your reaction, given that the reaction appears to proceed very well over a long time period? Is this not H2-removal? It would be nice to see a calculation of estimates of net H2-yield, after subtracting relative loss in energy (expressed as H2) as a result of energetic costs related to H2-removal (if this indeed is the major driving force?). Bruce Logan and co-workers did a similar analysis of the BEAMER-process, a process where electricity had to be applied in order to allow oxidation of acetate to take place, and then back-calculated the relative loss in potential H2 as a result of energy-loss (from the need to supply a constant current). See Liu et al. (Environ. Sci. Technol. 2005, 39, 4317-4320). What happens if you turn off the sparging and carry out the reaction under closed batch conditions?

4) It would also be interesting to see a discussion regarding the relative merit of carrying out synthetic biology reactions in vitro as opposed to in vivo. You mentioned the concept of introducing your system to a minimum genome organism, although it would be difficult to see how the outcome would be much different from introduction to a standard biotechnologically applicable microorganisms? For example, even a minimum genome organism would still most likely rely on glycolysis and glycolytic and PPP intermediates for their basic metabolic needs, and the NADP(H) co-factor couple would possibly be restrained to limit NADPH-dependent H2-synthesis to a much lower maximum partial headspace H2-pressure than what you have in the in vitro system, thereby perhaps requiring even more dilution of H2 in order to maintain sufficiently low pH2-levels to allow the reaction to proceed at reasonable rates? (If this is an issue or not is another question). Then, the possibility of ever achieving a reaction which could be regarded as near-futile (except for the loss of CO2), ie. cyclic PPP, in vivo, would be interesting to also consider. With respect to in vivo vs. in vitro, a more extensive discussion of issues and potentials for either case would be nice to see. For example, broadening your discussion of cost of enzyme production to stability and regeneration, including (in)stability of NADPH, and relating this to issues associated with in vivo H2-production?

Please excuse the long comments, but the topic is interesting and I would be interested to read your response, and yet I realize that no one paper can contain everything. Perhaps next ones?

Cheers,

Patrik


RE: Comments in relation to papers by Woodward, Yoshida and energetic analysis

ypzhang replied to Patrik_Jones on 07 Oct 2007 at 03:20 GMT

Patrik,

I would like to answer your questions and comments briefly. I like this kind of open scientific discussion.

1. Reaction rate. We clearly know that the enzymatic hydrogen rate is much lower than other reported processes because we never optimize enzyme components, their ratio, etc. At current stage, the hydrogen yield is no. 1 (demonstration of proof-of-concept). Now we are working on increasing reaction rates. From engineers' view, in vitro process should have higher reaction rates than in vivo because of no cell membrane slowing substrate and product transfer.

2. The carrier gas was helium. It is too costly, and we will use nitrogen in the future. The removal of hydrogen from aqueous reactants is very important for increasing reaction rates and decreasing product inhibition. For practical application, we will not use carrier gas but use fast product removal. The carrier also dilutes hydrogen concentration so to reduce fuel cell efficiency.

3. The overall reaction has a negative Gibbs energy. Each component reaction is reversible (well-recorded in the literature). The removal of gaseous products is important to drive the overall reaction forward. The close system will result in an equilibrium between products and substrates. The reaction cannot go to a completion.

4. In vivo system can NEVER reach the yield of 12 H2/glucose based on thermodynamics. In vivo system could be a sub-optimal system. Personally I like cell free system. But an in vivo system could save a lot of costs for enzyme production, etc. Two parallel methods will compete for a long time.

We are seeking the funding to go ahead. We cannot go as fast as we expect. (Until now, no significant grants are supporting our research. I am a new assistant professor) Any way, the two main obstacles to this new technology are slow reaction rates and high enzyme costs. We believe that we will solve them one by one.

Best

Percival