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Virginia Tech team engineers optimized synthetic enzymatic pathway for high-yield production of H2 directly from biomass

A team of Virginia Tech researchers and colleagues has demonstrated the complete conversion of glucose and xylose from pretreated plant biomass to H2 and CO2 based on an in vitro synthetic enzymatic pathway crafted from more than 10 purified enzymes. Glucose and xylose were simultaneously converted to H2 with a yield of two H2 per carbon, the maximum possible yield.

The researchers used a nonlinear kinetic model fitted with experimental data to identify the enzymes that had the greatest impact on reaction rate and yield. After optimizing enzyme loadings using this model, volumetric H2 productivity was increased 3-fold to 32 mmol H2⋅L−1⋅h−1. The productivity was further enhanced to 54 mmol H2⋅L−1⋅h−1 by increasing reaction temperature, substrate, and enzyme concentrations—an increase of 67-fold compared with the initial studies using this method.

The gaseous hydrogen can be separated from aqueous substrates easily, greatly decreasing product separation costs, and avoid reconcentrating sugar solutions. This greatly reduces processing costs from biomass hydrolyzate sub- strates and avoids inhibition from fermentation products and/or potentially toxic compounds from biomass hydrolyzate.

In an open access paper describing the technology published in Proceedings of the National Academy of Sciences (PNAS), the team suggests that distributed hydrogen production based on evenly distributed less-costly biomass could accelerate the implementation of a hydrogen economy.

Pathway depicting the enzymatic conversion of biomass to hydrogen and CO2. Full names of enzymes used are polyphosphate glucokinase (PPGK), xylose isomerase (XI), xylulokinase (XK), glucose 6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), ribose 5-phosphate isomerase (R5PI), ribulose 5-phosphate epimerase (Ru5PE), transketolase (TK), transaldolase (TAL), triose phosphate isomerase (TIM), aldolase (ALD), fructose 1,6- bisphosphate (FBP), phosphoglucose isomerase (PGI), hydrogenase (H2ase), and Cellic Ctec2 cellulase (Ctec2). source: Rollins et al. Click to enlarge.

In this pathway cellulose and hemicellulose were first completely converted to glucose and xylose, which in turn served as substrates for phosphorylation and hydrogen generation, using an enzyme mixture. In preliminary experiments we attempted simultaneous saccharification and hydrogen production but this required compromising the pH optimum of either the hydrogen-producing enzyme mixture (pH optimum of 7.5) or the cellulase–hemicellulase mixture (pH optimum of 4.8). Separating the two processes allowed for more careful control of each system. However, combining these process steps is a logical step forward to minimize production costs.

A more advanced and likely more efficient integrated approach could make use of cello-oligomer phosphorylases. For example, a cellulase phosphorylase could use the energy of glycosidic bond hydrolysis to catalyze phosphorylation of long chain cellulose molecules, although so far such an enzyme has yet to be discovered or engineered.

—Rollin et al.

The team already has received significant funding for the next step of the project, which is to scale up production to a demonstration size.

Lonnie O. Ingram, director of the Florida Center for Renewable Chemicals and Fuels at the University of Florida, who is familiar with the work but not associated with the team, commented that the work represents a revolutionary approach that offers many new advantages.

These researchers have certainly broadened the scope of our thinking about metabolism and how it plays into the future of alternative energy production.

—Lonnie Ingram

Joe Rollin, a former doctoral student of Zhang’s at Virginia Tech and co-founder with Zhang of the start-up company Cell-free Bioinnovations, is the lead author on the paper.

This work builds upon previous studies Zhang’s team has done with xylose, the most abundant simple plant pentose sugar, to produce hydrogen yields that previously were attainable only in theory.

We believe this exciting technology has the potential to enable the widespread use of hydrogen fuel cell vehicles around the world and displace fossil fuels.

—Joe Rollin

The project was funded in part by the Shell GameChanger initiative and the National Science Foundation’s Small Business Technology Transfer program.


