Tag: fusel oil

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Bioelectrically engineered fuel produced by yeasts

Yeasts such as Saccharomyces cerevisiae are, as the name suggests, used for large scale production of beer and other alcoholic beverages. Their high salt and ethanol tolerance not only makes them useful for the production of beverages, but also suitable for the production of combustion fuels at high alcohol concentrations. Besides ethanol, long-chain fusel alcohols are of high interest for biofuel production as well. Bioethanol is already mixed with gasoline and thus improves the CO2 balance of internal combustion engines. This liquid biofuel is made from either starch or lignocellulose. The production and use of bioethanol supports local economies, reduces CO2 emissions and promotes self-sufficiency. The latter is especially important for resource-depleted landlocked countries.

In order to efficiently produce ethanol and other alcohols from lignocellulose hydrolysates, yeasts must use both glucose and pentoses such as xylose and arabinose. This is because biomass is rich in both lignocellulose and thus glucose and xylose. However, this is also the main disadvantage of using Saccharomyces cerevisiae because it does not ferment xylose. Consequently, the identification of another yeast strains capable of fermenting both these sugars could solve the problem. Highly efficient yeasts can be grown in co-cultures with other yeasts capable of lignocellulose fermentation for ethanol production. Such a yeast is, for example, Wickerhamomyces anomalous.

To further improve ethanol production, bioelectric fermentation technology supporting traditional fermentation can be used. The microbial metabolism can thus be controlled electrochemically. There are many benefits of this technology. The fermentation process becomes more selective due to the application of an electrochemical potential. This, in turn, increases the efficiency of sugar utilization. In addition, the use of additives to control the redox equilibrium and the pH is minimized. Ultimately cell growth can be stimulated, further increasing alcohol production.

Such bioelectric reactors are galvanic cells. The electrodes used in such a bioelectric reactor may act as electron acceptors (anodes) or source (cathodes). Such electrochemical changes affect the metabolism and cell regulation as well as the interactions between the yeasts used. Now, a research group from Nepal (a resource-depleted landlocked country) has used new yeast strains of Saccharomyces cerevisiae and Wickerhamomyces anomalous in a bioelectric fermenter to improve ethanol production from biomass. The results were published in the journal Frontiers in Energy Research.

For their study, the researchers chose Saccharomyces cerevisiae and Wickerhamomyces anomalus as both are good ethanol producers. The latter is to be able to convert xylose to ethanol. After the researchers applied a voltage to the bioelectrical system, ethanol production doubled. Both yeasts formed a biofilm on the electrodes, making the system ideal for use as a flow-through system because the microorganisms are not washed out.

Saccharomyces cerevisiae cells in a brightfield microscopic image of 600-fold magnification (Foto: Amanda Luraschi)

The researchers speculated that the increased ethanol production was due to the better conversion of pyruvate to ethanol − the yeast’s central metabolic mechanism. The researchers attributed this to accelerated redox reactions at the anode and cathode. The applied external voltage polarized the ions present in the cytosol, thus facilitating the electron transfer from the cathode. This and the accelerated glucose oxidation probably led to increased ethanol production.

Normally, pyruvate is converted into ethanol in fermentation yeast. External voltage input can control the kinetics of glucose metabolism in Saccharomyces cerevisiae under both aerobic and anaerobic conditions. Intracellular and transplasmembrane electron transfer systems play an important role in electron transport across the cell membrane. The electron transfer system consists of cytochromes and various redox enzymes, which confer redox activity to the membrane at certain sites.

The authors also found that an increased salt concentration improved conductivity and therefore ethanol production. The increased ethanol production from lignocellulosic biomass may have been also be due to the presence of various natural compounds that promoted yeast growth. When the cellulose acetate membrane was replaced by a Nafion™ membrane, ethanol production also increased. This was perhaps due to improved transport of xylose through the Nafion™ membrane as well as the decrease of the internal resistance. A further increase of ethanol production was observed when the bioelectrical reactor was operated with fine platinum particles coated on the platinum anode and neutral red deposited on the graphite cathode.

Several yeast cultures from left to right: Saccharomyces cerevisiae, Candida utilis, Aureobasidium pullulans, Trichosporum cutaneum, Saccharomycopsis capsularis, Saccharomycopsis lipolytica, Hanseniaspora guilliermondii, Hansenula capsulata, Saccharomyces carlsbergensis, Saccharomyces rouxii, Rhodotorula rubra, Phaffia rhodozyba, Cryptococcus laurentii, Metschnikowia pulcherrima, Rhodotorula pallida

At Frontis Energy, we think that the present study is promising. However, long-chain fusel alcohols should be considered in the future as they are less volatile and better compatible with current internal combustion engines. These can also be easily converted into the corresponding long-chain hydrocarbons.

