Earth Day: Biophysics Research on Biofuels

April 22 is Earth Day. The goal of Earth Day, established in 1970, is to draw public attention to issues affecting the environment. In honor of the event, we spoke with Biophysical Society member Gnana S. Gnanakaran of Los Alamos National Laboratory (LANL) about his research on biofuels and the role of LANL in pioneering biofuel research.


What is the connection between your research and biofuels?

Cellulose, an assembly of glucose polymers, is a vital renewable energy resource originating from plants. A major barrier for biofuel production is the efficient extraction of cellulose fibers from biomass and their degradation to glucose. We conduct theoretical studies on biomass in order to obtain a molecular level understanding of the resistant properties of the cell wall components. Our studies probe these properties using computational techniques at varying levels of resolution comprising quantum chemical calculations, all atom and coarse-grained molecular dynamics simulations, polymer and statistical mechanical models, and agent based and mechanistic kinetic models. Our multiscale approach towards the molecular level understanding of biomass architecture and breakdown has offered significant new strategies to improve biomass conversion.

Why is your research important to the public?

Renewable liquid biofuel for transportation promises to replace some of our non-renewable fossil fuel and reduce greenhouse gas emissions. This is evident from the rising popularity of ethanol as a biofuel, particularly in blends with gasoline and diesel. To be competitive, the production cost of ethanol needs to be reduced, including the cost of raw materials as well as their conversion to fuel. Many key issues in lowering the cost are related to the cellulose-to-ethanol conversion technologies, which are hampered by uncertainties in the physical properties of the feedstock. The conversion of cellulosic biomass, such as agricultural waste products and energy crops, into ethanol involves the extraction and pretreatment of biomass components (cellulose, hemicellulose and lignin) and the enzymatic breakdown of crystalline cellulose fibers into monomer glucose by combined action of many enzymes.  Then, a conventional fermentation process is utilized to obtain ethanol.

Over the course of evolution, plant cell walls developed great strength in their architecture and molecular design in order to deal with environmental stresses and pathogens. With biofuels, we are looking for a way to break down these strong plant cell walls so that we can access the cellulose. As biophysicists, we are probing the biomass, including the unusually high thermal and mechanical stability of cellulose, to identify the weakest links that can be targeted during the conversion process. Also, optimal synergistic action of various enzymes known as cellulases is critical for efficient digestion of cellulose. It is an interesting and challenging biophysical problem, because heterogeneous catalysis occurs on crystalline cellulose surfaces where numerous factors play a role in overall hydrolysis. We have developed spatial models of cellulose degradation that can capture effects including chemical crowding and surface heterogeneity that have been shown to cause a reduction in hydrolysis rates.

How did you get into this area of research?

It was nearly eight years ago when two senior scientists at the Los Alamos National Laboratory (LANL), Cliff Unkefer and Paul Langan, introduced me to biofuels. Back then, I was working on protein dynamics and folding/misfolding problems. We decided to try using computational biophysics to gain a better understanding of carbohydrates and aromatic polymers associated with biomass. A local LANL grant headed by Paul Langan, currently the director of the Spallation Neutron Source at Oak Ridge National Laboratory, provided the initial funding to probe the different forms of cellulose crystalline structures. Several X-ray and neutron structures from Paul Langan’s team were instrumental in many of the computational problems that we handled.  Initially, we learned a great deal about the structural aspects of oligomers of glucose.  An effective collaboration with a USDA Scientist, Al French, helped us to fast-track almost two decades worth of structural knowledge on carbohydrates.  Also, the Center for Nonlinear Studies (CNLS) has supported several postdocs who worked on many of the biofuel projects.

How long have you been working on it?

Initial efforts that began in late 2007 focused primarily on cellulose. Subsequently, we also expanded our research portfolio to cover lignin and the enzymatic degradation of cellulose. Recently, we also started to look at biophysical problems linked to photosynthesis in algae. Making fuel from algae has now gained renewed interest, as it has the potential to produce valuable byproducts in addition to fuels. Algal fuels, unlike lignocellulosic ethanol, can also be used as fuels for aviation and don’t directly compete with food demands. We are fortunate to be working with Richard Sayre, a renowned crop researcher and molecular biologist. A few years back, he moved with his large research team to LANL in order to work on algal problems.

Have you had any surprise findings thus far?

We’ve had several surprises over the years. The first one relates to the nature of cellulose. In textbooks, the remarkable stability of cellulose fiber is often explained in terms of inter- and intra-chain hydrogen bonds. However, this didn’t quite explain why we needed to heat the cellulose fiber to almost 500K to even separate the sheets. By using a statistical mechanical, two-dimensional lattice model, we were able to show that multiple, alternative hydrogen bond patterns can exist within the crystalline layer, and that this “plasticity” of the hydrogen bond network greatly contributes to the stability of the layers over a wide range of temperatures. This was published in the Biophysical Journal in 2009 (image above). It was followed by a replica exchange molecular dynamics study that showed – in contrast to what was typically observed in non-interacting polymers – that cellulose oligomers become more rigid as the degree of polymerization increases.