  • Joseph A. Rollin, Julia Martin del Campo, Suwan Myung, Fangfang Sun, Chun You, Allison Bakovic, Roberto Castro, Sanjeev K. Chandrayan, Chang-Hao Wu, Michael W. W. Adams, Ryan S. Senger, and Y.-H. Percival Zhang (2015) “High-yield hydrogen production from biomass by in vitro metabolic engineering: Mixed sugars coutilization and kinetic modeling” PNAS doi: 10.1073/pnas.1417719112



Since it is more directly relevant here I will replicate a post I made on another thread, where EP queried how significant biomass compared to fuel needs:

Biomass production is put here at 140 billion tons a year:

They give that as the energy equivalent of 50 billion tons of oil.

Oil production runs just over 4 billion tons a year:

Clearly not all the biomass will be used, and the efficiencies will typically give the overall numbers a big hit.

Equally clearly the potential is substantial relative to our use, as other resources such as solar will also take up a large part of the load.

For the most common simple sugar in plants, xylose:

'The team liberates the high-purity hydrogen under mild reaction conditions at 122 degrees and normal atmospheric pressure. The biocatalysts used to release the hydrogen are a group of enzymes artificially isolated from different microorganisms that thrive at extreme temperatures, some of which could grow at around the boiling point of water.

The researchers chose to use xylose, which comprises as much as 30 percent of plant cell walls. Despite its abundance, the use of xylose for releasing hydrogen has been limited. The natural or engineered microorganisms that most scientists use in their experiments cannot produce hydrogen in high yield because these microorganisms grow and reproduce instead of splitting water molecules to yield pure hydrogen.

To liberate the hydrogen, Virginia Tech scientists separated a number of enzymes from their native microorganisms to create a customized enzyme cocktail that does not occur in nature. The enzymes, when combined with xylose and a polyphosphate, liberate the unprecedentedly high volume of hydrogen from xylose, resulting in the production of about three times as much hydrogen as other hydrogen-producing microorganisms.

The energy stored in xylose splits water molecules, yielding high-purity hydrogen that can be directly utilized by proton-exchange membrane fuel cells. Even more appealing, this reaction occurs at low temperatures, generating hydrogen energy that is greater than the chemical energy stored in xylose and the polyphosphate. This results in an energy efficiency of more than 100 percent — a net energy gain. That means that low-temperature waste heat can be used to produce high-quality chemical energy hydrogen for the first time. Other processes that convert sugar into biofuels such as ethanol and butanol always have energy efficiencies of less than 100 percent, resulting in an energy penalty.

In his previous research, Zhang used enzymes to produce hydrogen from starch, but the reaction required a food source that made the process too costly for mass production.

The commercial market for hydrogen gas is now around $100 billion for hydrogen produced from natural gas, which is expensive to manufacture and generates a large amount of the greenhouse gas carbon dioxide. Industry most often uses hydrogen to manufacture ammonia for fertilizers and to refine petrochemicals, but an inexpensive, plentiful green hydrogen source can rapidly change that market.

“It really doesn’t make sense to use non-renewable natural resources to produce hydrogen,” Zhang said. “We think this discovery is a game-changer in the world of alternative energy.”'


So while this might not be a total solution to all transport needs, it could potentially provide all the power needed for agriculture, in, for instance, the US Midwest, and in addition provide the hydrogen for the production of ammonia fertilizer.

For transport it would seem to have great potential to provide low carbon hydrogen for fuel cell vehicles, perhaps in a PHEV configuration, so that every day running around is done on electricity, perhaps from solar, and the cars only need a ~12kwh battery instead of a very large pack such as the Tesla's have, as longer runs are done on hydrogen.

A billion tons of oil equivalent here, and a billion there, can really add up!