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Producing liquid bio-electrically engineered fuels from CO2

At Frontis Energy we have spent much thought on how to recycle CO2. While high value products such as polymers for medical applications are more profitable, customer demand for such products is too low to recycle CO2 in volumes required to decarbonize our atmosphere to pre-industrial levels. Biofuel, for example from field crops or algae has long been thought to be the solution. Unfortunately, they require too much arable land. On top of their land use, biochemical pathways are too complex to understand by the human brain. Therefore, we propose a different way to quickly reach the target of decarbonizing our planet. The proce­dure begins with a desired target fuel and suggests a mi­crobial consortium to produce this fuel. In a second step, the consortium will be examined in a bio-electrical system (BES).

CO2 can be used for liquid fuel production via multiple pathways. The end product, long-chain alcohols, can be used either directly as fuel or reduced to hydrocarbons. Shown are examples of high level BEEF pathways using CO2 and electricity as input and methane, acetate, or butanol as output. Subsequent processes are 1, aerobic methane oxida­tion, 2, direct use of methane, 3 heterotrophic phototrophs, 4, acetone-butanol fermentation, 5, heterotrophs, 6, butanol di­rect use, 7, further processing by yeasts

Today’s atmospheric CO2 imbalance is a consequence of fossil carbon combus­tion. This real­ity requires quick and pragmatic solutions if further CO2 accu­mulation is to be prevented. Direct air capture of CO2 is moving closer to economic feasibility, avoid­ing the use of arable land to grow fuel crops. Producing combustible fuel from CO2 is the most promis­ing inter­mediate solution because such fuel integrates seamlessly into existing ur­ban in­frastructure. Biofuels have been ex­plored inten­sively in re­cent years, in particular within the emerging field of syn­thetic biol­ogy. How­ever tempt­ing the application of genetically modified or­ganisms (GMOs) ap­pears, non-GMO technology is easier and faster to im­plement as the re­quired microbial strains al­ready exist. Avoiding GMOs, CO2 can be used in BES to produce C1 fu­els like methane and precursors like formic acid or syngas, as well as C1+ com­pounds like ac­etate, 2-oxybut­yrate, bu­tyrate, ethanol, and butanol. At the same time, BES inte­grate well into urban in­frastructure without the need for arable land. However, except for meth­ane, none of these fuels are readily com­bustible in their pure form. While elec­tromethane is a com­mercially avail­able al­ternative to fossil natu­ral gas, its volumetric energy den­sity of 40-80 MJ/m3 is lower than that of gasoline with 35-45 GJ/m3. This, the necessary technical modifications, and the psychological barrier of tanking a gaseous fuel make methane hard to sell to automobilists. To pro­duce liq­uid fuel, carbon chains need to be elongated with al­cohols or better, hy­drocarbons as fi­nal prod­ucts. To this end, syngas (CO + H2) is theoreti­cally a viable option in the Fischer-Tropsch process. In reality, syngas pre­cursors are ei­ther fossil fu­els (e.g. coal, natural gas, methanol) or biomass. While the for­mer is ob­viously not CO2-neu­tral, the latter com­petes for arable land. The di­rect con­version of CO2 and electrolytic H2 to C1+ fuels, in turn, is catalyzed out by elec­troactive microbes in the dark (see title figure), avoid­ing food crop com­petition for sun-lit land. Unfortunately, little re­search has been under­taken beyond proof of con­cept of few electroactive strains. In stark con­trast, a plethora of metabolic studies in non-BES is avail­able. These studies often pro­pose the use of GMOs or complex or­ganic sub­strates as precur­sors. We propose to systemati­cally identify metabolic strategies for liquid bio-electrically engineered fuel (BEEF) production. The fastest approach should start by screening meta­bolic data­bases using es­tablished methods of metabolic modeling, fol­lowed by high throughput hypothesis testing in BES. Since H2 is the intermediate in bio-electrosynthesis, the most efficient strategy is to focus on CO2 and H2 as di­rect pre­cursors with as few in­termediate steps as pos­sible. Scala­bility and energy effi­ciency, eco­nomic feasibil­ity that is, are pivotal elements.

First, an electrotrophic acetogen produces acetate, which then used by heterotrophic algae in a consecutive step.