The national recognition of our work became evident recently when one of the DOE bioenergy centers, Great Lakes Bioenergy Center (GLBRC), approached our team for help with theoretical calculations involving their core technology, liquid ammonia pretreatment. One of my former postdocs, Giovanni Bellesia, was instrumental in directing fundamental research work in biofuels at LANL. Shishir Chundawat, a former postdoc of Bruce Dale and currently on the faculty at the Rutgers University, played a pivotal role in establishing a collaborative research effort involving LANL and the GLBRC. Our theoretical studies targeted the pretreatment of liquid ammonia to catalyze the structural crossover from natural crystalline cellulose to another crystalline cellulose allomorph named cellulose III(I).  Experiments have shown an enhancement in enzymatic hydrolysis rates by up to five fold for cellulose III compared to natural crystalline cellulose. We looked at this enhancement from multiple angles to understand and further improve it; simple coarse-grained models of cellulose, detailed all-atom comparative studies on different cellulose allomorphs, quantum mechanical calculations, and mechanistic and rule-based kinetic models. The resulting theoretical studies captured in a series of more than 7 papers over 5 years provided clues on improving this chemical pretreatment that may help lower the cost of producing biofuels from plants.  We were able to characterize the mechanistic details of how liquid ammonia penetrates and disrupts the hydrogen bonding network in native cellulose to create cellulose III and the differences between native and non-native celluloses from the perspective of enzymatic digestion. Also, we suggested – counter-intuitively – that increased binding of enzymes to cellulose polymers does not always lead to faster breakdown into simple sugars. In addition to the enhancement in cellulose digestibility, ammonia-based treatments have additional advantages over other chemical treatment technologies in terms of both economic feasibility and environmental impact.

What is particularly interesting about the work from the perspective of other researchers?

As stated earlier, crystalline cellulose exists in various allomorphic states, many of which don’t occur naturally.  A greater understanding of unnatural cellulose allomorphs, such as the case with cellulose III and their biodegradation, is bound to improve the efficiency of next-generation extractive ammonia based pre-treatments. We identified key structural and molecular features in cellulose III and the action of enzymes on this unnatural cellulose substrate that can be optimized.  Our investigation of substrate properties, especially on the structural transition to non-native form of cellulose II as a way for efficient biomass conversion, makes our research efforts unique. There are various theoretical and computational efforts ongoing at many universities, National labs and DOE bioenergy centers. The majority of them focus on the native cellulose and its degradation by biological and thermochemical conversions and pyrolysis techniques. Greg Beckham at NREL has carried out ground-breaking computational work on enzymatic digestion of cellulose and continues to make great advances.  Also, Blake Simmons and Seema Singh at JBEI have made breakthroughs and have come up with concepts towards efficient biomass treatment using ionic liquids by considering the problem with both the experimental and computational techniques. At Oak Ridge National Lab, Jeremy Smith and his team have been combining large-scale computing with neutron scattering to look at plant cell walls with molecular machines such as cellulosomes, helping to overcome the problem of resistant cell walls.

Our theoretical and computational studies on cellulosic biofuels complement a larger effort on algal biofuels at LANL. LANL was a key player in the National Alliance for Advanced Biofuels and Bioproducts (NAABB), an algal biofuels research consortium consisted of 39 partner institutions. It was formed to understand the impact of algae on overall biomass and liquid transportation fuel production.  The partners of this consortium came with expertise in feedstock supply, feedstock logistics, and conversion/production pathways. The success of this consortium has led to many active programs on algal research at LANL. Recently, we started collaborations with Dick Sayre, who was the Chief Scientist of the NAABB consortium. Sayre is currently developing novel methods to increase algal performance. We are involved in his efforts that seek to genetically engineer algae to absorb light more efficiently for cost-effective algae-to-fuel production. My postdoc, Cesar Lopez, is heading the theoretical efforts that consider the non-photoquenching mechanisms involving photosynthetic antennae systems in a thylakoid membrane mimic.

What is particularly interesting about the LANL work on biofuels from the perspective of the public?

Let me focus on the achievements of NAABB consortium where LANL was a key player. In a matter of three years, NAABB managed to develop technologies that are likely to reduce the cost of algae-based biocrude from $240 (starting baseline) to $7.50 per gallon. They were able to achieve the cost reduction by two-orders of magnitude as follows (

  • Identified new, robust, high oil yield algal strains that can reduce the cost by 85%
  • Developed a new, efficient open pond system that reduces cost by 16%
  • Demonstrated the use of an electrocoagulation harvesting technology that reduces cost by 14%
  • Notably, they created a unique hydrothermal liquefaction system that combines extraction and conversion to deliver high biocrude yield without the need for extraction solvents, resulting in an 86% cost reduction

The above were accomplished with $48.6 million public funds from the American Recovery and Reinvestment Act of 2009 and $19.1 million in private funds.

Finally, I should point out that, since its inception in 1943 in a beautiful remote area of New Mexico, LANL has been making pioneering discoveries and transformational solutions for national security challenges.   Although primarily assigned to safeguard the United States’ stockpile of nuclear weapons, it is also home to talented and creative scientists who tackle burning security issues and challenges facing the nation, including cyber security, biosecurity and energy security.  I am glad to be part of it.

Do you have a cool image you want to share with the blog post related to this research?

The image at the top of this blog post was featured as a cover of Biophysical Journal in 2009. It depicts the following: In plants, cellulose is biosynthesized by polymerization of glucose into chains (green, dark blue). As the chains are produced, they are assembled into sheets (light blue) that stack on top of each other to form nanometer-thick crystalline microfibrils (blue rods) in the cell wall (gold). The microfibrils are encrusted in other polysaccharides and lignin. Cellulose stability is maintained by networks of hydrogen bonds (yellow dashes) within the sheets. These hydrogen bonds must be broken to release glucose for the production of biofuels.


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