The guys at Virginia tech are actually significantly more optimistic than the very conservative guesstimate I gave:

'The researchers used cellulosic materials isolated from wood chips, but crop waste or switchgrass could also be used. “If a small fraction – 2 or 3 percent – of yearly biomass production were used for sugar-to-hydrogen fuel cells for transportation, we could reach transportation fuel independence,” Zhang said. (He added that the 3 percent figure is for global transportation needs. The U.S. would actually need to convert about 10 percent of biomass – which would be 1.3 billion tons of usable biomass).'



Two memes which were bandied around are shown to be fallacious here:

1.Fuel cells and hydrogen is fossil fuel consumption by another name.

2. Fuel cell cars are hopelessly inefficient, with some really dodgy arithmetic often accepted as gospel based on the premise that the hydrogen comes from electrolysis, and the power for EVs from solar with storage problems solved by magical and unspecified means.

The widespread use of biomass would use a resource currently ploughed into the ground, which ain't a bad way to increase the efficiency provided that you can engineer around lugging the biomass itself too far, as it is heavy and bulky.

This would pretty well do this, although of course it is not a done deal.

But then neither are batteries cheap enough and energy dense enough to allow ~100kwh cars at reasonable prices.


Conclusions for tomorrow shouldn't be drawn from todays SOA. There are some very interesting and exciting innovations in the pipeline that are bound to bear fruit in the next 5 to 10 years.
Here are just two of many more:
Once these functional prototypes advance to SOA, any thoughts to H2 technology can be forgotten for mobility purposes.
Of course the world is big and harbors plenty of fools. Building fool cells and targeting this paticular group would still be a lucrative business deal.


The world certainly harbours plenty of fools, chiefly those who seek to dismiss whole fields of technology on the basis of their entirely speculative assumptions that those they happen to favour will get all the breaks, whilst those they don't suffer standstill.

'The trouble with the world is that the stupid are cocksure and the intelligent are full of doubt.'

Bertrand Russell


Perhaps instead of conducting Party rallies with lectures on the 'Inevitable Triumph of BEVs' which are about as soundly based as other semi-religious inevitable triumphs have been in the past, someone would lay out, with appeal to the numbers and references, why biomass can't work to produce large amounts of hydrogen and assist in a hydrogen economy.

Whilst they are at it, they could also give precise details on why the several approaches to direct solar to hydrogen which are presently making rapid advances are doomed.


This could be the prod that future H2 economy required to better compete with fossil fuels and batteries.

Canada, USA, Russia, Brazil and many other countries have and/or could grow enough biomass to produce enough H2 for FCEVs (and many other uses) to complement batteries and progressively replace ICEVs and CPPs.

FCEVs may become an option of choice in places with very long cold winters.

Multiple distributed large FCs could eventually make Solar and Wind energy a 24/7 solution (and replace CPPs) in many places.


I know this is the GreenCARcongress website, but Yoatman and others seem to totally dismiss the broader applications of fuel cells, including home heating and electric production, grid balancing of renewables, etc. Why does one technology have to be judged as the WINNER and for one purpose only?


Other viewpoints are a fine thing, but when it is simply a rehash of prejudice without any attempt at taking on board new information, and when the technologies changing leads to not the slightest dent in the pre-conceived position, or any gesture towards actually thinking about the issues, it is tedious.

Fine quote of someone's advertising slogan though.

“If a small fraction – 2 or 3 percent – of yearly biomass production were used for sugar-to-hydrogen fuel cells for transportation, we could reach transportation fuel independence,” Zhang said.

Dave, it was in discussion with you that I calculated that biomass-to-hydrogen was adequate to supply the US transportation market.  More than adequate, as I recall; my calculation came out about 3x basic requirements, and the biomass cost appeared quite reasonable.  But that doesn't mean that biomass-to-hydrogen would get any traction absent mandates, and there's a long way between the lab bench and industrial-scale production.  In the mean time SMR would rule the roost, and the purveyors of natural gas would work to keep it that way.

There's the basic issue that biomass is subject to bad growth years and is, at its root, taken from the limited productivity that nature is capable of making.  We need to be careful with this, especially promoting it as a panacea.  And again... what about everything else?