The biggest obstacle for BEEF production is lacking knowledge about pathways that use CO2 and electrolytic H2. This gap exists despite metabolic data­bases like KEGG and more recently KBase, making metabolic design and adequate BEEF strain selection a guessing game rather than an educated ap­proach. Nonetheless, metabolic tools were used to model fuel pro­duction in single cell yeasts and various prokaryotes. In spite of their shortcomings, metabolic data­bases were also employed to model species interactions, for example in a photo-het­erotroph consor­tium using software like ModelSEED / KBase (http://mod­elseed.org/), RAVEN / KEGG and COBRA. A first sys­tematic at­tempt for BEEF cul­tures produci­ng acetate demonstrated the usability of KBase for BES. This research was a bottom-up study which mapped ex­isting genomes onto existing BEEF consor­tia. The same tool can also be em­ployed in a top-down ap­proach, starting with the desired fuel to find the re­quired or­ganisms. Some possi­ble BEEF organisms are the following.

Possible pathways for BEEF production involving Clostridium, 3, or heterotrophic phototrophs, 7, further processing by yeasts

Yeasts are among the microorganisms with the greatest potential for liquid biofuel production. Baker’s yeast, (Saccha­romyces cerevisiae) is the most promi­nent exam­ple. While known for ethanol fermentat­ion, yeasts also produce fusel oils such as bu­tane, phenyl, and amyl derivate aldehy­des and alco­hols. Unlike ethanol, which is formed via sugar fer­mentation, fusel oil is syn­thesized in branched-off amino acid pathways followed by alde­hyde reduction. Many en­zymes involved in the re­duction of aldehydes have been identified, with al­cohol dehydro­genases be­ing the most commonly ob­served. The corre­sponding reduc­tion reactions require reduced NADH⁠ but it is not known whether H2 pro­duced on cathodes of BES can be in­volved.
Clostridia, for example Clostridium acetobutylicum and C. carboxidivo­rans, can pro­duce alcohols like butanol, isopropanol, hexanol, and ketones like acetone from complex sub­strates (starch, whey, cel­lulose, etc. ) or from syngas. Clostridial me­tabolism has been clarified some time ago and is dif­ferent from yeast. It does not necessar­ily require com­plex precursors for NAD+ reduction and it was shown that H2, CO, and cath­odes can donate elec­trons for alcohol production. CO2 and H2 were used in a GMO clostridium to produce high titers of isobu­tanol. Typi­cal representa­tives for acetate produc­tion from CO2 and H2 are C. ljungdahlii, C. aceticum, and Butyribac­terium methy­lotrophicum. Sporo­musa sphaeroides pro­duces acetate in BES. Clostridia also dominated mixed cul­ture BESs converting CO2 to butyrate. They are therefore prime targets for low cost biofuel production. Alcohols in clostridia are produced from acetyl-CoA. This reaction is re­versible, al­lowing ac­etate to serve as substrate for biofuel production with extra­cellular en­ergy sup­ply. Then, en­ergy con­servation, ATP syn­thesis that is, can be achieved from ethanol electron bifurca­tion or H2 oxida­tion via respi­ration. While pos­sible in anaero­bic clostridia, it is hitherto unknown whether elec­tron bifurca­tion or res­piration are linked to alcohols or ke­tone synthesis.
Phototrophs like Botryococcus produce C1+ biofuels as well. They synthesize a number of different hydro­carbons including high value alkanes and alkenes as well as terpenes. However, high titers were achieved by only means of ge­netic engineering, which is economically not feasible in many countries due to regulatory constrains. Moreover, aldehyde dehy­dration/deformylation to alkanes or alkenes requires molecular oxygen to be present. Also the olefin path­way of Syne­chococcus depends on molecular oxygen with the cytochrome P450 involved in fatty acid de­carboxylation. The presence of molecular oxygen affects BES performance due to immediate product degrada­tion and unwanted cathodic oxygen reduction. In contrast, our own preliminary experi­ments (see title photo) and a corrosion experi­ment show that algae can live in the dark using electrons from a cath­ode. While the en­zymes in­volved in the production of some algal biofuels are known (such as olefin and alde­hyde de­formylation), it is not known whether these pathways are connected to H2 utilization (perhaps via ferredox­ins). Such a con­nection would be a promising indicator for the possibility of growing hydrocar­bon produc­ing cyanobacteria on cathodes of BES and should be examined in future research.
At Frontis Energy we believe that a number of other microorganisms show potential for BEEF production and these deserve further investi­gation. To avoid GMOs, BES compatible co-cultures must be identified via in silico meta­bolic reconstruc­tion from existing databases. Possible inter-species intermediates are unknown but are prerequisite for suc­cessful BES operation. Finally, a techno-economical assessment of BEEF pro­duction, with and with­out car­bon taxes, and compared with chemical methods, will direct future research.