Any comments I made regarding the quality of debate were certainly not aimed in your direction, as I much appreciate your comments and analysis.

'We need to be careful with this, especially promoting it as a panacea.'

Which is why I said for instance:
'So while this might not be a total solution to all transport needs, it could potentially provide all the power needed for agriculture, in, for instance, the US Midwest, and in addition provide the hydrogen for the production of ammonia fertilizer.'

As for:
'In the mean time SMR would rule the roost, and the purveyors of natural gas would work to keep it that way.'

I don't see why this is more the case than that the NG industry (and the coal) industry will work to continue their present dominance of electricity production, so your criticism does not appear to me even handed.

I think the link I provided gives a good answer to your (and mine!) question about how substantial the resource is, and the answer is clearly that it is very large, even in relation to potential uses.

As my comments in reply to the analysis you did and which you linked make clear, I have had severe reservations about biomass for energy, most particularly about crops grown specifically for the purpose with their water use and displacement of crops for food.

This is different as it answers most of the issues.

It used waste not, crops grown for the purpose.

It can be processed locally, instead of heavy mass being transported long distances.

It is low temperature, reducing energy costs.

It is something which is clearly worth doing anyway, aside from its potential uses in transport, as it would revolutionise and localise energy for agriculture, including for farm machinery and fertiliser.

I would also see it has having the potential, if all goes well, to provide hydrogen to cover long distance transport, avoiding the need for very large batteries and away from home charging networks.


Many countries (Canada, Russia, Brazil, USA, Indonesia, etc) have enough natural biomass for most of the feed stock required to sustain a strong H2 economy.

New biomass, using less water and (less or no) food production land, could be developed.

Existing wastes could also become another source of essential feed stock.

Feed stock (and H2) transport could be limited with the installation of H2 transformers in the right places.

I think the link I provided gives a good answer to your (and mine!) question about how substantial the resource is, and the answer is clearly that it is very large, even in relation to potential uses.

This extensively referenced piece by The Breakthrough Institute documents just how small the resource actually is, how much damage is done in its harvest, and how the push toward biofuels is driven by romanticism.  Seriously, consider the example of Massachusetts given there.  Suppose you could convert the biomass to electricity with 100% efficiency.  You'd still be in a massive hole.

It used waste not, crops grown for the purpose.
It can be processed locally, instead of heavy mass being transported long distances.
It is low temperature, reducing energy costs.

Maybe that can ultimately make it successful in both economic and ecological terms, but given that the problem isn't the process but the feedstock I am betting the other way.


Using biomass and wastes to produce H2 for future FCEVs, for large FCs to produce electricity when needed to make REs 24/7, for continued production of essential fertilizers and other chemicals etc., is as strong possibility in many countries but probably not in Monaco.

It could be a much better solution than the current production of ethanol with corn and sugar cane for our gas guzzling polluting ICEVs and coal for our polluting CPPs.

Eventually, we will have to make better choices?


this process not only yields pure H2, but also pure CO2, which can be sequestered relatively easy. This would effectively result in carbon-negative fuel, compensating for the errors of yesterday.

Henry Gibson

Biomass energy failed in the UK centuries ago as it did in Spain and Iceland and as it is now doing in developing countries where much of the day is being spent destroying natural forests to collect or make fuel for merely cooking not driving automobiles. Every kind of biomass can be used to make food. Much biomass is now used by organisms in the soil to produce nitrogen compounds from the air to keep the soil fertile. Vast tracks of naturtal forests are bein converted to tree plantations for production of bio-oils demanded by european directives for biofuels. If counties dont have enough land for producing biofuels they should not import them. It is very informative to have the religeous nature of many adament beliefs about energy and other issues pointed out.

Any adament beliefs about dangers of the world should be put aside until the dangers of tobacco consumption is eliminated. This extends to the worries and laws about mercury, lead, cadmium and small amounts of radioactive materials; Bananas are radioactive; as all good foods have been for billions of years because all natural potassium is radioactive. Look up potassium. ..HG..

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